Process optimization for microencapsulation of probiotic yeasts

Background

Microencapsulation is a technique which improves the survival and viability of probiotics. We demonstrate encapsulation of five potential probiotic yeasts with alginate and gum as encapsulation matrices to improve their gastrointestinal transit.

Methods

Gum extracted from various cereals viz. rice, oats, barley, finger millet and pearl millet along with alginate have been used to encapsulate five potential probiotic yeasts. Screening was carried out by measuring swelling index, encapsulation efficiency and nutritional value of microcapsules encapsulated with alginate and gum. The concentration of OBG, sodium alginate and inoculum dosage of probiotic yeasts was optimized using response surface methodology (RSM). Efficiency of alginate OBG microcapsules with or without coating materials viz. whey protein and chitosan also tested. The mucoadhesion ability and storage stability of alginate OBG microcapsules with coating materials were tested.

Results

Highest encapsulation efficiency of probiotic yeasts was noted using oats bran gum (OBG) microcapsules along with alginate in all the five probiotic yeasts. Notably whey protein coated microcapsules showed maximum GIT tolerance (95%) and mucoadhesion (90%) for L. starkeyi VIT-MN03. The minimum loss of viability was observed in L. starkeyi VITMN03 microcapsules on 60th day of storage.

Conclusions

This is the first report on optimization and survival of microencapsulated probiotic yeasts under simulated GIT conditions using natural gum and alginate as encapsulation matrices and whey protein as coating material.

     

Progress technology in microencapsulation methods for cell therapy

Cell encapsulation in microcapsules allows the in situ delivery of secreted proteins to treat different pathological conditions. Spherical microcapsules offer optimal surface‐to‐volume ratio for protein and nutrient diffusion, and thus, cell viability. This technology permits cell survival along with protein secretion activity upon appropriate host stimuli without the deleterious effects of immunosuppressant drugs. Microcapsules can be classified in 3 categories: matrix‐core/shell microcapsules, liquid‐core/shell microcapsules, and cells‐core/shell microcapsules (or conformal coating). Many preparation techniques using natural or synthetic polymers as well as inorganic compounds have been reported. Matrix‐core/shell microcapsules in which cells are hydrogel‐embedded, exemplified by alginates capsule, is by far the most studied method. Numerous refinement of the technique have been proposed over the years such as better material characterization and purification, improvements in microbead generation methods, and new microbeads coating techniques. Other approaches, based on liquid‐core capsules showed improved protein production and increased cell survival. But aside those more traditional techniques, new techniques are emerging in response to shortcomings of existing methods. More recently, direct cell aggregate coating have been proposed to minimize membrane thickness and implants size. Microcapsule performances are largely dictated by the physicochemical properties of the materials and the preparation techniques employed. Despite numerous promising pre‐clinical results, at the present time each methods proposed need further improvements before reaching the clinical phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Isolation of human foetal myoblasts and its application for microencapsulation

Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18-24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology.     

微囊化干细胞及其应用研究进展

干细胞极强的自我更新能力和多向分化潜能使其可以成为绝佳的种子细胞来源,用于各种疑难疾病的治疗。微胶囊不仅可以为细胞提供三维生长微环境,而且具有良好的免疫隔离性能和生物相容性。微囊化干细胞技术为干细胞大规模、高活性体外培养及长期保存提供了新的技术支持,为细胞移植疗法开辟了新途径。以下首先简述了微囊化技术的发展情况,然后介绍了目前用于微囊化干细胞的材料、制备方法及其免疫隔离作用,重点阐述了近年来微囊化各种不同类型干细胞的研究和应用进展。最后,提出目前微胶囊化干细胞的问题所在并对此技术进行展望。     

Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms

Methods to microencapsulate enzyme, cells, and genetically engineered cells have been described in this article. More specific examples of enzyme encapsulation include the microencapsulation of xanthine oxidase for Lesch-Nyhan disease; phenylalanine ammonia lyase for pheny, ketonuria and microencapsulation of multienzyme systems with cofactor recycling for multistep enzyme conversions. Methods for cell encapsulation include the details for encapsulating hepatocytes for liver failure and for gene therapy. This also includes the details of a novel two-step method for encapsulation of high concentrations of smaller cells. Another new approach is the detailed method of the encapsulation of genetically engineered Escherichia coli DH5 cells for lowering urea, ammonia, and other metabolites in kidney or, liver failure and other diseases.     

