Effect of chitosan and chitin on the separation of membranes from proteins solubilized by pH shifts using cod (Gadus morhua)

Studies with isolated membranes and isolated membranes suspended in muscle proteins solubilized at pH 3 showed that mixing chitosan and membranes at this low pH followed by a pH adjustment to 10.5 could sediment membranes effectively at 4000 g. In the solubilized muscle homogenate, the effectiveness of membrane removal by chitosan at 4000 g for 15 min was molecular weight dependent. About 80% of the phospholipids and 28% of proteins were sedimented from solubilized muscle homogenate by mixing muscle homogenate (10 g of muscle tissue homogenized with 90 mL of distilled water) with 10 mL of MW 310-375 k chitosan (10 mg/mL in 0.1 N HCl) before solubilizing it at pH 10.5, whereas 55% of the phospholipids and 12% of proteins were sedimented by mixing muscle homogenate with the MW 310-375 k chitosan before solubilizing the homogenate at pH 3. Low molecular weight chitosans (at MW 1k or 33k) showed little effect on membrane sedimentation under the same conditions. Chitin was not useful for removing membranes at either pH 3 or 10.5, whether added before or after pH adjustment.     

Protein-binding affinity of leucaena condensed tannins of differing molecular weights

Depending on their source, concentration, chemical structure, and molecular weight, condensed tannins (CTs) form insoluble complexes with protein, which could lead to ruminal bypass protein, benefiting animal production. In this study, CTs from Leuceana leucocephala hybrid were fractionated into five fractions by a size exclusion chromatography procedure. The molecular weights of the CT fractions were determined using Q-TOF LC-MS, and the protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay with bovine serum albumin (BSA) as the standard protein. The calculated number-average molecular weights (M(n)) were 1348.6, 857.1, 730.1, 726.0, and 497.1, and b values (the b value represents the CT quantity that is needed to bind half of the maximum precipitable BSA) of the different molecular weight fractions were 0.381, 0.510, 0.580, 0.636, and 0.780 for fractions 1, 2, 3, 4, and 5, respectively. The results indicated that, in general, CTs of higher molecular weight fractions have stronger protein-binding affinity than those of lower molecular weights. However, the number of hydroxyl units within the structure of CT polymers also affects the protein-binding affinity.     

葵花籽壳纳米纤维素/壳聚糖/大豆分离蛋白可食膜制备工艺优化

为了研究具有良好性能的可食膜及其制备方法,该文以大豆分离蛋白(soy protein isolate,SPI)为成膜基材,向其中添加葵花籽壳纳米纤维素(nano-crystalline cellulose,NCC)和壳聚糖(chitosan,CS)制备得到共混可食膜。通过研究成膜材料配比、pH值和丙三醇质量浓度对可食膜抗拉强度(tensile strength,TS)、断裂伸长率(elongarion,E)、水蒸气透过系数(water vapor permeability,WVP)和氧气透过率(oxygen permeability,OP)的影响,以可食膜综合性能为响应值,各因素为自变量,利用响应面法对工艺参数进行优化,并建立了二次多项式回归模型,通过对模型的分析得到各因素对可食膜性能综合分影响的大小顺序为pH值成膜材料配比丙三醇质量浓度。结果表明:成膜材料质量比NCC:CS:SPI为1.25:0.75:2,pH值为3.59,丙三醇质量浓度为0.02 g/m L时,可食膜性能(抗拉强度、断裂伸长率、水蒸气透过系数和氧气透过率)的综合分达到最高为0.63。红外和扫描电镜结果表明成膜材料间具有良好的相容性。研究结果可为可食膜的生产应用提供参考。     

Characterization of a chitosanase isolated from a commercial ficin preparation

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).     

