双歧杆菌微胶囊的研究
摘要
双歧杆菌是益生菌当中最为重要的一大类，它们具有多种生理活性作用，但外界许多不利的因素会导致双歧杆菌死亡，从而降低其生理活性作用。微胶囊作为双歧杆菌的包埋技术手段之一，它常被用来提高双歧杆菌在不良环境中的存活率并实现双歧杆菌肠道释放的目的。海藻酸钠和蛋白质是目前制备双歧杆菌微胶囊最常采用的两种壁材，但这两种壁材在双歧杆菌的包埋上都有各自的不足之处，这也是目前双歧杆菌微胶囊化研究中亟待解决的问题。所以本论文以海藻酸钠和蛋白质为壁材，建立各自的双歧杆菌微胶囊包埋体系，探讨微胶囊在模拟胃液中对益生菌的保护机制，并采取相应的强化手段来提高微胶囊对益生菌的保护效果。
以海藻酸钠为壁材，采用乳化内源凝胶的方法制备包埋有两歧双歧杆菌F-35（Bifidobacteriumbifidum F-35）的微胶囊。为了提高微胶囊的保护效果，选取了四种常用的强化手段对海藻酸钠微胶囊进行改进：海藻酸钠与辅料混合（淀粉和果胶）和用多聚物（壳聚糖和多聚赖氨酸）对海藻酸钠微胶囊进行包衣，评价了不同强化手段对微胶囊特性和保护效果的影响作用。结果发现：强化手段对包埋效率影响不大（43-50%），淀粉的加入和壳聚糖的包衣能够将微胶囊的粒径分别增加至178μm和149μm。另外通过两歧双歧杆菌F-35的存活率实验发现，壳聚糖包衣的微胶囊对两歧双歧杆菌F-35的保护效果最好，包埋于其中的两歧双歧杆菌F-35在模拟胃液（2h）和连续的胃肠道中（胃1h，肠4h）分别死亡了4.5和3.7个对数值，并且它们还能将包埋细胞完全释放于人体肠道中。
以乳清蛋白为作用底物，转谷氨酰胺酶为催化剂，通过研究乳清蛋白溶液pH、乳清蛋白溶液浓度以及酶的添加量对凝胶强度的影响，得到了最佳的凝胶反应参数：乳清蛋白溶液pH为6.4、乳清蛋白溶液浓度为10%，酶的添加量为10U/g，并以此为依据建立了一套以乳清蛋白为包埋壁材的乳化凝胶包埋方法。将新方法制备得到的蛋白质微胶囊（WPMs-B）与传统喷雾干燥方法制备的蛋白质微胶囊（WPMs-A）进行比较后发现，在低酸环境中，两种蛋白质微胶囊都能显著地提高两歧双歧杆菌F-35的存活率，但是WPMs-B对两歧双歧杆菌F-35的保护效果要高于WPMs-A，它们的D值分别为103.6和82.0min，这和WPMs-B具有较大的粒径、较高的交联程度和较为致密的形态结构有关。在模拟胃液中，蛋白质微胶囊的保护效果被大大削弱，其D值仅略高于未经过包埋处理的两歧双歧杆菌F-35（D=13.2min），证实了胃蛋白的水解作用是影响蛋白质微胶囊保护效果的主要原因，虽然在模拟胃液中WPMs-B的降解速度要低于WPMs-A，但两者在模拟胃液中对两歧双歧杆菌F-35的保护效果并无明显差异。在高胆盐环境中，相比于未经过包埋处理的两歧双歧杆菌F-35，包埋于蛋白质微胶囊中的两歧双歧杆菌F-35的存活量提高了2个对数值。除此之外，还发现WPMs-B对两歧双歧杆菌F-35具有较高的包埋效率和储藏稳定性。
以大豆蛋白（S）、乳清蛋白（W）、酪蛋白（C）、明胶（G）为壁材通过乳化凝胶方法将两歧双歧杆菌F-35包埋于蛋白质胶囊中，通过测定两歧双歧杆菌F-35在人体模拟胃液中的存活率发现：相比于未经过包埋处理的两歧双歧杆菌F-35，包埋于蛋白质微胶囊中的两歧双歧杆菌F-35具有较高的存活率，并且还发现大豆蛋白具有好的保护效果，但大豆蛋白、乳清蛋白对益生菌的保护效果差别并不大，明胶微胶囊则最差,两歧双歧杆菌F-35在这四种蛋白质微胶囊中的D值分别为：31.7、24.2、22.7、18.7和124.3、103.5、97.6、47.8min。除此之外，本文还比较了蛋白质的五种理化特性（缓冲能力、凝胶强度、乳化稳定性、渗透性、在模拟胃液中的降解速度），其结果为：乳化能力（S> C> W> G），凝胶强度（G>S>C>W），拟胃液的渗透性（W>S=C>G），缓冲能力（W>S=C>G），在模拟胃液中的降解速度（G=C>W>S）。通过上述结果可以推测：蛋白质的缓冲能力和蛋白质微胶囊在酸性环境中的保护效果有很大的相关性，但缓冲能力并不是唯一决定因素，大豆蛋白其它优良的化性质（例如：较高的弹性储能模量，较低的渗透性、最好的乳化稳定性以及在模拟胃液中最慢的降解速度）有助于使得它成为保护效果最好的蛋白质壁材。
以两歧双歧杆菌F-35在模拟胃液中的存活率和微胶囊粒径为指标，得到了海藻酸钠对蛋白质微胶囊的最佳包衣工艺：海藻酸钠溶液pH5.5，海藻酸钠溶液浓度0.5%，搅拌速度500rpm，CaCl2浓度为0.07M。由该工艺制备而来的微胶囊平均粒径为132.6μm，它们能将两歧双歧杆菌F-35在2h模拟胃液中的存活率提高至14.5%。研究证实了该包衣方法能有效地将海藻酸钠包覆于乳清蛋白微胶囊表面，在胃部的酸性环境中，微胶囊外表的海藻酸钠能与乳清蛋白发生复合凝聚，从而提高微胶囊在模拟胃液中的稳定性，并且外部的这层海藻酸钠薄膜能够显著减少胃蛋白酶对乳清蛋白壁材的降解作用。相比于海藻酸钠微胶囊和蛋白质微胶囊，包衣后的微胶囊会延缓两歧双歧杆菌F-35在模拟肠液中的释放，但它们在连续的模拟胃肠道中对两歧双歧杆菌F-35保护效果最好，其最终的存活率高达51.0%
Bifidobacterium is an important sort of probiotics. They have various of physiological effect forhuman being However, there are many factors affecting the viability of Bifidobacterium duringdownstream processing, storage and eventually digestion..Microencapsulation is one of most commonlyused method to protect probiotics from adverse conditions and to achieve intestinal release. Althoughalginate and protein are the main