Fermentation optimization and enzyme characterization of a new ι-Carrageenase from Pseudoalteromonas carrageenovora ASY5

Background: A new ι-carrageenase-producing strain was screened from mangroves and authenticated as Pseudoalteromonas carrageenovora ASY5 in our laboratory. The potential application of this new strain was evaluated. Results: Medium compositions and culturing conditions in shaking flask fermentation were firstly optimized by single-factor experiment. ι-Carrageenase activity increased from 0.34 U/mL to 1.08 U/mL after test optimization. Optimal fermentation conditions were 20°C, pH 7.0, incubation time of 40 h, 15 g/L NaCl, 1.5% (w/v) yeast extract as nitrogen source, and 0.9% (w/v) ι-carrageenan as carbon source. Then, the crude ι-carrageenase was characterized. The optimum temperature and pH of the ι-carrageenase were 40°C and 8.0, respectively. The enzymatic activity at 35­40°C for 45 min retained more than 40% of the maximum activity. Meanwhile, The ι-carrageenase was inhibited by the addition of 1 mmol/L Cd2+ and Fe3+ but increased by the addition of 1 mmol/L Ag+, Ba2+, Ca2+, Co2+, Mn2+, Zn2+, Fe2+, and Al3+. The structure of oligosaccharides derived from ι-carrageenan was detected using electrospray ionization mass spectrometry (ESI-MS). The ι-carrageenase degraded ι-carrageenan, yielding disaccharides and tetrasaccharides as main products. Conclusions: The discovery and study of new ι-carrageenases are beneficial not only for the production of ι-carrageenan oligosaccharides but also for the further utilization in industrial production.

Tannase application in secondary enzymatic processing of inferior Tieguanyin oolong tea

Background: Inferior Tieguanyin oolong tea leaves were treated with tannase. The content and bioactivity of catechins in extracts from the treated tea leaves were investigated to assess the improvement in the quality of inferior Tieguanyin oolong tea. Results: Analysis showed that after treatment, the esterified catechin content decreased by 23.5%, whereas non-galloylated catechin and gallic acid contents increased by 15.3% and 182%, respectively. The extracts from tannase-treated tea leaves showed reduced ability to bind to BSA and decreased tea cream levels. The extracts also exhibited increased antioxidant ability to scavenge OH and DPPH radicals, increased ferric reducing power, and decreased inhibitory effects on pancreatic α-amylase and lipase activities. Conclusions: These results suggested that tannase treatment could improve the quality of inferior Tieguanyin oolong tea leaves.

Efficient immobilization of agarase using carboxyl-functionalized magnetic nanoparticles as support

Background: A simple and efficient strategy for agarase immobilization was developed with carboxyl-functionalized magnetic nanoparticles (CMNPs) as support. The CMNPs and immobilized agarase (agarase-CMNPs) were characterized by transmission electron microscopy, dynamic light scattering, vibrating sample magnetometry, scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, and zeta-potential analysis. The hydrolyzed products were separated and detected by ESI-TOF-MS. Results: The agarase-CMNPs exhibited a regular spherical shape with a mean diameter of 12 nm, whereas their average size in the aqueous solution was 43.7 nm as measured by dynamic light scattering. These results indicated that agarase-CMNPs had water swelling properties. Saturation magnetizations were 44 and 29 emu/g for the carriers and agarase-CMNPs, respectively. Thus, the particles had superparamagnetic characteristics, and agarase was successfully immobilized onto the supports. Agaro-oligosaccharides were prepared with agar as substrate using agarase-CMNPs as biocatalyst. The catalytic activity of agarase-CMNPs was unchanged after six reuses. The ESI-TOF mass spectrogram showed that the major products hydrolyzed by agarase-CMNPs after six recycle uses were neoagarotetraose, neoagarohexaose, and neoagarooctaose. Meanwhile, the end-products after 90 min of enzymatic treatment by agarase-CMNPs were neoagarobiose and neoagarotetraose. Conclusions: The enhanced agarase properties upon immobilization suggested that CMNPs can be effective carriers for agarase immobilization. Agarase-CMNPs can be remarkably used in developing systems for repeated batch production of agar-derived oligosaccharides.

