Separation of chitin from shrimp shells enabled by transition metal salt aqueous solution and ionic liquid

Chitin is a widely used important industrial polymer mainly from shrimp shells, but its commercial preparation is under the great challenge of serious pollution due to the requirement of HCl and Na OH.Herein, we demonstrated that high purity chitin can be obtained from waste shrimp shells(WSSs) by cascade separation with transition metal salt aqueous solution and ionic liquid(IL). Firstly, calcium carbonate of WSSs was effectively removed in the metal salt aqueous solution driven by the ion exchange interaction. Subsequently, 1-butyl-3-methylimidazolium chloride([Bmim]Cl) had bifunctional abilities to remove residual protein and introduced metal salts simultaneously by hydrogen bonding and coordination interactions. The key experimental factors affecting the separation process were systematically studied, including the type of metal salts, temperature, and [Bmim]Cl loading. After sequential treatment with a 20%(mass) Ni SO4aqueous solution at 130 ℃ and [Bmim]Cl at 150 ℃, the purity of a-chitin can be up to 96.5%(mass) that meets commercial requirements. The use of metal salts with higher coordination ability makes the preparation of chitin no longer depend on the commonly acid-base reaction, which is conducive to the preservation of chitin structure.

Extraction and Characterization of Litopenaeus vannamei’s Shell as Potential Sources of Chitosan Biopolymers

Chitin is the second most abundant polysaccharide,produced mainly as an industrial waste stream during crustacean processing.Chitin can be derived into chitosan through the deacetylation process.Conversion of shrimp waste into chitosan via the deacetylation process could be considered a practical approach for shell waste remediation.In this study,chitosan’s physicochemical characteristics extracted from two types of Pacific white leg shrimp,L.vannamei’s shell(i.e.,rough and smooth),were compared with commercial chitosan.The yield,moisture,ash,solubility,water and fat binding capacity were measured.The degree of deacetylation(DDA)was calculated using FTIR,and their chemical Structure was confirmed using XRD and SEM-EDS.Both extracted chitosan showed no significant difference in yield,moisture,ash,solubility and water binding capacity but showed a significant difference with commercial chitosan.Moreover,the fat binding capacity of commercial chitosan showed the lowest percentage(408.34±0.83%)as compared to extracted chitosan(smooth shell 549.59±12.48%;rough shell 500.55±12.10%).The DDA indicated that extracted chitosan from the smooth and rough shell was considered good chitosan as compared to commercial chitosan with 84.08±1.27%,80.78±0.79%and 74.99±1.48%,respectively.Additionally,the presence of hydroxyl and amino groups from FTIR and a good crystallinity index was recorded using XRD of extracted chitosan.Based on observed characteristics,shrimp shell waste from L.vannamei can achieve chitosan standard quality as a biopolymer and highly potential to be applied in various industrial applications.

在养殖水体中添加甲壳素发酵液对鲢幼鱼生长的影响

为研究甲壳素(Chitin,β-(1,4)-2-乙酰氨基-2-脱氧-D-葡萄糖)发酵后对鲢(Hypophthalmichthys molitrix)幼鱼生长的影响,该研究选用6300尾体质量为(50.0±1.2)g的鲢幼鱼为研究对象,对其随机分为3组,即对照组、发酵液组和壳聚糖组。每组2100尾,分3个平行,每个重复700尾。对照组养殖水体中不添加任何物质;发酵液组在养殖水体中添加甲壳素发酵液;壳聚糖组在养殖水体中添加壳聚糖液。整个饲喂试验为期8周。试验过程中称量鲢幼鱼的始末体重情况。结果发现,甲壳素发酵液组的试验末期体重、相对增重率显著地高于壳聚糖组(P<0.05),极显著地高于对照组(P<0.01)。研究表明,鲢幼鱼的生长性能甲壳素发酵液组>壳聚糖组>对照组。