coating materials used for microencapsulation of probiotics, they havetheir own limitilations. So the remaining challenge is to overcome these limitilations posed by alginate andprotein and to further improve the protective effect provided by microcapsules. The improvedmicroencapsulation system have been developed based on alginate and protein, the mechanism ofprotective effect provided by microcapsules was disscussed. Meanwhile some reinforcement were adoptedto futher improve the protective effect.
Alginate microspheres containing Bifidobacterium bifidum F-35prepared by emulsification/internalgelation were reinforced by blending with pectin or starch, or coating with chitosan or poly-L-lysine toprovide extra protection for the strain. The influence of these treatments on the size of microspheres,encapsulation yield (EY) and protective effect of microencapsulation on the cells was studied. Nodifference were detected in EY with different type of reinforcement, which was approximately43%-50%.The mean diameter of reinforced alginante microspheres ranged from117to178μm, reaching a maxiumvalue when starch was incorporated in the alginate matrix. It was observed that the protective effects variedwith the type of reinforcement. However, chitosan-coated alginate microspheres provided the bestprotection for microencapsulated cells in simulated gastro-intestinal tract and during one month storage at4℃, and this system could be the comparatively effective vector of bifidobacteria for intestinal delivery.
Bifidobacterium bifidum F-35was microencapsulated into whey protein microcapsules (WPMs) by atransglutaminase (TGase)-induced method after optimization of gelation conditions. The performance ofthese WPMs was compared with that produced by a spray drying method (WPMs-A). WPMs produced bythe TGase-induced gelation method (WPMs-B) had larger and denser structures in morphologicalexaminations. Native gel and SDS-PAGE analyses showed that most of the polymerization observed inWPMs-B was due to stable covalent crosslinks catalyzed by TGase. The degradation properties of theseWPMs were investigated in simulated gastric juice (SGJ) with or without pepsin. In the presence of pepsin,WPMs-A degraded more quickly than did WPMs-B. Finally, survival rates of the microencapsulated cellsin both WPMs were significantly better than that of free cells and varied with the microencapsulationmethod. However, WPMs-B produced by TGase-induced gelation could provide better protection formicroencapsulated cells in low pH conditions and during one month of storage at4oC or at ambienttemperature.