Effects and mechanism of Shexiang Baoxin Pills on insulin resistance in patients with coronary heart disease / 药物评价研究

Objective To investigate the effects and mechanism of Shexiang Baoxin Pills on insulin resistance (IR) in patients with coronary heart disease.Methods From January 2016 to March 2017,82 patients with coronary heart disease in The Second People's Hospital of Nanyang were selected as the research object.All the patients were divided into observation group and control group with 41 cases in each group according to the principle of random envelope drawing.The control group was given conventional treatment,and the observation group was treated with Shexiang Baoxin Pills on the basis of the treatment of the control group.The observation periods of the two groups were 3 months.Results The total effective rates of the observation group and control group were 97.6％ and 82.9％,respectively,and the observation group was better than that of the control group (P ＜ 0.05).The IR indexes of the observation group and control group after treatment were (1.79 ± 0.45) and (2.44 ± 0.51),respectively,which were significantly lower than before treatment of (4.01 ± 0.44) and (4.00 ± 0.56) (P ＜ 0.05),and the IR index in the observation group after treatment was significantly higher than that in the control group (P ＜ 0.05).The serum CRP levels of the observation group and control group were significantly lower than those before treatment (P ＜ 0.05),and the level of serum CRP in the observation group after treatment was also significantly lower than that in the control group (P ＜ 0.05).Atter treatment,the coronary artery remodeling,no reconstruction and negative reconstruction in the observation group were 4 cases,25 cases and 13 cases,while the control group were 15 cases,10 cases and 16 cases,the difference between the two groups was statistically significant (P ＜ 0.05).Conclusion Shexiang Baoxin Pill can improve the therapeutic effect and improve the IR status in patients with coronary heart disease,and its mechanism may be related to inhibiting the expression of inflammatory factors and improving coronary artery remodeling.

Effects and mechanism of Shexiang Baoxin Pills on insulin resistance in patients with coronary heart disease / 药物评价研究

Objective To investigate the effects and mechanism of Shexiang Baoxin Pills on insulin resistance (IR) in patients with coronary heart disease.Methods From January 2016 to March 2017,82 patients with coronary heart disease in The Second People's Hospital of Nanyang were selected as the research object.All the patients were divided into observation group and control group with 41 cases in each group according to the principle of random envelope drawing.The control group was given conventional treatment,and the observation group was treated with Shexiang Baoxin Pills on the basis of the treatment of the control group.The observation periods of the two groups were 3 months.Results The total effective rates of the observation group and control group were 97.6％ and 82.9％,respectively,and the observation group was better than that of the control group (P ＜ 0.05).The IR indexes of the observation group and control group after treatment were (1.79 ± 0.45) and (2.44 ± 0.51),respectively,which were significantly lower than before treatment of (4.01 ± 0.44) and (4.00 ± 0.56) (P ＜ 0.05),and the IR index in the observation group after treatment was significantly higher than that in the control group (P ＜ 0.05).The serum CRP levels of the observation group and control group were significantly lower than those before treatment (P ＜ 0.05),and the level of serum CRP in the observation group after treatment was also significantly lower than that in the control group (P ＜ 0.05).Atter treatment,the coronary artery remodeling,no reconstruction and negative reconstruction in the observation group were 4 cases,25 cases and 13 cases,while the control group were 15 cases,10 cases and 16 cases,the difference between the two groups was statistically significant (P ＜ 0.05).Conclusion Shexiang Baoxin Pill can improve the therapeutic effect and improve the IR status in patients with coronary heart disease,and its mechanism may be related to inhibiting the expression of inflammatory factors and improving coronary artery remodeling.

