Motility and fertility of alginate encapsulated boar spermatozoa

Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 degrees C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16-18 degrees C, and then were stored at 5 degrees C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 degrees C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 degrees C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8h incubation at 37 degrees C after storing for over three days (P<0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability.     

Microencapsulation of bovine liver arginase: Characterization and in vivo evaluation of its effect on the growth of the L1210 murine leukaemia

Bovine liver arginase (EC 3.5.3.1) was encapsulated within nylon membrane microcapsules by the process of interfacial polymerization. The effect of microencapsulation on the properties of arginase was investigated. The K_m, pH optimum and temperature stability at 37°C of arginase, were not significantly altered. The microencapsulated enzyme was much less stable at temperatures between 50 and 70°C than the free enzyme. Native arginase was inactivated by exposure to proteolytic enzymes, whereas microencapsulated arginase was much more resistant to proteolysis. A preliminary investigation of the effectiveness of the microencapsulated enzyme against the L1210 murine leukaemia in vivo has been made.     

包裹于PLGA微球中用于蛋白控释的多糖纳米颗粒

目的:研究包裹在PLGA微球中的多糖纳米颗粒在保护蛋白稳定性和改善药物体外释放行为方面的作用。方法:将模型药物BSA用低温诱导相分离方法担载于多糖纳米颗粒之中,后将其用水包油包固复乳法包裹于PLGA微球内。应用体积排阻色谱(SEC-HPLC)和红外色谱(FTIR)表征蛋白的稳定性,而且也研究了样品的体外释放行为。结果:这种方法能够很好的保护蛋白的稳定性,保持蛋白结构在制备过程中不会改变,而且改善了体外释放行为,减少了突释。结论:多糖纳米颗粒结合PLGA微球能够提供一种有效的解决蛋白控释的途径。     

Accelerated Polymer Biodegradation of Risperidone Poly(d, l-Lactide-Co-Glycolide) Microspheres

The influence of a tertiary amine, namely risperidone (pKa = 7.9) on the degradation of poly(d, l lactide-co-glycolide) (PLGA) microspheres was elucidated. Risperidone and blank microspheres were fabricated at two lactide/glycolide ratios, 65:35 and 85:15. The microspheres were characterized for drug loading by high-performance liquid chromatography, particle size by laser diffractometry, and surface morphology by scanning electron microscopy. Polymer degradation studies were carried out with drug-loaded microspheres and blank microspheres in presence of free risperidone in 0.02 M PBS containing 0.02% Tween®80 at 37°C. Molecular weight was monitored by gel permeation chromatography. Risperidone and blank microspheres had similar size distribution and were spherical with a relatively nonporous smooth surface. The presence of risperidone within the microspheres enhanced the hydrolytic degradation in both polymeric matrices with faster degradation occurring in 65:35 PLGA. The molecular weight decreased according to pseudo-first-order kinetics for all the formulations. During the degradation study, the surface morphology of drug-loaded microspheres was affected by the presence of risperidone and resulted in shriveled microspheres in which there appeared to be an intrabatch variation with the larger microspheres being less shriveled than the smaller ones. When blank microspheres were incubated in free risperidone solutions, a concentration-dependent effect on the development of surface porosity could be observed. Risperidone accelerates the hydrolytic degradation of PLGA, presumably within the microenvironment of the drug-loaded particles, and this phenomenon must be taken into consideration in designing PLGA dosage forms of tertiary amine drugs.Key words: mass loss, microencapsulation, PLGA microspheres, polymer degradation, risperidone, tertiary amine drug     

Microencapsulation of Bacteriophage Felix O1 into Chitosan-Alginate Microspheres for Oral Delivery

This paper reports the development of microencapsulated bacteriophage Felix O1 for oral delivery using a chitosan-alginate-CaCl₂ system. In vitro studies were used to determine the effects of simulated gastric fluid (SGF) and bile salts on the viability of free and encapsulated phage. Free phage Felix O1 was found to be extremely sensitive to acidic environments and was not detectable after a 5-min exposure to pHs below 3.7. In contrast, the number of microencapsulated phage decreased by 0.67 log units only, even at pH 2.4, for the same period of incubation. The viable count of microencapsulated phage decreased only 2.58 log units during a 1-h exposure to SGF with pepsin at pH 2.4. After 3 h of incubation in 1 and 2% bile solutions, the free phage count decreased by 1.29 and 1.67 log units, respectively, while the viability of encapsulated phage was fully maintained. Encapsulated phage was completely released from the microspheres upon exposure to simulated intestinal fluid (pH 6.8) within 6 h. The encapsulated phage in wet microspheres retained full viability when stored at 4°C for the duration of the testing period (6 weeks). With the use of trehalose as a stabilizing agent, the microencapsulated phage in dried form had a 12.6% survival rate after storage for 6 weeks. The current encapsulation technique enables a large proportion of bacteriophage Felix O1 to remain bioactive in a simulated gastrointestinal tract environment, which indicates that these microspheres may facilitate delivery of therapeutic phage to the gut.     