Bifidobacterium bifidum F-35were encapsulated into microcapsules with four types of protein-basedcoating material, soy protein isolation (S), whey protein isolation (W), sodium caseinate (S), gelatin (G)through transglutaminase-induced emusification/gelation method. The survivability of microencapsulatedcells in the four protein-based microcapsules was conducted in simulated gastric juice (SGJ) with orwithout pepsin. SPI-based microcapsules provided the best protection for microencapsulated cells in alltreatments. As for Bifidobacterium bifidum F-35, in SGJ with or without pepsin, the the D-value ofencapsulated cells in these four protein-based microcapsules were31.7,24.2,22.7,18.7and124.3,103.5,97.6,47.8min, respectively. Additionally, this research also investigated some physical properties of theseproteins for difference in protective effect on encapsulated cells in SGJ without pepsin. The result showedthat emulsion stability was S> C> W> G; gel strength was G>S>C>W; permeability to SGJ wasW>S=C>G, buffer capacity was W>S=C>G. It could be deduced that buffer capacity have great influenceon the protective effect in low pH condtion, other potential reasons for difference in the protective effectinclude difference in permeability of protein gels, emulsion stability and gel strength can also affect theprotective effect.
Taking survival rate of Bifidobacterium bifidum F-35in2-h simulated gastric juice, particle size ofmicrocapsules, the process conditions of coating alginate on whey protein microcapsules were optimalized.The optimum coating conditions were established: pH5.5alginate solution,0.5%concentration of alginatesolution,0.07M concentration of CaCl2, and500rpm agitation rate. The particle size of microcapsules were132.6μm, the survival rate of Bifidobacterium bifidum F-35in simulated gastric juice was improved to14.5%at the optimal coating condition. The study also showed that the alginate could be effectively coatedon the surface of whey protein microcapsules. The outer alginate membrane could significantly reduce thedegradation role of pepsin on whey protein matrix. Beside, the coated microcapsules could delay therelease rate of Bifidobacterium bifidum F-35in simulated intestinal juice, but they provided the best protecti on for microencapsulated cel ls in sequential si mulated gastrointest inal j uice, t he survival rate wasup to51%。
以海藻酸钠为壁材，采用乳化内源凝胶的方法制备包埋有两歧双歧杆菌F-35（Bifidobacteriumbifidum F-35）的微胶囊。为了提高微胶囊的保护效果，选取了四种常用的强化手段对海藻酸钠微胶囊进行改进：海藻酸钠与辅料混合（淀粉和果胶）和用多聚物（壳聚糖和多聚赖氨酸）对海藻酸钠微胶囊进行包衣，评价了不同强化手段对微胶囊特性和保护效果的影响作用。结果发现：强化手段对包埋效率影响不大（43-50%），淀粉的加入和壳聚糖的包衣能够将微胶囊的粒径分别增加至178μm和149μm。另外通过两歧双歧杆菌F-35的存活率实验发现，壳聚糖包衣的微胶囊对两歧双歧杆菌F-35的保护效果最好，包埋于其中的两歧双歧杆菌F-35在模拟胃液（2h）和连续的胃肠道中（胃1h，肠4h）分别死亡了4.5和3.7个对数值，并且它们还能将包埋细胞完全释放于人体肠道中。