Chitosan derivatives killed bacteria by disrupting the outer and inner membrane

Six kinds of water-soluble chitosan were prepared by grafting aminofunctionality onto chitosan at the C-6 position, and their antimicrobial activities were investigated against three Gram-negative and three Gram-positive bacteria. Among the derivatives, dimethylaminoethyl-chitosan (DMAEC) has the highest potential to suppress the growth of bacteria. To elucidate detailed antimicrobial modes of action against bacteria, cell integrity, outer membrane (OM), and inner membrane (IM) permeabilization assays were investigated. When treated with DMAEC, the release of 260 nm absorbing materials quickly increased for both Escherichia coli and Staphylococcus aureus, but the absorbance value was different due to the difference in cell structures. In OM and IM permeabilization assays, DMAEC rapidly increased 1-N-phenylnaphthylamine uptake and the release of cytoplasmic beta-galactosidase via an increase in the permeability of OM and IM. Moreover, DMAEC90 prepared from 90% deacetylated chitosan had more activity than DMAEC50 prepared from 50% deacetylated chitosan, and these results revealed that the antimicrobial action of water-soluble chitosans was dependent on the degree of deacetylation and the substituted group.     

Occurrence of Major Whey Proteins in the pH 4.6 Insoluble Protein Fraction from UHT-Treated Milk

A clear picture of the protein rearrangement in milk following UHT-treatment was drawn by a comparative analysis of the pH 4.6 soluble protein fraction (SPF) and the pH 4.6 insoluble protein fraction (IPF) recovered from raw and UHT-treated milk samples. The two protein fractions were analyzed by mono- or bidimensional gel electrophoresis under reducing and nonreducing conditions, and protein bands were identified by specific immunostaining. Results showed that bovine serum albumin, β-lactoglobulin, and, to a lesser extent, α-lactalbumin coprecipitated with caseins in UHT-treated milk samples at pH 4.6. These proteins were almost exclusively involved in high molecular weight aggregates held together by disulfide bonds. Partition of α-lactalbumin and bovine serum albumin in the protein fractions obtained upon acidification of milk at pH 4.6 was evaluated by competitive immunoassays. The ELISA-based results suggested the possibility of using pH 4.6 insoluble α-lactalbumin and bovine serum albumin, in addition to pH 4.6 insoluble β-lactoglobulin, as indicators of the intensity of the heat treatment applied to milk.     

Effect of chitosan, O-carboxymethyl chitosan, and N-[(2-hydroxy-3-N,N-dimethylhexadecyl ammonium)propyl] chitosan chloride on overweight and insulin resistance in a murine diet-induced obesity

Two water-soluble chitosan derivatives, O-carboxymethyl chitosan (O-CM-chitosan) and N-[(2-hydroxy-3-N,N-dimethylhexadecyl ammonium)propyl] chitosan chloride (N-CQ-chitosan), were prepared, and the therapeutic effects of chitosan, O-CM-chitosan, and N-CQ-chitosan on insulin resistance were simultaneously evaluated by rats fed on a high-fat diet. The parameters of high-fat diet-induced rats indicated that chitosan and its two derivatives not only have low cytotoxicity but can control overnutrition by fat and achieve insulin resistance therapy. However, the results in experiment in vivo showed that the therapeutic degree varied by the molecular weight and surface charge of chitosan, O-CM-chitosan, and N-CQ-chitosan. N-CQ-chitosan with a MW of 5 × 10(4) decreased body weight, the ratio of fat to body weight, triglyceride, fasting plasma glucose, fasting plasma insulin, free fatty acid, and leptin by 11, 17, 44, 46, 44, 87, and 64% and increased fecal lipid by 95%, respectively.     

Use of Biopolymeric Membranes for Adsorption of Paraquat Herbicide from Water

The use of membranes prepared with alginate and chitosan to adsorb paraquat aqueous solution was evaluated as a potential alternative technique for remediation of contaminated water. Production of bilayer membranes was based on the electrostatic interaction between alginate (a polyanion) and chitosan (a polycation). Herbicide adsorption experiments were performed using three different membranes, consisting of pure alginate, pure chitosan, and a chitosan/alginate bilayer. Adsorption was characterized using the Langmuir and Freundlich isotherm models, as well as by applying pseudo-first order and pseudo-second order kinetic models. The potential use of the membranes in environmental applications was evaluated using water collected from the Sorocabinha River in S?o Paulo State, Brazil. The results indicated that interactions between the membranes and the herbicide were strongly related to the type of biopolymer and the physical?Cchemical characteristics of the herbicide.     