Stem cell microencapsulation for phenotypic control,bioprocessing, and transplantation

Cell microencapsulation has been utilized for decades as a means to shield cells from the external environment while simultaneously permitting transport of oxygen, nutrients, and secretory molecules. In designing cell therapies, donor primary cells are often difficult to obtain and expand to appropriate numbers, rendering stem cells an attractive alternative due to their capacities for self‐renewal, differentiation, and trophic factor secretion. Microencapsulation of stem cells offers several benefits, namely the creation of a defined microenvironment which can be designed to modulate stem cell phenotype, protection from hydrodynamic forces and prevention of agglomeration during expansion in suspension bioreactors, and a means to transplant cells behind a semi‐permeable barrier, allowing for molecular secretion while avoiding immune reaction. This review will provide an overview of relevant microencapsulation processes and characterization in the context of maintaining stem cell potency, directing differentiation, investigating scalable production methods, and transplanting stem cells for clinically relevant disorders. Biotechnol. Bioeng. 2013; 110: 667–682. © 2012 Wiley Periodicals, Inc.     

Mitigation of quantum dot cytotoxicity by microencapsulation

When CdSe/ZnS-polyethyleneimine (PEI) quantum dots (QDs) are microencapsulated in polymeric microcapsules, human fibroblasts are protected from acute cytotoxic effects. Differences in cellular morphology, uptake, and viability were assessed after treatment with either microencapsulated or unencapsulated dots. Specifically, QDs contained in microcapsules terminated with polyethylene glycol (PEG) mitigate contact with and uptake by cells, thus providing a tool to retain particle luminescence for applications such as extracellular sensing and imaging. The microcapsule serves as the "first line of defense" for containing the QDs. This enables the individual QD coating to be designed primarily to enhance the function of the biosensor.     

Differential role of microenvironment in microencapsulation for improved cell tolerance to stress

The effect of the microenvironment in alginate–chitosan–alginate (ACA) microcapsules with liquid core (LCM) and solid core (SCM) on the physiology and stress tolerance of Sacchromyces cerevisiae was studied. The suspended cells were used as control. Cells cultured in liquid core microcapsules showed a nearly twofold increase in the intracellular glycerol content, trehalose content, and the superoxide dismutase (SOD) activity, which are stress tolerance substances, while SCM did not cause the significant physiological variation. In accordance with the physiological modification after being challenged with osmotic stress (NaCl), oxidative stress (H₂O₂), ethanol stress, and heat shock stress, the cell survival in LCM was increased. However, SCM can only protect the cells from damaging under ethanol stress. Cells released from LCM were more resistant to hyperosmotic stress, oxidative stress, and heat shock stress than cells liberated from SCM. Based on reasonable analysis, a method was established to estimate the effect of microenvironment of LCM and SCM on the protection of cells against stress factors. It was found that the resistance of LCM to hyperosmotic stress, oxidative stress, and heat shock stress mainly depend on the domestication effect of LCM’s microenvironment. The physical barrier of LCM constituted by alginate–chitosan membrane and liquid alginate matrix separated the cells from the damage of oxidative stress and ethanol stress. The significant tolerance against ethanol stress of SCM attributed to the physical barrier consists of solid alginate–calcium matrix and alginate–chitosan membrane.     

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.     

History, challenges and perspectives of cell microencapsulation

Cell microencapsulation continues to hold significant promise for biotechnology and medicine. The controlled, and continuous, delivery of therapeutic products to the host by immunoisolated cells is a potentially cost-effective method to treat a wide range of diseases. Although there are several issues that need to be addressed, including capsule manufacture, properties and performance, in the past few years, a stepwise analysis on the essential obstacles and limitations has brought the whole technology closer to a realistic proposal for clinical application. This paper summarizes the current situation in the cell encapsulation field and discusses the main events that have occurred along the way.     

益生菌微胶囊化壁材的研究进展

目前国内的益生菌囊化材料主要为天然高分子聚合物,而合成和半合成的高分子聚合物的研究很少.为此,本文从囊化技术和功效比较两方面讨论了海藻酸钠、明胶和壳聚糖等聚合材料在益生菌微胶囊化方面应用的国内研究进展,以其寻找具有更有功效的、对微生物细胞更有相容性的高分子包囊工艺技术.     

Design of high-throughput-compatible protocols for microencapsulation, cryopreservation and release of bovine spermatozoa

With a rate exceeding 90% in cattle, artificial insemination (AI) is the prime reproduction technology in stock farming. AI success is expected to increase with extended persistence of sperms in utero. In order to enable controlled sperm release during artificial insemination we have designed two strategies for the automated microencapsulation of bovine spermatozoa in either alginate-Ca2+ or cellulose sulfate (CS)-poly-diallyldimethyl ammonium chloride (pDADMAC) capsules using standard encapsulation hardware. Animal protein- and citric acid-free sperm extenders and encapsulation protocols have been developed to ensure encapsulation compatible with sperm physiology. Bovine spermatozoa have showed high motility rates inside CS-pDADMAC-based capsules, were preserved by standard cryoconservation and rescued with high viability/motility following disintegration of the thawed capsules. CS-pDADMAC-based capsules break up within 72 h after addition of either purified cellulase or cellulase-filled alignate-Ca2+ capsules. The controlled release, associated with the microencapsulation of bovine spermatozoa, may be a promising approach to increase the success rate of artificial insemination.     

