Biochemical characterization of a novel glucose-tolerant GH3 ß-glucosidase (Bgl1973) from Leifsonia sp. ZF2019.

Beta-glucosidase (Bgl) is an enzyme with considerable food, beverage, and biofuel processing potential. However, as many Bgls are inhibited by their reaction end product glucose, their industrial applications are greatly limited. In this study, a novel Bgl gene (Bgl1973) was cloned from Leifsonia sp. ZF2019 and heterologously expressed in E. coli. Sequence analysis and structure modeling revealed that Bgl1973 was 748 aa, giving it a molecular weight of 78 kDa, and it showed high similarity with the glycoside hydrolase 3 (GH3) family Bgls with which its active site residues were conserved. By using pNPGlc (p-nitrophenyl-ß-D-glucopyranoside) as substrate, the optimum temperature and pH of Bgl1973 were shown to be 50 °C and 7.0, respectively. Bgl1973 was insensitive to most metal ions (12.5 mM), 1% urea, and even 0.1% Tween-80. This enzyme maintained 60% of its original activity in the presence of 20% NaCl, demonstrating its excellent salt tolerance. Furthermore, it still had 83% residual activity in 1 M of glucose, displaying its outstanding glucose tolerance. The Km, Vmax, and kcat of Bgl1973 were 0.22 mM, 44.44 µmol/min mg, and 57.78 s-1, respectively. Bgl1973 had a high specific activity for pNPGlc (19.10 ± 0.59 U/mg) and salicin (20.43 ± 0.92 U/mg). Furthermore, molecular docking indicated that the glucose binding location and the narrow and deep active channel geometry might contribute to the glucose tolerance of Bgl1973. Our results lay a foundation for the studying of this glucose-tolerant ß-glucosidase and its applications in many industrial settings. KEY POINTS: • A novel ß-glucosidase from GH3 was obtained from Leifsonia sp. ZF2019. • Bgl1973 demonstrated excellent glucose tolerance. • The glucose tolerance of Bgl1973 was explained using molecular docking analysis.

Selective transformation of crocin-1 to crocetin-glucosyl esters by ß-glucosidase (Lf18920) from Leifsonia sp. ZF2019: Insights from molecular docking and point mutations.

Crocetin di/mono-glucosyl esters (crocin-4 and crocin-5) are rarely distributed in nature, limiting their potential applications in the food and pharmaceutical industries. In the present study, a novel GH3 family ß-glucosidase Lf18920 was identified from Leifsonia sp. ZF2019, which selectively hydrolyzed crocin-1 (crocetin di-gentiobiosyl ester) to crocin-5 and crocin-4, but not to its aglycone, crocetin. Under the optimal condition of 40 °C and pH 6.0 for 120 min, Lf18920 almost completely hydrolyzed crocin-1, yielding 73.50±5.66 % crocin-4 and 16.19±1.38 % crocin-5. Molecular docking and point mutation studies revealed that Lf18920 formed a narrow binding channel that facilitated crocin-1 binding. Five single amino acid variants (D50A, D53A, W274A, G420A, and Q421A) were constructed, all of which showed reduced hydrolytic activity. Mutations at D50 and D53, located distal to the active site, increased binding energy and decreased hydrolytic activity, while mutations at W274, G420, and Q421, proximal to the active site, disrupted hydrolytic function. These findings suggest that the narrow binding channel and specific enzyme-substrate interactions are crucial for Lf18920's selective hydrolytic activity. Overall, this study is the first to report a ß-glucosidase capable of selectively transforming crocin-1 to crocetin di/mono-glucosyl esters, offering potential for synthesizing crocin-4 and crocin-5.

Determination of oxidative deterioration in edible oils by high-pressure photoionization time-of-flight mass spectrometry.

Since lipid oxidation often causes serious food safety issues worldwide, determination of oil's oxidative deterioration becomes quite significant, which still calls for efficient analytical methods. In this work, high-pressure photoionization time-of-flight mass spectrometry (HPPI-TOFMS) was firstly introduced for rapid detection of oxidative deterioration in edible oils. Through non-targeted qualitative analysis, oxidized oils with various oxidation levels were successfully discriminated for the first time by coupling HPPI-TOFMS with the orthogonal partial least squares discriminant analysis (OPLS-DA). Furthermore, by targeted interpretation of the HPPI-TOFMS mass spectra and the subsequent regression analysis (signal intensities vs TOTOX values), good linear correlations were observed for several predominant VOCs. Those specific VOCs were promising oxidation indicators, which would play important roles as TOTOX to judge the oxidation states of tested samples. The proposed HPPI-TOFMS methodology can be used as an innovative tool for accurate and effective assessment of lipid oxidation in edible oils.