固定化微球降解土壤中PAEs效果及影响因素

旨为研究土壤邻苯二甲酸酯污染修复中,固定化微球降解土壤中邻苯二甲酸酯的效果及影响因素。以海藻酸钠为载体,采用包埋法对课题组前期提取的微小杆菌进行固定化,比较固定化微球和游离菌降解土壤中邻苯二甲酸酯(Phthalates esters,PAEs)的效果及pH、温度、重金属、无机盐等对降解菌降解目标物的影响。结果显示:(1)在土壤环境相同条件下,固定化微球对邻苯二甲酸二甲酯(Dimethyl ortho-phthalate,DMP)、邻苯二甲酸二正丁酯(Di-n-butyl ortho-phthalate,DnBP)和邻苯二甲酸二(2-乙基己)酯(Bis(2-ethylhexyl)ortho-phthalate,DEHP)的降解效果高于游离菌,DMP在7 d可降解完全,DnBP在10 d内可降解完全,DEHP在20 d降解率63.73%;而游离菌则在15 d内完全降解DMP,20 d内完全降解DnBP,DEHP在20 d降解率48.77%;(2)不同pH值时,固定化微球对DMP、DnBP、DEHP的降解率均高于游离菌,pH9时,固定化微球对于DMP、DnBP、DEHP的降解率最高分别为96.81%、89.39%、58.35%;(3)不同温度,固定化微球对DMP、DnBP、DEHP的降解率也均高于游离菌,温度为30℃时,固定化微球对于DMP、DnBP、DEHP的降解效率达到最高,分别为96.27%、89.19%、59.01%;(4)重金属使游离菌对DMP、DnBP、DEHP降解率下降较多,而使固定化微球对DMP、DnBP的降解率仅下降了16.35%、9.95%,DEHP不仅没有降低,反而增加2.49%,说明重金属对游离菌起到很强的抑制作用,但对于固定化微球的降解效果影响较小;(5)盐碱条件下,中性盐极大降低了游离菌和固定化微球降解DMP、DnBP、DEHP的降解能力,碱性盐和混合盐对降解菌影响较小,且增强了固定化微球对DnBP、DEHP的降解能力。固定化微球降解PAEs效果明显高于游离菌,对外界环境有更好的适应能力,且对重金属、无机盐污染环境有一定的抵御能力。     

Proteolysis of procollagen I.

1. Digestion of procollagen I which trypsin, pepsin or pronase performed at 20 degrees C causes the release of acidic non-collagenous fragments and hydroxyproline-rich fraction. Enzymatic proteolysis performed at 41 degrees C (above the temperature of denaturation) results in degradation of procollagen I to low-molecular peptides. 2. The hydroxyproline-rich fraction obtained by limited proteolysis of procollagen I with pepsin (at 20 degrees C) contains a material corresponding to alpha and beta subunits of tropocollagen. Reduction of the hydroxyproline-rich fraction released by trypsin or pronase (at 20 degrees C) causes the appearance of polypeptides similar to pro-alpha subunits.     

Immunochemical studies on thermal denaturation of ovomucoid

The thermal denaturation of ovomucoid was investigated by immunochemical methods, namely immunoprecipitation analyses and antibody-Sepharose 4B column chromatography. In the immunoprecipitation analyses, heated ovomucoid (90 degrees C, 90 min, pH 7.2) required about twice the antigen addition of the native protein to approach maximal precipitation with specific antibody, and the maximal immunoprecipitation was decreased to 80% of that by native ovomucoid. However, heated protein inhibited the binding of antibody with native ovomucoid, and 100% inhibition was attained at about 4-times the antigen addition necessary for the native protein. Heated ovomucoid (100 degrees C, 120 min) showed little immunoprecipitation and inhibition. To ovomucoid antigenicity was diminished more slowly than the trypsin inhibitory activity by heating, e.g., heated ovomucoid (90 degrees C, 120 min) retained more than 30% of the antigenicity but little trypsin inhibitory activity. By passing through the immunoaffinity column, heated ovomucoid (90 degrees C, 90 min) was separated into two fractions, either with (fraction II) or without (fraction I) antigenicity. Fraction II contained smaller fractions of ordered secondary structure than native ovomucoid, and trypsin inhibitory activity of fraction II was only 24% of the native one. These results indicated that thermally denatured ovomucoid was heterogeneous regarding the conformational damage caused by heating, and the structure around some antigenic sites in an ovomucoid molecule was retained even after the backbone conformation was partially destroyed and trypsin inhibitory activity was lost.     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Preparation and characterization of temperature-sensitive poly(N-isopropylacrylamide)-b-poly(D,L-lactide) microspheres for protein delivery