Use of dicarboxylic acids to improve and diversify the material properties of porous chitosan membranes

Several nontoxic dicarboxylic acid solutions (oxalic acid, succinic acid, malic acid, and adipic acid solutions) instead of an acetic acid solution were used as solvents for chitosan dissolution. The amount of free amino groups of the chitosan in the solution decreased due to the ionic cross-linking of the dicarboxylic acids with chitosan. These solutions were used to fabricate porous chitosan membranes. Replacing acetic acid with these dicarboxylic acids for membrane preparation improved the water uptake (by 35% at most), tensile strength (by 110% at most), and elongation capability (by 50% at most) of the membranes. These dicarboxylic acid solutions not only act as solvents but also improve the material properties of the chitosan membranes due to the ionic cross-linking and hydrogen bond formation. In brief, a nontoxic and straightforward cross-linking method has been developed for chitosan material; this method does not result in a brittle product, thus making it better than the use of toxic cross-linking reagents.     

Effect of cationic membrane permselectivity on the efficiency of skim milk electroacidification

Bipolar membrane electroacidification (BMEA) uses the property of bipolar membranes to split water and the demineralization action of cation-exchange membranes (CEM). As milk mineral salt content is very sensitive to ionic strength and pH changes, the aim of this study was to better understand the effect of changes in mineral content during pH decrease and demineralization of skim milk. The objectives were to investigate the effect of different cationic permselective membranes (CSV and CMX membranes) on skim milk cation migration and protein precipitation during BMEA. The permselectivity of both membranes tested does not influence the final efficiency of BMEA. The purity of the bovine milk casein isolates produced was similar to or higher (97-98% versus 93.4-96.7) than those of commercial isolates, due to a reduced ash content (1.2 versus 2.0-3. 8%) resulting from the CEM demineralizing phenomenon. For both membranes, the main ionic species to migrate was the potassium ions.     

Interactions of Protein and Starch Studied Through Amyloglucosidase Action

The access of amyloglucosidase to the carbohydrate molecule was taken as a measure of interactions occurring among starch, amylose, amylopectin, β-dextrin and purified gluten, gliadins, high molecular weight glutenin subunits, or bovine serum albumin when they were mixed together and gelatinized before digestion. The most relevant decrease in liberated glucose, denoting coverage of some reaction sites for amyloglucosidase, occurred when gliadins were mixed with the carbohydrates. Other proteins were not as effective as gliadins. Amylopectin was not affected by any protein. Comparison of results allows some hypothesis to be formulated about the influence of structure on molecular interactions.     

Characterization of the Carbohydrates of Nonreduced Glutenin Fractionated by Multistacking SDS-PAGE from Two Hard Red Spring Wheat Flours

Native (nonreduced) glutenin aggregates of two hard red spring wheat flours (Len of good quality and line 205 of poor quality) containing the same high molecular weight glutenin subunits (2*, 7+9, and 5+10) were investigated for the possibility of glycosylation. Glutenins isolated by pH-precipitation were separated and purified under nonreducing conditions into five different molecular weight species by multistacking SDS-PAGE and by transfer to polyvinylidene difluoride (PVDF) membranes by electroblotting. Carbohydrate compositions of the total glutenin fraction and of the different molecular weight glutenin species separated on the stacking gels (4–12% acrylamide) were determined. More total carbohydrates were found in the total glutenin of the line 205 flour (2.33%) than in that of the Len flour (1.87%), with glucose and xylose contributing to the greater total amount in line 205. However, the total glutenin of Len had approximately twice as much of the total of arabinose, mannose, and galactose as line 205 had. After purification by electrophoresis, smaller amounts of monosaccharides (glucose, xylose, arabinose, galactose, and mannose) in the different molecular species were detected. After electroblotting to PVDF membrane to increase purification, no arabinoxylans were found. Following extraction of glutenin from membranes, the β-elimination procedure in mild base under reducing conditions was used. In that procedure, the mono- or oligosaccharide side chains are released from the protein core, and the sugars originally involved in the protein-sugar linkage are reduced to the sugar alcohol. After derivatization (with trimethylsilyl), samples were analyzed by gas chromatography and mass spectrometry. Glucose, galactose, and a small amount of mannitol were found in most of the different glutenin aggregates, except that mannitol was not found in the 8% stacking gel glutenin fraction. The content of mannitol was greater in the higher molecular weight glutenin species at the 4 and 6% origins. These results support the hypothesis that the carbohydrates and protein in the glutenin macropolymer may be covalently linked.     