血浆甲壳质酶蛋白40和内皮微粒与老年急性冠状动脉综合征的相关性研究

目的:探讨血浆甲壳质酶蛋白40(YKL-40)和内皮微粒(EMP)与老年急性冠状动脉综合征(ACS)之间的相关性。方法:选择行冠状动脉造影检查且年龄≥65岁者180例,随机分为ACS组(n=96)和对照组(n=84)。应用酶联免疫吸附法检测血浆YKL-40水平;应用流式微球技术检测血浆EMP水平,以CD31^+/CD42b-EMPs为标志;采用Gensini评分对每支血管病变狭窄程度进行定量计算。结果:ACS组血浆YKL-40和CD31^+/CD42b-EMPs水平均明显高于对照组(P<0.01)。ACS组患者Gensini评分值与血浆YKL-40和CD31^+/CD42b-EMPs水平均呈正相关关系(r=0.522,P<0.01;r=0.561,P<0.01)。ACS组患者血浆YKL-40水平和CD31^+/CD42b-EMPs水平呈正相关关系(r=0.625,P<0.01)。结论:老年ACS患者血浆YKL-40和EMP水平明显升高,且升高水平与冠脉病变程度有关,提示两者有可能成为诊断ACS的一个新的生化标志物,并为冠状动脉病变程度及危险分层提供参考依据。

苦瓜几丁质酶基因的克隆、表达及酶学特性分析

采用一步法逆转录聚合酶链反应（RT—PCR），以苦瓜幼叶为材料克隆几丁质酶基因的cDNA序列，序列分析表明其包含了基因完整的编码序列，编码由333个氨基酸组成的几丁质酶，对所克隆的基因进行了原核表达和纯化．纯化的几丁质酶具有分解几丁质的生物活性．当底物质量浓度在100～400mg·L^-1时，几丁质酶分解底物的能力随底物浓度增加而增大；大于400mg·L^-1时，几丁质酶分解底物的能力反而降低；当底物质量浓度固定时，几丁质酶分解底物的量随反应时间延长而增多．苦瓜几丁质酶基因的克隆和表达将为研究其结构和生物学功能奠定基础．

REINFORCEMENT OF CALCIUM PHOSPHATE CEMENTS WITH PHOSPHORYLATED CHITIN

Phosphorylated chitins (P-chitins) as the additives of calcium phosphate cements (CPCs) were prepared by the phosphorylation of chitin with phosphorus pentoxide in methanesulfonic acid. Their physical properties and effects on CPCs from monocalcium phosphate monohydrate (MCPM) and calcium oxide (CaO) or dicalcium phosphate dihydrate (DCPD) and calcium hydroxide [Ca(OH)(2)] were investigated. Addition of P-chitin (M-w = 2.60 x 10(4); degree of substitution, DS = 0.68) to the liquid phase in amounts up to 3 wt.% for MCPM and CaO cements or 1.5 wt.% for DCPD and Ca(OH)(2) cements could enhance the mechanical strength considerably, while little influence on the setting time was observed. However, further addition of P-chitin will cause no setting.

肌钙蛋白Ⅰ的可溶性表达及纯化研究

目的:提高肌钙蛋白Ⅰ(troponinⅠ,TnⅠ)在大肠杆菌中的可溶性表达并进行分离纯化。方法和结果:将质粒CBD-intein/pTWI N1和TnⅠ/pET28a分别用NcoⅠ及XhoⅠ双酶切,回收。回收后的载体CBD-intein/pTWIN1与TnⅠ片段用T4连接酶连接,得到重组质粒CBD-intein-TnⅠ/pTWIN1。将重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达得到融合蛋白CBD-intein-TnⅠ。经chitin亲和层析和intein介导的融合蛋白自我裂解,纯化得到重组TnⅠ。结论:通过CBD-intein融合表达系统一步式裂解分离纯化能方便快捷地获得可溶性的TnⅠ。

Activities of the Deriviatives of Chitin on the Osbeoblast Proliferation and the Effect on Bone Strength in Ovariectomized Rats

This study intends to examine the effects of different concentrations of four kinds of degradations of chitin： glucosamine （GLC）, N-acetyl-D-glucosamine （NAG）, chitooligosaccharide（COS）, CM-chitooligosaccharide （CM-COS）—on the proliferation of MC3T3-E1 cell line cultured in vitro. Results suggest that all of the glucoses mentioned above promoted the proliferation of osteoblast, and various concentrations have different effects： the proliferation was remarkable when the concentration of GLC, NAG, COS, CM-COS was 100 , 100, 500, 500 μg/mL ,respectively. Furthermore we choose the glucosamine as the material and study the effect on the bone strength in ovariectomized rats. The results showed that the middle does of glucosamine can significantly increase the hone strength of femur in ovariectomized rats.