Preparation and characterization of κ-carrageenase immobilized onto magnetic iron oxide nanoparticles

Background Carboxyl-functionalized magnetic nanoparticles were synthesized via chemical co-precipitation method and modified with oleic acid which was oxidized by potassium permanganate, and κ-carrageenase from Pseudoalteromonas sp. ASY5 was subsequently immobilized onto them. The immobilization conditions were further optimized, and the characterizations of the immobilized κ-carrageenase were investigated. Results The κ-carrageenase was immobilized onto magnetic iron oxide nanoparticles, and the bonding was verified by Fourier transform infrared spectroscopy. The optimal conditions for κ-carrageenase immobilization were 2.5% (w/v) glutaraldehyde, 13.9 U κ-carrageenase for 20 mg of magnetic nanoparticles, a 2-h cross-linking time, and a 2-h immobilization time at 25°C. Under these conditions, the activity of the immobilized enzyme and the enzyme recovery rate were 326.0 U · g- 1 carriers and 46.9%, respectively. The properties of the immobilized κ-carrageenase were compared with those of the free enzyme. The optimum temperatures of the free and immobilized κ-carrageenase were 60 and 55°C, respectively, and the optimum pH of κ-carrageenase did not change before and after immobilization (pH 7.5). After immobilization, κ-carrageenase exhibited lower thermal stability and improved pH stability, as well as better storage stability. The immobilized κ-carrageenase maintained 43.5% of the original activity after being used 4 times. The kinetic constant value (Km) of κ-carrageenase indicates that the immobilized enzyme had a lower binding affinity for the substrate. Conclusions Under optimal conditions, the activity of the immobilized enzyme and enzyme recovery rate were 326.0 U · g- 1·κ-carrageenase-CMNPs and 46.9%, respectively. The thermal, pH, and storage stabilities of κ-carrageenase-CMNPs were relatively higher than those of free κ-carrageenase.

Statistical optimization for tannase production by Aspergillus tubingensis in solid-state fermentation using tea stalks

Background A sequential statistical strategy was used to optimize tannase production from Aspergillus tubingensis using tea stalks by solid-state fermentation. Results First, using a Plackett-Burman design, inoculum size and incubation time (among seven tested variables) were identified as the most significant factors for tannase yield. The effects of significant variables were further evaluated through a single steepest ascent experiment and central composite design with response surface analysis. Under optimal conditions, the experimental value of 84.24 units per gram of dry substrate (U/gds) closely matched the predicted value of 87.26 U/gds. Conclusions The result of the statistical approach was 2.09 times higher than the basal medium (40.22 U/gds). The results were fitted onto a second-order polynomial model with a correlation coefficient (R²) of 0.9340, which implied an adequate credibility of the model.

Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation

Background To study the relationship between intracellular anabolism and astaxanthin production, the influence of intracellular protein and fatty acids on astaxanthin production by four mutant Phaffia rhodozyma strains and their variations was investigated in this research. Results First, the content of astaxanthin in cells showed a reverse fluctuation in contrast to that of protein during the whole fermentation process. Moreover, compared with the three other strains, the astaxanthin-overproducing mutant strain of the yeast P. rhodozyma, called JMU-MVP14, had the highest specific productivity of astaxanthin as 6.8 mg/g, whereas its intracellular protein and fatty acid contents were the lowest. In addition, as a kind of sugar metabolic product, ethanol was only produced by P. rhodozyma JMU-VDL668 and JMU-7B12 during fermentation. Conclusions The results indicated that the accumulation of ethanol, intracellular protein, and fatty acids had competition effects on astaxanthin synthesis. This condition may explain why the P. rhodozyma strains JMU-VDL668 and JMU-7B12 achieved relatively lower astaxanthin production (1.7 and 1.2 mg/L) than the other two strains JMU-MVP14 and JMU-17W (20.4 and 3.9 mg/L).