Temperature-sensitive diblock copolymers, poly(N-isopropylacrylamide)-b-poly(D,L-lactide) (PNIPAAm-b-PLA) with different PNIPAAm contents were synthesized and utilized to fabricate microspheres containing bovine serum albumin (BSA, as a model protein) by a water-in-oil-in-water double emulsion solvent evaporation process. XPS analysis showed that PNIPAAm was a dominant component of the microspheres surface. BSA was well entrapped within the microspheres, and more than 90% encapsulation efficiency was achieved. The in vitro degradation behavior of microspheres was investigated using SEM, NMR, FTIR, and GPC. It was found that the microspheres were erodible, and polymer degradation occurred in the PLA block. Degradation of PLA was completed after 5 months incubation in PBS (pH 7.4) at 37 degrees C. A PVA concentration of 0.2% (w/v) in the internal aqueous phase yielded the microspheres with an interconnected porous structure, resulting in fast matrix erosion and sustained BSA release. However, 0.05% PVA produced the microspheres with a multivesicular internal structure wrapped with a dense skin layer, resulting in lower erosion rate and a biphasic release pattern of BSA that was characterized with an initial burst followed by a nonrelease phase. The microspheres made from PNIPAAm-b-PLA with a higher portion of PNIPAAm provided faster BSA release. In addition, BSA release from the microspheres responded to the external temperature changes. BSA release was slower at 37 degrees C (above the LCST) than at a temperature below the LCST. The microspheres fabricated with PNIPAAm-b-PLA having a 1:5 molar ratio of PNIPAAm to PLA and 0.2% (w/v) PVA in the internal aqueous phase provided a sustained release of BSA over 3 weeks in PBS (pH 7.4) at 37 degrees C.     

Ammonolysis-induced solvent removal: a facile approach for solidifying emulsion droplets into PLGA microspheres

An ammonolysis-based microencapsulation technique useful for the preparation of biodegradable microspheres was described in this study. A dispersed phase consisting of poly- d, l-lactide- co-glycolide, progesterone, and methyl chloroacetate was emulsified in an aqueous phase. Upon addition of ammonia solution, the emulsion droplets were quickly transformed into poly- d, l-lactide- co-glycolide microspheres laden with progesterone. Rapid solvent removal was accompanied by ammonolysis. The chemical reaction converted water-immiscible methyl chloroacetate to water-miscible chloroacetamide and methanol. Chloroacetamide formation was proved by (1)H NMR and ESI-MS studies. Thermogravimetric analysis showed that the microspheres contained only small amounts of residual methyl chloroacetate. Incorporation efficiencies of progesterone ranged from 64.3 +/- 1.1 to 72.8 +/- 0.3%, depending upon microsphere formulations. X-ray powder diffractometry analysis substantiated that no polymorphic transition of progesterone occurred during microencapsulation. To evaluate the feasibility of this new method against the commonly used microencapsulation method, microspheres were also prepared by a typical dichloromethane-based solvent evaporation process. The important attributes of microspheres prepared from both methods were characterized for comparison. The new ammonolysis-based microencapsulation process showed interesting features distinct from those of the solvent evaporation process. The microencapsulation process reported in this study might be applicable in loading pharmaceuticals into various polymeric microspheres.     