Investigation of isoflavone constituents from tuber of Apios americana Medik and its protective effect against oxidative damage on RIN-m5F cells.

In our previous study, AI-3, a mixture of isoflavones, was obtained from Apios Americana Medik tuber and showed strong protective ability on oxidative damaged RIN-m5F cells. This study aimed to identify the main compounds of AI-3 and elucidate their activities and underlying mechanism. In results, eleven compounds were purified from AI-3. Among them, Compound 8 (2'-Hydroxy, 5-methoxy genistein-7-O-glucoside, HMG) was the most effective compound against H2O2-induced injury in RIN-m5F cells (stronger than positive control α-LA). Further RNA-seq analysis found that compared with H2O2 group, 388 differentially expressed genes (DEGs) were identified in HMG group. The enrichment analyses revealed fluid shear stress and atherosclerosis pathway and hepatocellular carcinoma pathway enriched the most DEGs, in which HOX-1, GST, NQO1, SQSTM, TrxR1 were significantly up-regulated. The finding indicated Keap1-Nrf2-ARE signaling pathway may play essential role in the protective effect of HMG on oxidative damaged RIN-m5F cells.

Seasonal variations of the antioxidant composition in ground bamboo Sasa argenteastriatus leaves.

Sasa argenteastriatus, with abundant active compounds and high antioxidant activity in leaves, is a new leafy bamboo grove suitable for exploitation. To utilize it more effectively and scientifically, we investigate the seasonal variations of antioxidant composition in its leaves and antioxidant activity. The leaves of Sasa argenteastriatus were collected on the 5th day of each month in three same-sized sample plots from May 2009 to May 2011. The total flavonoids (TF): phenolics (TP) and triterpenoid (TT) of bamboo leaves were extracted and the contents analyzed by UV-spectrophotometer. Our data showed that all exhibited variations with the changing seasons, with the highest levels appearing in November to March. Antioxidant activity was measured using DPPH and FRAP methods. The highest antioxidant activity appeared in December with the lowest in May. Correlation analyses demonstrated that TP and TF exhibited high correlation with bamboo antioxidant activity. Eight bamboo characteristic compounds (orientin, isoorientin, vitexin, homovitexin and p-coumaric acid, chlorogenic acid, caffeic acid, ferulic acid) were determined by RP-HPLC synchronously. We found that chlorogenic acid, isoorientin and vitexin are the main compounds in Sasa argenteastriatus leaves and the content of isovitexin and chlorogenic acid showed a similar seasonal variation with the TF, TP and TT. Our results suggested that the optimum season for harvesting Sasa argenteastriatus leaves is between autumn and winter.

Microemulsion Delivery System Improves Cellular Uptake of Genipin and Its Protective Effect against Aß1-42-Induced PC12 Cell Cytotoxicity.

Genipin has attracted much attention for its hepatoprotective, anti-inflammatory, and neuroprotection activities. However, poor water solubility and active chemical properties limit its application in food and pharmaceutical industries. This article aimed to develop a lipid-based microemulsion delivery system to improve the stability and bioavailability of genipin. The excipients for a genipin microemulsion (GME) preparation were screened and a pseudo-ternary phase diagram was established. The droplet size (DS), zeta potential (ZP), polydispersity index (PDI), physical and simulated gastrointestinal digestion stability, and in vitro drug release properties were characterized. Finally, the effect of the microemulsion on its cellular uptake by Caco-2 cells and the protective effect on PC12 cells were investigated. The prepared GME had a transparent appearance with a DS of 16.17 ± 0.27 nm, ZP of -8.11 ± 0.77 mV, and PDI of 0.183 ± 0.013. It exhibited good temperature, pH, ionic strength, and simulated gastrointestinal digestion stability. The in vitro release and cellular uptake data showed that the GME had a lower release rate and better bioavailability compared with that of free genipin. Interestingly, the GME showed a significantly better protective effect against amyloid-ß (Aß1-42)-induced PC12 cell cytotoxicity than that of the unencapsulated genipin. These findings suggest that the lipid-based microemulsion delivery system could serve as a promising approach to improve the application of genipin.