Bridging peripheral nerves using a deacetyl chitin conduit combined with short-term electrical stimulation

Previous studies have demonstrated that deacetyl chitin conduit nerve bridging or electrical stimulation can effectively promote the regeneration of the injured peripheral nerve. We hypoth-esized that the combination of these two approaches could result in enhanced regeneration. Rats with right sciatic nerve injury were subjected to deacetyl chitin conduit bridging combined with electrical stimulation （0.1 ms, 3 V, 20 Hz, for 1 hour）. At 6 and 12 weeks after treatment, nerve conduction velocity, myelinated axon number, ifber diameter, axon diameter and the thickness of the myelin sheath in the stimulation group were better than in the non-stimulation group. The results indicate that deacetyl chitin conduit bridging combined with temporary electrical stimu-lation can promote peripheral nerve repair.

Recovery of Chitin and Protein from Shrimp Head Waste by Endogenous Enzyme Autolysis and Fermentation

The industrial processing of shrimp produces massive quantities of solid waste that is a notable source of animal protein, chitin, carotenoids, and other bioactive compounds that are not appropriately utilized. In the present study, chitin and protein extraction from shrimp head with autolysis and fermentation using Bacillus licheniformis were investigated. The results showed that when shrimp heads were autolyzed with a natural pH at 50℃ for 4 h, the total amino acid nitrogen in the supernatant was 5.01 mg mL^-1. Then, when a 50%(v/m) inoculum of the hydrolysate was incubated at 60℃ for 10 h, a deproteinization rate of 88.3% could be obtained. The fermented supernatant was processed into a dry protein powder, while the residues were demineralized by 10% citric acid for chitin. The recovered protein powder contained 5.5% moisture, 11.5% ash, and 66.7% protein, while the chitin contained 3.5% moisture, 2.1% ash, and 3.1% protein. In addition, amino acids, minerals, heavy metals, the degree of acetylation, microstructure, and Fourier-transform infrared(FT-IR) spectroscopy results were analyzed. Furthermore, the statistics of the large scale trial after treatment with 20 kg of shrimp heads were analyzed. Thus, this work made the shrimp waste utilization environmentally sound and valuable.

Synthesis of Novel Benzoylphenylurea Chitin Inhibitors from Chlorothalonil

Twenty-six novel benzoylphenylurea chitin inhibitor derivatives have been synthesized in over 30~50% yield from chlorothalonil 1 via sequential fluorine exchange, aminolysis, hydrolysis, decarboxylation and acylation reactions.

Chitin scaffold combined with autologous small nerve repairs sciatic nerve defects

Although autologous nerve transplantation is the gold standard for treating peripheral nerve defects,it has many clinical limitations.As an alternative,various tissue-engineered nerve grafts have been developed to substitute for autologous nerves.In this study,a novel nerve graft composed of chitin scaffolds and a small autologous nerve was used to repair sciatic nerve defects in rats.The novel nerve graft greatly facilitated regeneration of the sciatic nerve and myelin sheath,reduced atrophy of the target muscle,and effectively restored neurological function.When the epineurium of the small autogenous nerve was removed,the degree of nerve regeneration was similar to that which occurs after autogenous nerve transplantation.These findings suggest that our novel nerve graft might eventually be a new option for the construction of tissue-engineered nerve scaffolds.The study was approved by the Research Ethics Committee of Peking University People's Hospital(approval No.2019 PHE27)on October 18,2019.