以乳清蛋白为作用底物，转谷氨酰胺酶为催化剂，通过研究乳清蛋白溶液pH、乳清蛋白溶液浓度以及酶的添加量对凝胶强度的影响，得到了最佳的凝胶反应参数：乳清蛋白溶液pH为6.4、乳清蛋白溶液浓度为10%，酶的添加量为10U/g，并以此为依据建立了一套以乳清蛋白为包埋壁材的乳化凝胶包埋方法。将新方法制备得到的蛋白质微胶囊（WPMs-B）与传统喷雾干燥方法制备的蛋白质微胶囊（WPMs-A）进行比较后发现，在低酸环境中，两种蛋白质微胶囊都能显著地提高两歧双歧杆菌F-35的存活率，但是WPMs-B对两歧双歧杆菌F-35的保护效果要高于WPMs-A，它们的D值分别为103.6和82.0min，这和WPMs-B具有较大的粒径、较高的交联程度和较为致密的形态结构有关。在模拟胃液中，蛋白质微胶囊的保护效果被大大削弱，其D值仅略高于未经过包埋处理的两歧双歧杆菌F-35（D=13.2min），证实了胃蛋白的水解作用是影响蛋白质微胶囊保护效果的主要原因，虽然在模拟胃液中WPMs-B的降解速度要低于WPMs-A，但两者在模拟胃液中对两歧双歧杆菌F-35的保护效果并无明显差异。在高胆盐环境中，相比于未经过包埋处理的两歧双歧杆菌F-35，包埋于蛋白质微胶囊中的两歧双歧杆菌F-35的存活量提高了2个对数值。除此之外，还发现WPMs-B对两歧双歧杆菌F-35具有较高的包埋效率和储藏稳定性。
以大豆蛋白（S）、乳清蛋白（W）、酪蛋白（C）、明胶（G）为壁材通过乳化凝胶方法将两歧双歧杆菌F-35包埋于蛋白质胶囊中，通过测定两歧双歧杆菌F-35在人体模拟胃液中的存活率发现：相比于未经过包埋处理的两歧双歧杆菌F-35，包埋于蛋白质微胶囊中的两歧双歧杆菌F-35具有较高的存活率，并且还发现大豆蛋白具有好的保护效果，但大豆蛋白、乳清蛋白对益生菌的保护效果差别并不大，明胶微胶囊则最差,两歧双歧杆菌F-35在这四种蛋白质微胶囊中的D值分别为：31.7、24.2、22.7、18.7和124.3、103.5、97.6、47.8min。除此之外，本文还比较了蛋白质的五种理化特性（缓冲能力、凝胶强度、乳化稳定性、渗透性、在模拟胃液中的降解速度），其结果为：乳化能力（S> C> W> G），凝胶强度（G>S>C>W），拟胃液的渗透性（W>S=C>G），缓冲能力（W>S=C>G），在模拟胃液中的降解速度（G=C>W>S）。通过上述结果可以推测：蛋白质的缓冲能力和蛋白质微胶囊在酸性环境中的保护效果有很大的相关性，但缓冲能力并不是唯一决定因素，大豆蛋白其它优良的化性质（例如：较高的弹性储能模量，较低的渗透性、最好的乳化稳定性以及在模拟胃液中最慢的降解速度）有助于使得它成为保护效果最好的蛋白质壁材。
以两歧双歧杆菌F-35在模拟胃液中的存活率和微胶囊粒径为指标，得到了海藻酸钠对蛋白质微胶囊的最佳包衣工艺：海藻酸钠溶液pH5.5，海藻酸钠溶液浓度0.5%，搅拌速度500rpm，CaCl2浓度为0.07M。由该工艺制备而来的微胶囊平均粒径为132.6μm，它们能将两歧双歧杆菌F-35在2h模拟胃液中的存活率提高至14.5%。研究证实了该包衣方法能有效地将海藻酸钠包覆于乳清蛋白微胶囊表面，在胃部的酸性环境中，微胶囊外表的海藻酸钠能与乳清蛋白发生复合凝聚，从而提高微胶囊在模拟胃液中的稳定性，并且外部的这层海藻酸钠薄膜能够显著减少胃蛋白酶对乳清蛋白壁材的降解作用。相比于海藻酸钠微胶囊和蛋白质微胶囊，包衣后的微胶囊会延缓两歧双歧杆菌F-35在模拟肠液中的释放，但它们在连续的模拟胃肠道中对两歧双歧杆菌F-35保护效果最好，其最终的存活率高达51.0%
Bifidobacterium is an important sort of probiotics. They have various of physiological effect forhuman being However, there are many factors affecting the viability of Bifidobacterium duringdownstream processing, storage and eventually digestion..Microencapsulation is one of most commonlyused method to protect probiotics from adverse conditions and to achieve intestinal release. Althoughalginate and protein are the main coating materials used for microencapsulation of probiotics, they havetheir own limitilations. So the remaining challenge is to overcome these limitilations posed by alginate andprotein and to further improve the protective effect provided by microcapsules. The improvedmicroencapsulation system have been developed based on alginate and protein, the mechanism ofprotective effect provided by microcapsules was disscussed. Meanwhile some reinforcement were adoptedto futher improve the protective effect.