枯草芽胞杆菌微生态制剂的研制

采用液体发酵工艺,确定枯草芽胞杆菌的最适发酵条件为:发酵温度30℃,初始pH值7.2,并以1%海藻酸钠和3%明胶组成的混合胶体溶液为囊壁材料,以4%氯化钙作固化剂将枯草芽胞杆菌制成微胶囊剂,稳定性试验结果显示经微胶囊包埋的枯草芽胞杆菌制剂,室温下保存1个月,活菌存活率为98.8%,保存3个月,活菌存活率为50.6%,保存6个月,活菌存活率为15.7%,均高于未经微胶囊化的样品;在4℃冷藏下保存3个月,未经微胶囊化的样品活菌存活率仅为经微胶囊包埋制剂的66.2%。该微胶囊制剂提高了活菌存活率,延长了活菌常温保存期。     

Properties of immobilized pepsin on Modified PMMA microspheres

In this work we use micro-size poly(methyl methacrylate)/acrylaldehyde microspheres as a support for pepsin immobilization. The aldehyde groups on the microspheres offer a very simple, mild and firm combination for enzyme immobilization. The amount of enzyme we can bind to this support reaches 82 mg/g, which is much higher than for other supports (mostly less than 10 mg/g). Compared to free enzyme, the Km of immobilized enzyme is increased, whereas the Vmax is decreased. Further, the Vmax/Km value for immobilized pepsin is about 50% of the value for free enzyme. This is better than values reported previously, generally lower than 35%. The optimum temperature shifts from 43 degrees C for free pepsin to 47 degrees C. However, the optimum pH does not change between free and immobilized enzyme. This improved resistance of the immobilized enzyme towards changes in temperature and pH also shows that the aldehyde modified poly(methyl methacrylate)/acrylaldehyde microspheres can be a valuable support for pepsin immobilization.     

Metal chelates of 2-hydroxy-4-methylthiobutanoic acid in animal feeding. Part 2: Further characterizations, in vitro and in vivo investigations

The alpha-hydroxyacid 2-hydroxy-4-methylthiobutanoic acid (the so-called methionine hydroxy-analogue, MHA), largely used in animal nutrition as a source of methionine, forms stable metal chelates with divalent metals of formula [{CH(3)SCH(2) CH(2)CH(OH)COO}(2)M].nH(2)O. Protonation and iron(III) and copper(II) complex formation constants have been determined by potentiometry at 25 degrees C. Distribution diagrams show that no free Fe(3+) cations are present in solution at pH>2.5. ESI-MS (Electron-Spray Ionization Mass Spectrometry) investigations carried out both on iron and zinc complexes in solution have evidenced various species with different MHA/metal ratios. In vivo trials were carried out with rats. After receiving a zinc-deficient diet for 3 weeks, animals were fed the same diet added with zinc sulfate or zinc/MHA chelate; the zinc content of faeces was higher (+45%; P<0.05) in sulfate fed rats, whereas zinc retention was higher (+61%; P<0.05) in the Zn/MHA diet. Experiments in vitro with human intestinal Caco-2 cells indicated that the MHA/Fe chelate was taken up by the cells without any apparent toxic effect. The iron uptake was higher than that of iron nitrilotriacetate (Fe(3+)NTA), an effective chelate for delivering iron to milk diets. In conclusion, these data indicate that the use of MHA chelates could be a valuable tool to increase bioavailability of trace minerals and reduce the environmental impact of animal manure.     

Microencapsulated sodium selenite supplementation in dairy cows: effects on selenium status

The objective of this study was to compare the efficiency of transfer of selenium (Se) to plasma and milk from inorganic sodium selenite, either free or microencapsulated, and from selenized yeast in dairy cows. The study consisted of an in situ-nylon bags incubation, and in an in vivo experiment to compare the Se status of cows supplemented with either sodium selenite, microencapsulated sodium selenite, or Se yeast. Thirty dairy cows, divided in five groups, were fed the following diets: the control group (CTR) received a total mixed ration supplemented with sodium selenite in order to have 0.3 mg/kg DM of total Se; 0.3M and 0.5M groups received the same control diet supplemented with lipid microencapsulated sodium selenite to provide 0.3 and 0.5 mg/kg DM of total Se, respectively; 0.3Y and 0.5Y groups received selenized yeast to provide 0.3 and 0.5 mg/kg of total Se, respectively. Cows were fed the supplements for 56 days during which milk, blood, and fecal samples were collected weekly to conduct analysis of Se and glutathione peroxidase (GSH-px) activity. Se concentration in the nylon bags was assessed to 72%, 64%, and 40% of the initial value (time 0) after 4, 8, and 24 h of incubation, respectively. In vivo, cows supplemented with 0.3 mg/kg of microencapsulated Se had higher milk Se concentration compared to CTR. The increment was more pronounced at the highest inclusion rate (0.5 mg/kg, 0.5M group). GSH-px activity was not significantly affected by treatments. The results indicate that lipid microencapsulation has the potential to protect nutrients from complete rumen reduction and that Se from microencapsulated selenite is incorporated in milk more efficiently than the free form. Microencapsulated sodium selenite was shown to be comparable to Se-yeast in terms of availability and incorporation in milk when fed at 0.3 mg/kg DM, whereas the inclusion in the diet at 0.5 mg/kg DM resulted in higher plasma and milk concentrations than selenized yeast.     

