Simultaneous Quantification of κ-Carrageenan Oligosaccharides of DP 3, 5 and 7 by LC-MS/MS: Application to an in vitro Absorption Study

Carrageenans are widely utilized in many commercial applications such as the food and pharmaceutical industry, due to their excellent functional properties. In this study, a sensitive LC-MS/MS method was developed to determine κ-3, κ-5, and κ-7 carrageenan oligosaccharides simultaneously. Optimum MRM transitions for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were(645.079→565.111, [M-H]-),(515.137→474.946, [M-2H]^2-), and(471.484→445, [M-3H]^3-), respectively. Chromatographic separation was performed on an Amide column coupled with a guard column operated at 60℃ under stepwise gradient elution. The linearity of the LC-MS/MS method for κ-3, κ-5, and κ-7 carrageenan oligosaccharides, evaluated over the concentration range of 0.10- 20.0 μmol L^-1, was excellent. The precisions of the method for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were from 0.91% to 9.66%, and the inter-day precisions were from 0.92% to 10.5%. Validation of the LC-MS/MS method indicated that the method was precise and in line with the CFDA guidance. This method has been successfully applied to an in vitro absorption study.

Preparation of κ-carra-Oligosaccharides with Microwave Assisted Acid Hydrolysis Method

A rapid method of microwave assisted acid hydrolysis was established to prepare κ-carra-oligosaccharides. The optimal hydrolysis condition was determined by an orthogonal test. The degree of polymerization （DP） of oligosaccharides was detected by high performance thin layer chromatography （HPTLC） and polyacrylamide gel electrophoresis （PAGE）. Considering the results of HPTLC and PAGE, the optimum condition of microwave assisted acid hydrolysis was determined. The concentration of κ-carrageenan was 5 mgmL-1; the reaction solution was adjusted to pH 3 with diluted hydrochloric acid; the solution was hydrolyzed under microwave irradiation at 100 ℃ for 15 min. Oligosaccharides were separated by a Superdex 30 column （2.6 cm × 90 em） using AKTA Purifier UPC100 and detected with an online refractive index detector. Each fraction was characterized by electrospray ioni- zation mass spectrometry （ESI-MS）. The data showed that odd-numbered κ-carra-oligosaccharides with DP ranging from 3 to 21 could be obtained with this method, and the structures of the oligosaccharides were consistent with those obtained by traditional mild acid hydrolysis. The new method was more convenient, efficient and environment-friendly than traditional mild acid hydrolysis. Our results provided a useful reference for the preparation of oligosaceharides from other polysaccharides.

A New κ-Carrageenase Cgk S from Marine Bacterium Shewanella sp. Kz7

A new κ-carrageenase gene cgkS was cloned from marine bacterium Shewanella sp. Kz7 by using degenerate and site-finding PCR. The gene was comprised of an open reading frame of 1224bp, encoding 407 amino acid residues, with a signal peptide of 24 residues. Based on the deduced amino acid sequence, the κ-carrageenase CgkS was classified into the Glycoside Hy- drolase family 16. The cgkS gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity with a specific activity of 716.8 U mg-1 and a yield of 69%. Recombinant CgkS was most active at 45 ℃ and pH 8.0. It was stable at pH 6.0-9.0 and below 30℃. The enzyme did not require NaCl for activity, although its activity was enhanced by NaCI. CgkS degraded κ-carrageenan in an endo-fashion releasing tetrasaccharides and disaccharides as main hydrolysis products.

Capillary Zone Electrophoresis Investigation of Interactions between Granulocyte-colony Stimulating Factor and Dextran Sulfate/Carrageenan Oligosaccharide

The interactions between granulocyte-colony stimulating factor (G-CSF) and dextran sulfate / κ-carrageenan oligosaccharide were studied by capillary zone electrophoresis. Dextran sulfate could strongly interact with G-CSF and the complex was detected. The binding constant and stoichiometry were determined to be 1.2×106 (mol/L)-1 and 3:1, respectively. However, the interaction between κ-carrageenan oligosaccharide and G-CSF was not found.