Myelin-associated glycoprotein combined with chitin conduit inhibits painful neuroma formation after sciatic nerve transection

Studies have shown that myelin-associated glycoprotein(MAG)can inhibit axon regeneration after nerve injury.However,the effects of MAG on neuroma formation after peripheral nerve injury remain poorly understood.In this study,local injection of MAG combined with nerve cap made of chitin conduit was used to intervene with the formation of painful neuroma after sciatic nerve transfection in rats.After 8 weeks of combined treatment,the autotomy behaviors were reduced in rats subjected to sciatic nerve transfection,the mRNA expression of nerve growth factor,a pain marker,in the proximal nerve stump was decreased,the density of regenerated axons was decreased,the thickness of the myelin sheath was increased,and the ratio of unmyelinated to myelinated axons was reduced.Moereover,the percentage of collagen fiber area and the percentage of fibrosis marker alpha-smooth muscle actin positive staining area in the proximal nerve stump were decreased.The combined treatment exhibited superior effects in these measures to chitin conduit treatment alone.These findings suggest that MAG combined with chitin conduit synergistically inhibits the formation of painful neuroma after sciatic nerve transection and alleviates neuropathic pain.This study was approved by the Animal Ethics Committee of Peking University People’s Hospital(approval No.2019PHE027)on December 5,2019.

Comparison of the defluoridation efficiency of calcium phosphate and chitin in the exoskeleton of Antarctic krill

Calcium （Ca）, phosphorus （P）, and chitin are the main components of the exoskeleton of krill. Defluoridation of a solution of sodium fluoride （NaF） using calcium phosphate （Ca3（PO4）2） and chitin as defluoridation agents was studied. Orthogonal experiments were designed to find the optimum reaction conditions for defluoridation, to obtain the maximum defluoridation efficiency and fluoride removal capacity of calcium phosphate and chitin. At the same time, a comparison of the capacity of the two defluoridation agents was made. The results suggest that calcium phosphate has a far greater capability than chitin for the removal of fluoride （F） from water under similar reaction conditions. It is also suggested that Antarctic krill is likely to adsorb fluoride via compounds such as calcium phosphate, hydroxyapatite, and other compounds of Ca and P with the general form （Ca, X）x（PO4, HPO4, Y）y（OH, Z）z, in addition to chitin.

Ionic Liquid as Useful Media for Dissolution, Derivatization, and Nanomaterial Processing of Chitin

This paper reviews studies on dissolution, derivatization, and nanomaterial processing of chitin using an ionic liquid as useful media. Because chitin is the second most abundant polysaccharide on the earth after cellulose, there is major interest in conversion of native chitin resources into various useful materials after proper dissolution in suitable solvents. For the derivatization and nanomaterial processing of chitin, the author has been focusing on ionic liquids because which have been found to be used as good solvents for cellulose in a past decade. The author found that an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), dissolved chitin in concentrations up to 4.8 wt% and mixtures of the higher amounts of chitin with AMIMBr gave ion gels. Acetylation, the simplest derivatization, of chitin using acetic anhydride was achieved in the AMIMBr solvent under mild conditions. Furthermore, the chitin nanofibers were fabricated by regeneration technique from the chitin ion gel with AMIMBr using methanol. Moreover, filtration of the chitin nanofiber dispersion with methanol was carried out to give a chitin nanofiber film. The chitin nanofiber-poly(vinyl alcohol) composite film was also prepared from the ion gel by co-regeneration method.

A Review of Various Sources of Chitin and Chitosan in Nature

Chitin was first discovered by its name from the Greek word“chiton”,which means“mail coat”.It is indeed a polysaccharide made up of naturally occurring acetyl-D-glucosamine monomers.Hatchett was the first researcher who extracted chitin from the shells of mollusks(crabs and lobsters),prawns,and crayfish in 1799.Later in 1811,Henri Braconnot discovered chitin in the cell walls of mushrooms and called it“fungine”.Chitin and chitosan are abundant in the biosphere as essential components of many organisms’exoskeletons and as by-products of the global seafood industry.The biopolymer must be deacetylated before chitosan can be produced.It can also be extracted using microbes in a biological extraction procedure.The development of products that take advantage of the bioactivities of the existing primary commercial source of chitin(crustacean)has lagged expectations.Also,the disadvantages of the present commercial source such as seasonality and competition for other uses among others has been one of the driving forces towards seeking alternative sources of chitin and chitosan in nature.This review highlights some of the efforts made by environmental scholars to locate possible commercial sources of chitin and chitosan in nature over time.