Microencapsulated Chromatophores for the Production of ATP

Chromatophores, organelles for photophosphorylation in non-sulfur purple photosynthetic bacteria, were microencapsulated and utilized in ATP production. The microcapsules were formed by photocrosslinking with trimethylolpropane triacrylate (TMPTA). In batch experiments chromatophores microencapsulated in TMPTA capsules were repeatedly used in ATP production for more than 5 times. Continuous ATP production was then undertaken. ATP was produced at a production rate of 14 μmol h^-1 L^-1 over 200 hrs. The yield (from ADP to ATP) was 35%. The total amount of ATP produced was 0.7 mM (μM Bchl)^-1. Therefore, this microencapsulation method was found to be suitable for the continuous ATP production using chromatophores.     

Membrane formation by interfacial cross-linking of chitosan for microencapsulation of Lactococcus lactis

Lactic acid bacteria were microencapsulated within cross-linked chitosan membranes formed by emulsification/interfacial polymerization. The technique was modified and optimized to provide biocompatible conditions during encapsulation involving the use of mineral oils as the continuous phase and chitosan as the membrane material. Chitosan cross-linked with hexamethylene diisocyanate or glutaraldehyde resulted in strong membranes, with a narrow size distribution about a mean diameter of 150 mum. Cell viability and activity was demonstrated by the acidification of milk. Loss of acidification activity during microencapsulation was recovered in subsequent fermentations to levels similar to that of free cell fermentations. (c) 1993 John Wiley & Sons, Inc.     

Functional stabilization of trypsin by conjugation with beta-cyclodextrin-modified carboxymethylcellulose

Bovine pancreatic trypsin was chemically modified by a beta-cyclodextrin-carboxymethylcellulose polymer using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as coupling agent. The conjugate retained 110% and 95% of the initial esterolytic and proteolytic activity, respectively, and contained about 2 mol of polymer per mol of trypsin. The optimum temperature for trypsin was increased to 8 degrees C after conjugation. The thermostability of the enzyme was increased to about 16 degrees C after modification. The conjugate prepared was also more stable against thermal incubation at different temperatures ranging from 45 degrees C to 60 degrees C. In comparison with native trypsin, the polymer-enzyme complex was more resistant to autolytic degradation at pH 9.0, retaining about 65% of the initial activity after 3h incubation. In addition, modification protected trypsin against denaturation in the presence of sodium dodecylsulfate.     

Microencapsulated Chromatophores for the Production of ATP

Chromatophores, organelles for photophosphorylation in non-sulfur purple photosynthetic bacteria, were microencapsulated and utilized in ATP production. The microcapsules were formed by photocrosslinking with trimethylolpropane triacrylate (TMPTA). In batch experiments chromatophores microencapsulated in TMPTA capsules were repeatedly used in ATP production for more than 5 times. Continuous ATP production was then undertaken. ATP was produced at a production rate of 14 μmol h^?1 L^?1 over 200 hrs. The yield (from ADP to ATP) was 35%. The total amount of ATP produced was 0.7 mM (μM Bchl)^?1. Therefore, this microencapsulation method was found to be suitable for the continuous ATP production using chromatophores.     

静电喷雾法制备乳酸菌微胶囊的实验研究

目的为解决乳酸菌产品活菌数的不稳定性,对乳酸菌进行微胶囊化包埋。方法用海藻酸钠和明胶的混合体系作为壁材,对乳酸菌进行静电喷雾包埋处理,并让微胶囊化乳酸菌在模拟胃肠液的环境中进行耐酸性和肠溶性实验。结果混合体系的壁材与乳酸菌具有较好的生物相容性,优选得出当芯壁材为12时包埋率最高（96．3％）,微胶囊化乳酸菌在经人工胃液处理2h后,活菌数比未经微胶囊化的对照组高出2个数量级,且在经人工肠液处理40min后,乳酸菌几乎全部释放。结论静电喷雾法制备的乳酸菌微胶囊具有一定耐酸性和肠溶性。