关于端基分析法测定壳低聚糖的相对数均分子质量问题的探讨

为使端基分析法更具合理性 ,文中以铁氰化钾为显色剂 ,以盐酸氨基葡萄糖和N 乙酰基 D 葡萄糖胺为还原糖的标准品 ,建立了端基分析法测定低聚糖相对数均分子质量的标准曲线。实验结果证明 :通过不同浓度的标准溶液绘制的标准曲线 ,测得壳低聚糖的相对数均分子质量差别比较大。以凝胶色谱法测定的相对数均分子质量为对照 ,经分析得 :以盐酸氨基葡萄糖为标准品建立的标准曲线方程和以N 乙酰基 D 葡萄糖胺为标准品建立的标准曲线方程更为合理。

κ-卡拉胶寡糖抗凝血作用的研究

目的:研究κ-卡拉胶寡糖的抗凝血作用。方法:通过体外实验的方法,考察κ-卡拉胶寡糖对家兔的凝血时间(CT)、凝血酶时间(TT)、凝血酶原时间(PT)以及活化部分凝血酶时间(APTT)的作用。结果:质量浓度为0.25%、0.5%、1%、2%、4%、8%的κ-卡拉胶寡糖显著延长家兔的凝血时间(CT),对凝血酶时间(TT)和活化部分凝血酶时间(APTT)均有明显的抑制作用,而对凝血酶原时间(PT)的抑制作用相对较小。结论:κ-卡拉胶寡糖具有抗凝血的作用。

肿瘤标志物与新型寡糖链检测在原发性肝癌诊断中的应用研究

目的探究肿瘤标志物甲胎蛋白(alpha fetal protein,AFP)、异常凝血酶原(PIVKA-Ⅱ)和寡糖链组分的相对含量(G-test)分别在原发性肝癌检测中的临床应用价值。方法选取2017年10月至2017年12月首都医科大学附属北京佑安医院临床诊断原发性肝癌(hepatoma carcinoma cell,HCC)组共165例,非肝癌组共142例,分别用仪器检测两组患者AFP、PIVKA-Ⅱ及G-test,通过统计学方法来对肿瘤标志物AFP、PIVKA-Ⅱ和G-test诊断的灵敏度、特异性和准确度进行分析。结果在灵敏度上,G-tes的灵敏度最高(83.6%),AFP的灵敏度最低(16.4%),且差异有统计学意义;在特异性上,AFP的特异性最高(97.9%),G-test的特异性最低(73.2%),且差异有统计学意义;在准确度上,G-test的准确度最高(78.8%),AFP的准确度最低(54.1%),且差异有统计学意义。联合检测时G-test+PIVKA-Ⅱ的ROC曲线下面积(area under the curve,AUC)与G-tesT+AFP的AUC一样(86.3%),高于AFP+PIVKA-Ⅱ的AUC(68.3%),G-test+AFP+PIVKA-Ⅱ的AUC为86.4%,灵敏度为93.3%。结论G-test诊断原发性肝癌的价值较AFP、PIVKA-Ⅱ高,AFP诊断灵敏度最高,联合检测时可提高检测率,G-test值较传统的原发性肝癌肿瘤标志物对原发性肝癌的检出率更高,意义更大。

壳聚糖/k-卡拉胶寡糖温敏水凝胶的制备与表征

利用k-卡拉胶寡糖(KOS)和壳聚糖(CS)带有不同电荷的特点,以KOS为交联剂,通过离子交联制备可以缓释KOS和可控降解的温敏性水凝胶。将CS酸性溶液与KOS水溶液在室温下混合,制备KOS与CS质量比为2.25:1.0~2.75:1.0的系列凝胶体系。使用试管倒置法测凝胶时间,差值法测相变体积变化,称重法测溶胀度和降解率,苯酚-硫酸法测凝胶对KOS的缓释,在电子扫描显微镜(SEM)下观察凝胶表面结构,MTT法检测中枢神经免疫效应细胞(N9)在凝胶上的粘附和增殖情况。结果表明KOS/CS系列水凝胶具有温度响应性,在4℃呈现为液态的溶胶状态,在生理温度条件下(37℃)可相变为固形水凝胶,凝胶时间为(300±50)~(17±5)min。凝胶后具有明显的相变体积变化,孔隙率为(78.7±2.5)%~(92.9±2.1)%,溶胀率为(558.7±5.046)%~(853.9±6.143)%,降解率为(77.46±3.520)%~(70.68±4.080)%以及KOS缓释程度为(30.38±0.9523)%~(24.24±3.988)%;该凝胶在SEM下呈纤维多孔结构,且支持N9细胞的黏附和增殖。KOS/CS水凝胶具有良好的温敏性、溶胀性、可控降解性、KOS缓释能力和细胞相容性,可为药物递送和组织工程领域提供较好的生物材料。