Synthesis of Novel Benzoylphenylurea Chitin Inhibitors From Chlorothalonil(Ⅱ)

Six novel benzoylphenylurea chitin inhibitor derivatives were synthesized in the yields of 30%_50% from the readily available starting material chlorothalonil 1 viasequential fluorine exchange, aminolysis, hydrolysis and acylation reactions.

Chitinolytic Assay and Identification of Bacteria Isolated from Shrimp Waste Based on 16S rDNA Sequences

Shrimp waste contains 20% - 60% chitin and possible to be source of chitinolytic bacteria. Chitinolytic bacteria are capable of hydrolyzing of chitin progressively to produce N-acetylglucosamine monomer which can be used to overcome the shrimp waste. The objectives of this research were to identify species of bacteria with high activity of chitin degradation in shrimp waste and to analyze their potency as chitin degradation agent. The research consists of screening of chitinolytic bacteria based on chitinolytic index, activity assay of chitinase using colorimetric method, and molecular identification of bacteria based on 16S rDNA sequences. Two of eighteen isolates of chitinolytic bacteria (PBK 2 and SA 1.2 isolates) showed the highest chitinolytic index, which were 2.069 and 2.084, whereas chitinase activity was 0.213 and 0.219 U/ml respectively. Based on 16S rDNA sequences, isolate of PBK 2 was identified as Acinetobacter johnsonii 3-1, whereas SA 1.2 was identified as Bacillus amyloliquefaciens GR53 with 99.78% similarity.

Chitin oligosaccharides alleviate atherosclerosis progress in ApoE;mice by regulating lipid metabolism and inhibiting inflammation

Atherosclerosis is driven both by hyperlipidemia and inflammation. Chitin oligosaccharides(NACOS) have shown pharmacological effects on multiple diseases via hypolipidemic and/or anti-inflammatory activities. The present study aims to evaluate whether NACOS treatment can prevent atherosclerosis induced by a highfat-diet(HFD) in Apolipoprotein E-knockout(Apo E;) mice. Results showed that 300 and 900 mg/kg b.w./day NACOS supplementation for 14 weeks significantly decreased atherosclerotic lesions up to 45% and 67% in compared with the HFD(P < 0.05), as measured in the valve area of the aortic root. Further, NACOS supplementation significantly reduced the serum hyperlipidemia and circulating proinflammatory cytokines including interleukin-1β, interleukin-6, monocyte chemoattractant protein-1 and tumor necrosis factor-α. NACOS decreased the hepatic Hmgcr to reduce cholesterol synthesis, activated the genes involved in reverse cholesterol transport to enhance cholesterol efflux and excretion, and reduced the intestinal Npc1L1 to lower cholesterol absorption. Additionally, NACOS enhanced cecum short chain fatty acids production and intestinal integrity. Thus, NACOS supplementation ameliorated atherosclerosis via altering lipid metabolism and reducing inflammation. These findings indicate that NACOS may be a potential functional food material for attenuating atherosclerosis development.

Diradical Character and Controllable Magnetic Properties in Chitin Through Radical Modification

The parent and radical-modified chitin units are computationally studied and the parent chitin unit possesses closed-shell singlet(CS)state as its ground state.Two radicals(CH_(3)·and CH_(3)CH_(2)·)are chosen to modify the parent chitin unit.Calculations predict that CH_(3)CH_(2)·-modified chitin unit possesses the open-shell broken-symmetry(BS)singlet diradical ground state,different from that CH_(3)·-modified chitin unit is a triplet diradical.That is to say,the magnetic conversion can occur from nonmagnetic exchange coupling to antiferromagnetic(AFM)or ferromagnetic(FM)exchange coupling by means of the radical modification method.The present work helps to get a basic understanding of structures and electronic properties of chitin and its radical-modified derivatives at the atomistic level.Moreover,it also provides a feasible way to design new chitin-based magnetic materials and organic magnetic switches.