Rapid Identification of Adulteration in Extra Virgin Olive Oil via Dynamic Headspace Sampling and High-Pressure Photoionization Time-of-Flight Mass Spectrometry.

High-pressure photoionization time-of-flight mass spectrometry (HPPI-TOFMS) combined with dynamic headspace sampling was developed for rapid identification of adulteration in extra virgin olive oil (EVOO). The volatile organic compound (VOC) fingerprints of EVOO, refined rapeseed oil (r-RO), peanut oil (PO), corn oil (CO), fragrant rapeseed oil (f-RO), and sunflower oil (SO) were obtained in just 1.5 min, which enabled satisfactory classification of different edible oils. 1,4-Bis(methylene)cyclohexane and dimethyl disulfide were unique VOCs in r-RO and f-RO, respectively, while 2,5-dimethylpyrazine and 2-methylpyrazine were distinctive VOCs in PO. Percentages as low as 3% r-RO, 1% PO, and 1% f-RO in r-RO-EVOO, PO-EVOO, and f-RO-EVOO mixtures, respectively, were successfully identified based on the characteristic VOCs. Linear regression equations of these VOCs were established and utilized for predicting the adulteration proportions. The good agreements between the actual adulteration proportions and the predicted ones demonstrated that HPPI-TOFMS was reliable for the quantification of EVOO adulteration.

Accelerated Solvent Extraction of Antioxidant Compounds from Gardeniae Fructus and Its Acetylcholinesterase Inhibitory and PC12 Cell Protective Activities.

Gardeniae fructus is a common neuroprotective medicinal food in China, however the extraction efficiency and mixture activities are rarely mentioned. In this study, accelerated solvent extraction (ASE) parameters were optimized by a response surface methodology to extract antioxidants from Gardeniae fructus. Neuroprotective activity was evaluated using H2O2 and amyloid-ß25-35 peptide-treated PC12 cells. By comparing with three other extract methods (i.e., heated refluxing extraction (HRE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE)), it was found that the yield (35.10%), total iridoids (27.69%), total flavonoid (6.12%) content, antioxidant activities (IC50 on DPPH, 164.46 µg/mL; FRAP value 4703.54 µmol/L), and acetylcholinesterase inhibitory ability (IC50 92.58 µg/mL) of ASE extract under the optimal condition (150 °C temperature, 10 min static time, 60% ethanol, 2 extract cycles) were significantly higher than other extract methods. The strongest ability to protect PC12 cells from damage was also present in ASE extract, as evidenced by decreasing lactate dehydrogenase and malondialdehyde levels, elevating superoxide dismutase and glutathioneperoxidase activities. Compositional analysis indicated that the extremely high crocetin level in ASE extract (1.30 µg/mg) may offer great potential. Our results indicated that ASE is a proper extraction method that could offer great potential for finding the neuroprotective ability of Gardeniae fructus for the treatment of AD.

Molecular size of collagen peptide reverses the permeability of Caco-2 cells.

Intestinal permeability to size-classified fish collagen peptides (FCPs) was examined using Caco-2 monolayers. Minimum-sized FCPs were most efficiently transported across the Caco-2 monolayers. Permeability depended on peptide size. It was independent of the H(+)-coupled peptide transport system but was associated with tight junction permeability, suggesting that the paracellular pathway is responsible for transepithelial transport of collagen peptides.

Physicochemical and rheological characterization of pectin-rich polysaccharides from Gardenia jasminoides J. Ellis flower.

Gardenia (Gardenia jasminoides J. Ellis) is regarded as an edible medicine plant in China. Here, gardenia flower polysaccharide fraction (GFPF) was extracted by water at 90°C and its chemical composition, rheological properties, and antioxidant activities of GFPF were investigated. The GFPF extraction yield was 18.04 ± 1.81% (W/W) and mainly comprised neutral sugars (46.83 ± 3.14%), uronic acid (35.21 ± 0.17%), protein (1.63 ± 0.34%), and total phenol (9.49 ± 0.08 mgGAE/g). Galacturonic acid (41.05 ± 0.59%) was the main monosaccharide, and galactose, glucose, arabinose, rhamnose, xylose, mannose, and glucuronic acid were also detected in GFPF. Its degree of esterification was 32.76 ± 1.52%. FT-IR spectra analysis showed a similar absorption pattern between GFPF and pectin from apple. The results suggested that GFPF was low methoxy pectin. Thermogravimetric analysis and zeta potential analysis indicated that the pectin was stable under high temperature and alkaline condition. Steady rheology showed that the GFPF dispersion was a shear thinned pseudoplastic fluid with high apparent viscosities at concentration above 2%. The degree of pseudoplasticity of the solutions increased with the concentrations increased and the temperatures decreased. DPPH and ABTS free radical scavenging assay indicated that GFPF had relatively high antioxidant activity. The results showed that gardenia flower was rich in pectin polysaccharides with low methoxy pectin. It had high apparent viscosities at concentration above 2% and had good antioxidant activity. The data suggested that GFPF can be a new resource of low methoxy pectin with potential application as thicker or gelling agents in food industry.