The neuroprotective efects of peracetylated chitosan oligosaccharides againstβ‑amyloid‑induced cognitive defcits in rats

Chitosan oligosaccharides(COSs)have been reported to possess a broad range of activities such as antitumor,antioxidant and neuroprotective activities.In this study,the protective efects and mechanisms of peracetylated chitosan oligosaccharides(PACOs)against Aβ-induced cognitive defcits were investigated in Sprague–Dawley(SD)rats.PACOs treatment signifcantly improved the learning and memory function of Alzheimer’s disease(AD)rats and attenuated the neuron cell damage caused by Aβ.PACOs also markedly reduced the levels of lactate dehydrogenase(LDH)and Malondialdehyde(MDA)and decreased the phosphorylation of Tau protein to inhibit oxidative injury and infammatory responses in AD rats.Further studies indicated that PACOs may promote the repair of Aβinduced nerve damage and inhibit neuronal apoptosis mainly through regulating PI3K/Akt/GSK3βsignaling pathway.Consistently,the transcriptome analysis verifed that the diferentially expressed genes(DEGs)were mainly involved in neuron development and the PI3K-Akt signaling pathway.Taken together,peracetylated chitosan oligosaccharides(PACOs)have the potential to be developed into novel anti-AD agents targeting the cellular PI3K/Akt/GSK3βsignaling pathway.

Combined effects of chitosan and microencapsulated Enterococcus faecalis CG1.0007 probiotic supplementation on performance and diarrhea incidences in enterotoxigenic Escherichia coli K88^+ challenged piglets

The aim of this study was to investigate the combined effects of chitosan oligosaccharide(COS) and a microencapsulated Enterococcus faecalis CG1.0007 probiotic(PRO) on growth performance and diarrhea incidences in enterotoxigenic Escherichia coli(ETEC) K88^+ challenged piglets in a 14-d study. Thirty piglets,7.19 ± 0.52 kg initial BW weaned at 21 ± 1 d.were allotted to 5 treatment groups(n = 6)consisting of a corn-soybean meal diet with no additive(negative control, NC), NC + 0.25% chlortetracycline(positive control, PC), NC + 400 mg/kg COS(COS), NC + 100 mg/kg PRO(PRO) and NC + a combination of COS and PRO(CPRO). Pigs were individually housed in cages, acclimated to treatments for a 7-d period and had ad libitum access to feed and water throughout the study, On d 8, pigs were weighed, blood samples were collected, and then orally challenged with 6 mL(1 ×10^(11) cfu/mL) of freshly grown ETEC inoculum. During post-challenge period, blood was sampled at 24 and 48 h to determine plasma urea nitrogen(PUN), and diarrhea incidences and fecal consistency scores were recorded from d 9 to 12. On d 14, all pigs were weighed and then euthanized to obtain intestinal tissue samples for histomorphometric measurements. Growth performance responses were similar among treatments during the pre-and post-challenge periods. There were no significant differences in PUN content, incidences of diarrhea, and fecal consistency scores among treatments. The intestinal histomorphology results did not differ significantly among treatments except for PC with increased(P = 0.0001) villus:crypt ratio compared with the NC. Under the conditions of the present study, it can be concluded that supplementation of piglet diets with 400 mg/kg COS, 100 mg/kg microencapsulated PRO or their combination did not significantly improve piglet growth performance both during the pre-and post-ETEC K88+ oral inoculation. Also, there were no significant reduction of incidences and severity of diarrhea after challenge compared with the control group.

Metabolic engineering of Escherichia coli for the production of Lacto-N-neotetraose(LNnT)

Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these methods still face some problems such as low yield and high cost.The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E.coli K12 MG1655.The lgtA and lgtB were first expressed by a plasmid,resulting in a LNnT titer of 0.04 g/L.To improve the yield of LNnT on substrate lactose,lacZ and lacI were knocked out,and lacY was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,the pathway was regulated using the titer of Lacto-N-triose II(LNTII)as a measure,and obtained a high titer strain of LNnT for 1.04 g/L.Finally,the gene expressions were fine-tuned,the titer of LNnT reached 1.2 g/L,which was 93%higher than the control strain,and the yield on lactose reached 0.28 mol/mol.The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E.coli.