Ultraviolet protection of nucleopolyhedrovirus through microencapsulation with different polymers

A microencapsulated formulation of Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) was produced by emulsion technique to improve its stability under ultraviolet (UV) radiation. The polymers used include sodium alginate, gelatin and starch at concentration levels of 3% and 5% w/v. Except for the starch microencapsulated formulation (3% w/v), the difference in mortality of treated insects between microencapsulated and non-microencapsulated suspensions before irradiation was not significant according to variance analysis (Duncan test, P < 0.05, df = 6). This indicates that microencapsulation of HaNPV does not affect viral activity. Among the three polymers, gelatin performed the best and provided the most stable formulation. The Original Activity Remaining (OAR) percentage for the gelatin formulations did not change from its initial value after 24 h of irradiation. There was no significant difference between the OAR percentage values of 3% and 5% gelatin formulations after 72 h of UVA exposure (90 and 94, respectively; Duncan test, P < 0.05, df = 6). The OAR percentage for the gelatin microencapsulated formulation was 90 after 30 minutes of exposure to UVC radiation. However, for the non-microencapsulated virus suspension, the OAR percentage value declined sharply to 16 after 30 minutes of exposure to UVC radiation. Concerning the in vitro release behaviour of gelatin microparticles (MPs), virus release initiated quickly, but continued at a slower rate until it reached 100% after 1 h of exposure to the release media. The experimental data for the gelatin MPs showed good correlations with the Korsmeyer–Peppas semi-empirical model, indicating that the transport mechanism is primarily consistent with Fickian diffusion.     

紫玉米花青素的微胶囊化研究

花青素作为植物界广泛存在的一种天然食用色素,安全、无毒、资源丰富,而且具有一定营养和药理作用,然而花青素对pH值、氧气、温度、光、金属离子等十分敏感。拟利用微胶囊化技术,保护花青素的抗氧化特性,并比较了2种常用的微胶囊方法（喷雾干燥法和锐孔法）的包埋效果,以期为花青素的使用提供一定参考。结果表明,啧雾干燥法制备紫玉米花青素微胶囊效果较锐孔法更好,而且当选择麦芽糊精和阿拉伯胶1：1作喷雾干燥的壁材,芯材（花青素）与壁材按1：16制备时,其包埋率可达34％,效果最佳。     

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。     

Irradiation depolymerized guar gum as partial replacement of gum Arabic for microencapsulation of mint oil

Spray dried microcapsules of mint oil were prepared using gum Arabic alone and its blends with radiation or enzymatically depolymerized guar gum as wall materials. Microcapsules were evaluated for retention of mint oil during 8-week storage during which qualitative changes in encapsulated mint oil was monitored using principal component analysis. The microcapsules with radiation depolymerized guar gum as wall material component could better retain major mint oil compounds such as menthol and isomenthol. The t(1/2) calculated for mint oil in microcapsules of gum Arabic, gum Arabic:radiation depolymerized guar gum (90:10), gum Arabic:enzyme depolymerized guar gum (90:10) was 25.66, 38.50, and 17.11 weeks, respectively. The results suggested a combination of radiation depolymerized guar gum and gum Arabic to show better retention of encapsulated flavour than gum Arabic alone as wall material.     

Novel biopolymer matrices for microencapsulation of phages: enhanced protection against acidity and protease activity

Phage therapy by oral administration requires enhanced resistance of phages to the harsh gastric conditions. The aim of this work is the microencapsulation of phages in natural biopolymeric matrices as a protective barrier against the gastric environment. Alginate and pectin are used as base polymers. Further emulsification with oleic acid or coating with a different biopolymer is also studied. Emulsified pectin shows the maximum encapsulation efficiency and the highest protection against acidity, leaving more than 10(3) active phages after 30?min exposure at pH?=?1.6, and protects phage from pepsin activity (4.2?mg?mL(-1) ). Non-encapsulated phages are fully inactivated at pH?=?1.6 or with pepsin (0.5?mg?mL(-1) ) after 10?min.     

Separation of empty microcapsules after microencapsulation of porcine neonatal islets

Pancreatic islet transplantation is used to treat diabetes mellitus that has minimal complications and avoids hypoglycemic shock. Conformal microencapsulation of pancreatic islets improves their function by blocking immunogenic molecules while protecting fragile islets. However, production of empty alginate capsules during microencapsulation causes enlargement of the transplantation volume of the encapsulated islets and interferes with efficient transfer of nutrients and insulin. In this study, empty alginate capsules were separated after microencapsulation of neonatal porcine islet-like cell clusters (NPCC) using density-gradient centrifugation. Densities of NPCC and alginate capsules were determined using Percoll. Encapsulation products following alginate removal were 97 % of products, with less than 10 % of the capsules remaining empty. The viability of this process compared with manually-selected encapsulated islets indicates the separation process does not harm islets.