Purification, characterization, and cDNA cloning of a Bowman-Birk type trypsin inhibitor from Apios americana Medikus tubers.

An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) M and 1.0 x 10(-6) M respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.

Postharvest chitosan-g-salicylic acid application alleviates chilling injury and preserves cucumber fruit quality during cold storage.

The effect of salicylic acid with and without chitosan, or a chitosan-g-salicylic acid complex, on chilling injury and post-harvest quality of cucumber stored at 2 °C for 12 days plus 2 days at 20 °C was investigated. The results showed the chitosan-g-salicylic acid coating inhibited chilling injury better than salicylic acid alone or with chitosan. Chitosan-g-salicylic acid also reduced weight loss and respiration rate, limited increases in malondialdehyde content and electrolyte leakage, and maintained higher total soluble solids, chlorophyll and ascorbic acid content. Furthermore, this coating increased the endogenous salicylic acid concentrations and antioxidant enzyme activities including superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase in cucumber during storage. Our study suggests that chitosan-g-salicylic acid alleviated chilling injury in cucumber through sustained-release of salicylic acid and the higher antioxidant enzymes concentrations.

Evaluation of reactive oxygen species scavenging activities and DNA damage prevention effect of Pleioblastus kongosanensis f. aureostriatus leaf extract by chemiluminescence assay.

Reactive oxygen species scavenging effect of Pleioblastus kongosanensis f. aureostriatus leaf extract against O2(-), OH and H2O2 were investigated by chemiluminescence methods in vitro. Bamboo grass leaves were extracted with 70% ethanol solution and sequentially partitioned with solvents in an order of increasing polarity. Among fractions of different polarity, BuOH and EtOAc fractions showed powerful scavenging activities than others, and showed better scavenging ability on OH than that of O2(-)and H2O2, with IC50 of 0.55 µg/mL and 0.60 µg/mL, respectively. Both OH-induced DNA damage model by chemiluminescence assay and plasmid pUC18 double-strand break model by agarose gel electrophoresis showed that BuOH and EtOAc fractions had remarkable concentration-dependent prevention effect on the OH-induced damage of DNA attribute to their good scavenging effects on ROS. Results from the compositional analysis of different fractions indicate that the flavonoids in the Pleioblastus kongosanensis f. aureostriatus leaf may be responsible for its ROS scavenging activity and DNA damage prevention ability.

Altitudinal variation of antioxidant components and capability in Indocalamus latifolius (Keng) McClure leaf.

Indocalamus latifolius (Keng) McClure leaf is a popular food material in East Asia due to its antioxidant and anticorrosive activities. To utilize it more effectively, we investigated the discrepancy of antioxidant activities and active compound content in Indocalamus latifolius leaf along with the altitude change. Total flavonoids, phenolics, titerpenoids and eight characteristic active constituents, i.e, orientin, isoorientin, vitexin, homovitexin, p-coumaric acid, chlorogenic acid, caffeic acid, and ferulic acid, were determined by UV-spectrophotometer and synchronous RP-HPLC, respectively. Antioxidant activity was measured using DPPH and FRAP methods. Our data showed that the content of TP and TF, DPPH radical scavenging ability and ferric reduction power of Indocalamus latifolius leaf changed as altitude altered, with the trends of decreasing gradually when lower than 700 m and then increasing to 1,000 m. Chlorogenic acid and orientin were the main characteristic compounds in Indocalamus latifolius leaf and were also affected by altitude. Our result indicated that higher altitude with an adverse environment is conducive to secondary metabolite accumulation for Indocalamus latifolius. It would provide a theoretical basis to regulate the leaf collection conditions in the industrial use of Indocalamus latifolius leaf.

Enhancement of amygdalin activated with ß-D-glucosidase on HepG2 cells proliferation and apoptosis.