Expression and purification of glycosylated bovine beta-casein (L70S/P71S) in Pichia pastoris

Post-translational glycosylation of bovine beta-casein (L70S/P71S) that results in Asn(68)-linked glycan on the protein was obtained in up to 30% of total beta-casein expressed in the methylotrophic yeast Pichia pastoris. Among the growth/induction media used, buffered minimal glycerol (BMG)/buffered minimal methanol (BMM) media were best for the production of glycosylated bovine beta-casein, indicating pH-dependent glycosylation. Glycosylated bovine beta-casein (L70S/P71S) expressed in P. pastoris was purified to homogeneity by the combination of ammonium sulfate fractionation, Concanavalin A--Sepharose affinity column, and Mono Q anion-exchange FPLC. The purified glycosylated bovine beta-casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated beta-casein was of high-mannose type. Unlike the hyperglycosylation that occurred in yeast, the majority of bovine beta-casein was not hyperglycosylated in P. pastoris, and its molecular weight was estimated to be 33.6 kDa. Glycosylated bovine beta-casein was normally phosphorylated to the same degree as native bovine beta-casein.     

Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.     

Comparison of physicochemical, binding, and antibacterial properties of chitosans prepared without and with deproteinization process

Physicochemical, binding, and antibacterial properties of chitosans prepared without and with deproteinization (DP) process (5, 10, 15, and 30 min at 15 psi/121 degrees C) were compared. Chitosan from DP 0 min had comparable nitrogen content, lower degree of deacetylation and solubility, but higher molecular weight and viscosity than chitosans from DP 5-30 min. The latter four chitosans showed differences only in molecular weight. Deproteinization treatment resulted in slightly decreased L values and increased a and b values compared with those of DP 0 min. Chitosan from DP 0 min had comparable water and fat-binding capacity (FBC) except for chitosan from DP 15 min, which had a higher FBC but lower dye-binding capacity than those of the four chitosans from DP 5-30 min. The antibacterial activities of chitosans against seven different bacteria showed that the inhibitory effects varied with the deproteinization time and the particular bacterium.     

Protein binding and astringent taste of a polymeric procyanidin, 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose, castalagin, and grandinin

The objective of the present investigation was to examine the oral astringency and protein-binding activity of four structurally well-defined tannins, namely, procyanidin [epicatechin16(4-->8)catechin], pentagalloyl glucose (1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose), castalagin, and grandinin, representing the three main structural categories of tannins, the proanthocyanidins, the gallotannins, and the ellagitannins. Astringency threshold and dos/response were determined by the half-tongue test using a trained human panel. Protein-binding stoichiometry and relative affinity were determined using radioiodinated bovine serum albumin in precipitation or competitive binding assays. Procyanidin and pentagalloyl glucose were perceived as highly astringent compounds and had relatively steep dose/response curves, but castalagin and grandinin had a lower mass threshold for detection. In vitro, procyanidin was the most effective protein-precipitating agent and grandinin the least. Increasing the temperature increased protein precipitation by the hydrolyzable tannins, especially grandinin. All four polyphenols had higher relative affinities for proline-rich proteins than for bovine serum albumin.     

Angiotensin I converting enzyme (ACE) inhibitory activity of hetero-chitooligosaccharides prepared from partially different deacetylated chitosans