Alginate microspheres containing Bifidobacterium bifidum F-35prepared by emulsification/internalgelation were reinforced by blending with pectin or starch, or coating with chitosan or poly-L-lysine toprovide extra protection for the strain. The influence of these treatments on the size of microspheres,encapsulation yield (EY) and protective effect of microencapsulation on the cells was studied. Nodifference were detected in EY with different type of reinforcement, which was approximately43%-50%.The mean diameter of reinforced alginante microspheres ranged from117to178μm, reaching a maxiumvalue when starch was incorporated in the alginate matrix. It was observed that the protective effects variedwith the type of reinforcement. However, chitosan-coated alginate microspheres provided the bestprotection for microencapsulated cells in simulated gastro-intestinal tract and during one month storage at4℃, and this system could be the comparatively effective vector of bifidobacteria for intestinal delivery.
Bifidobacterium bifidum F-35was microencapsulated into whey protein microcapsules (WPMs) by atransglutaminase (TGase)-induced method after optimization of gelation conditions. The performance ofthese WPMs was compared with that produced by a spray drying method (WPMs-A). WPMs produced bythe TGase-induced gelation method (WPMs-B) had larger and denser structures in morphologicalexaminations. Native gel and SDS-PAGE analyses showed that most of the polymerization observed inWPMs-B was due to stable covalent crosslinks catalyzed by TGase. The degradation properties of theseWPMs were investigated in simulated gastric juice (SGJ) with or without pepsin. In the presence of pepsin,WPMs-A degraded more quickly than did WPMs-B. Finally, survival rates of the microencapsulated cellsin both WPMs were significantly better than that of free cells and varied with the microencapsulationmethod. However, WPMs-B produced by TGase-induced gelation could provide better protection formicroencapsulated cells in low pH conditions and during one month of storage at4oC or at ambienttemperature.
Bifidobacterium bifidum F-35were encapsulated into microcapsules with four types of protein-basedcoating material, soy protein isolation (S), whey protein isolation (W), sodium caseinate (S), gelatin (G)through transglutaminase-induced emusification/gelation method. The survivability of microencapsulatedcells in the four protein-based microcapsules was conducted in simulated gastric juice (SGJ) with orwithout pepsin. SPI-based microcapsules provided the best protection for microencapsulated cells in alltreatments. As for Bifidobacterium bifidum F-35, in SGJ with or without pepsin, the the D-value ofencapsulated cells in these four protein-based microcapsules were31.7,24.2,22.7,18.7and124.3,103.5,97.6,47.8min, respectively. Additionally, this research also investigated some physical properties of theseproteins for difference in protective effect on encapsulated cells in SGJ without pepsin. The result showedthat emulsion stability was S> C> W> G; gel strength was G>S>C>W; permeability to SGJ wasW>S=C>G, buffer capacity was W>S=C>G. It could be deduced that buffer capacity have great influenceon the protective effect in low pH condtion, other potential reasons for difference in the protective effectinclude difference in permeability of protein gels, emulsion stability and gel strength can also affect theprotective effect.
Taking survival rate of Bifidobacterium bifidum F-35in2-h simulated gastric juice, particle size ofmicrocapsules, the process conditions of coating alginate on whey protein microcapsules were optimalized.The optimum coating conditions were established: pH5.5alginate solution,0.5%concentration of alginatesolution,0.07M concentration of CaCl2, and500rpm agitation rate. The particle size of microcapsules were132.6μm, the survival rate of Bifidobacterium bifidum F-35in simulated gastric juice was improved to14.5%at the optimal coating condition. The study also showed that the alginate could be effectively coatedon the surface of whey protein microcapsules. The outer alginate membrane could significantly reduce thedegradation role of pepsin on whey protein matrix. Beside, the coated microcapsules could delay therelease rate of Bifidobacterium bifidum F-35in simulated intestinal juice, but they provided the best protecti on for microencapsulated cel ls in sequential si mulated gastrointest inal j uice, t he survival rate wasup to51%。
引文
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