The growth inhibition and induction of apoptosis brought by amygdalin and activated with ß-D-glucosidase were tested for cytoactivity in HepG2 cells. The MTT viability assay showed that all samples had effects on HepG2 proliferation in dose and time response manners. IC50 of stand-alone amygdalin and activation with ß-D-glucosidase on the proliferation of HepG2 cells for 48 h were 458.10 mg/mL and 3.2 mg/mL, respectively. Moreover, apoptotic cells were determined by AO/EB (acridine orange/ethidium bromide) fluorescent staining method and Annexin V-FITC/PI staining flow cytometry cell cycle analysis. With increasing of amygdalin concentration and the incubation time, the apoptotic rate was heightened. Compared with the control, there was significant difference (p<0.01). Together, these findings indicate that amygdalin had no strong anti-HepG2 activity; however the ingredients of amygdalin activated with ß-D-glucosidase had a higher and efficient anti-HepG2 activity. It was therefore suggested that this combination strategy may be applicable for treating tumors with a higher activity.

Chicken collagen hydrolysate protects rats from hypertension and cardiovascular damage.

We previously reported that chicken collagen hydrolysate (CCH) has strong angiotensin I converting enzyme (ACE) inhibitory activity and antihypertensive effects on spontaneously hypertensive rats. Here, we investigated the chronic therapy effects of CCH on blood pressure and vascular relaxation in a cardiovascular damage model of Wistar-Kyoto rats induced by N-nitro-l-arginine methyl ester (L-NAME). Following co-treatment with CCH for 4 weeks, the increment of systolic blood pressure was suppressed significantly. At 8 weeks, the vasorelaxation of thoracic aorta increased significantly, and cardiovascular damage was ameliorated. The concentration of soluble intercellular adhesion molecule-1 (ICAM-1) in blood was reduced significantly by long-term administration of CCH, whereas the nitric oxide concentration was increased significantly at 1 hour post-treatment. The results suggest that beneficial effects of CCH result from antihypertensive function, but also from inhibition of cardiovascular damage to the endothelial cells via its ACE inhibitory activity and regulation of nitric oxide and ICAM-1, which suggests that CCH may be useful as a medicinal food for patients with cardiovascular disease.

Chicken collagen hydrolysate reduces proinflammatory cytokine production in C57BL/6.KOR-ApoEshl mice.

Effects of chicken collagen hydrolysate (CCH) on atherogenesis in apolipoprotein E-deficient C57BL/6.KOR-ApoE(shl) mice were investigated in this paper. The mice were fed on either a normal CE-2 diet (control group) or a diet containing 10% CCH (CCH group) for 12 wk. Compared with that of the control, the amount of total plasma cholesterol, total hepatic cholesterol and hepatic triglycerides in the CCH group was reduced by 14.4, 24.7 and 42.8%, respectively. Histological analysis results showed that the abundance of diffuse hepatic lipid droplets and fat vacuoles was decreased in the CCH group. Meanwhile, the concentration of proinflammatory cytokines in the CCH group plasma, including interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), and tumor necrosis factor-alpha (TNF-alpha), was downregulated by 43.4, 17.9 and 24.1%, respectively. The present results suggested that CCH treatment might help prevent atherosclerosis through not only its lipid-lowering effects but also inhibiting expression of inflammatory cytokines.

The bioavailable octapeptide Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro stimulates nitric oxide synthesis in vascular endothelial cells.

Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro (GAXGLXGP, X: Hyp), an octapeptide contained in chicken collagen hydrolysate, inhibits angiotensin I-converting enzyme activity in vitro. Intestinal Caco-2 and bovine aortic endothelial cells (BAECs) were used to investigate whether the transported GAXGLXGP improves vascular function. When GAXGLXGP was added to the apical side of Caco-2 monolayers, the intact form of GAXGLXGP was released to the basolateral side without incorporation into the cells. This transport was energy-independent but was associated with tight junction permeability. GAXGLXGP was then added to BAECs, and endothelial nitric oxide (NO) synthase (eNOS) activation was examined. GAXGLXGP at a concentration of 10 microM stimulated production of NO during a 1 h incubation. This event involved phosphorylation of eNOS at Ser(1179) without a change in the total eNOS protein level. These findings indicate that GAXGLXGP absorbed intact through the intestinal epithelium has direct effects on eNOS activity in vascular endothelial cells, leading to NO synthesis, thereby suggesting the potential for improvement in vascular function.