Angiotensin I converting enzyme (ACE) inhibitory activity of hetero-chitooligosaccharides (hetero-COSs) prepared from partially different deacetylated chitosans was investigated. Partially deacetylated chitosans, 90, 75, and 50% deacetylated chitosan, were prepared from crab chitin by N-deacetylation with 40% sodium hydroxide solution for durations. In addition, nine kinds of hetero-COSs with relatively high molecular masses (5000-10 000 Da; 90-HMWCOSs, 75-HMWCOSs, and 50-HMWCOSs), medium molecular masses (1000-5000 Da; 90-MMWCOSs, 75-MMWCOSs, and 50-MMWCOSs), and low molecular masses (below 1000 Da; 90-LMWCOSs, 75-LMWCOSs, and 50-LMWCOSs) were prepared using an ultrafiltration membrane bioreactor system. ACE inhibitory activity of hetero-COSs was dependent on the degree of deacetylation of chitosans. 50-MMWCOSs that are COSs hydrolyzed from 50% deacetylated chitosan, the relatively lowest degree of deacetylation, exhibited the highest ACE inhibitory activity, and the IC(50) value was 1.22 +/- 0.13 mg/mL. In addition, the ACE inhibition pattern of the 50-MMWCOSs was investigated by Lineweaver-Burk plots, and the inhibition pattern was found to be competitive.     

不同采后处理对翠冠梨果实品质的影响

为探讨不同采后处理对翠冠梨果实生理生化变化的影响,筛选出适宜翠冠梨果实常温贮藏的保鲜技术,本试验以翠冠梨果实为试验材料,研究0.25 μL·L^-1 1-甲基环丙烯(1-MCP)处理、2.0%氯化钙处理和1.5%壳聚糖处理对20℃模拟转货架条件下翠冠梨果实品质变化的影响。结果表明,0.25 μL·L^-1 1-MCP处理、2.0%氯化钙处理和1.5%壳聚糖涂膜处理能够显著降低货架期间翠冠梨果实腐烂率和失重率,推迟呼吸峰值的出现,使果实硬度、可溶性糖含量(TSC)、可滴定酸(TA)含量和维生素C(Vc)含量维持较高水平;同时,3种处理均能够有效减缓梨果实色泽褐变速度,抑制多酚氧化酶(PPO)活性,降低膜相对透性和丙二醛(MDA)含量,延缓过氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性的降低。主成分分析(PCA)表明,1.5%壳聚糖涂膜处理对翠冠梨常温货架期的保鲜效果最佳,其次为0.25 μL·L^-1 1-MCP处理、2.0%氯化钙处理。本研究结果为采后处理在翠冠梨果实常温贮藏的应用提供了理论依据。     

奶牛乳腺炎金黄色葡萄球菌临床分离株生化特性与菌体蛋白免疫原性分析

摘要：病原学调查发现金黄色葡萄球菌是奶牛乳腺炎的最主要致病菌之一。从53株金黄色葡萄球菌乳样分离株中挑选了培养和形态典型的六株进行生化试验和传代稳定性试验，进一步研究这六株菌的凝固酶、溶血素等致病性因子和耐药性。对菌体裂解产物进行蛋白电泳图谱分析发现菌体蛋白条带具有很高的相似性,TQ-1株电泳蛋白条带密度较其它几株高。用其中四株生化典型、传代稳定、致病因子较多的菌株免疫兔，制备高免血清，分析全菌抗原的蛋白转印图谱，发现XG-2株在52KD处有特殊保护性抗原条带。综合本文各试验结果，确定以TQ-1和XG-2株为疫苗候选株。     

秘鲁鱿鱼皮明胶-壳聚糖复合膜的性能与结构表征

为考察壳聚糖对鱿鱼皮明胶膜的改性效果,将壳聚糖添加到明胶溶液中,考察壳聚糖的添加量对鱿鱼皮明胶复合膜机械性能、水蒸气透过率、透光性及其结构的影响。结果表明,壳聚糖能有效改善鱿鱼皮明胶膜的有关性能指标,当鱿鱼皮明胶溶液与壳聚糖溶液以60∶40(v/v)比例混合,制得复合膜的断裂伸长率相比单一明胶膜下降,但其抗拉伸强度、透光率和水蒸气阻隔能力分别比单一明胶膜提高了652%、11%和48%;差示扫描量热仪、红外及扫描电镜分析结果显示,壳聚糖能与鱿鱼皮明胶相互作用,形成结构致密的均相体系,提高复合膜的热变性温度。综上可知,鱿鱼皮明胶与壳聚糖之间具有良好的相容性,壳聚糖是一种较理想的明胶膜改性材料。本试验结果为鱿鱼皮明胶作为食品保鲜膜的应用提了供依据。