脂质体模拟乳杆菌细胞膜筛选冻干保护剂研究
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摘要
乳酸菌细胞冻干保护剂研究涉及冻干损伤机理、冻干保护机理及冻干保护剂筛选等内容。目前,冻干保护剂筛选中依旧采用细胞活菌数作为主要指标,通过评价不同介质在冷冻干燥下保护细胞的效果来确定最优组合的保护体系。因而,乳酸菌细胞保护剂筛选是一项费时耗力、工作量很大的工作。本项研究利用脂质体来模拟乳酸菌细胞膜,首先在脂质体模拟细胞膜中包封β-半乳糖苷酶,以膜渗透率为研究指标,以β-半乳糖苷酶为检测对象,创建了一种用脂质体模型筛选冻干保护剂的新方法,从而确定适宜的保护介质参数；然后以瑞士乳杆菌9(Lactobacillus helveticus 9)来评价该模型在快速筛选乳杆菌冷冻干燥保护剂中的效果。主要研究结果如下：
1、β-半乳糖苷酶脂质体模型的构建与相关影响因素研究
对影响酶脂质体制备的相关因素的研究表明,逆向蒸发法适宜制备β-半乳糖苷酶脂质体,具体操作方法如下：即原材料选用大豆卵磷脂和十八胺,摩尔比10：1,共144μmol；选用乙醚为有机溶剂；超声破碎仪匀化粒径,超声功率240W,超声时间5minx2：包封β-半乳糖苷酶量为5ml稀释5倍后的市售酶液。制得β-半乳糖苷酶脂质体平均粒径为110nm,分散系数为0.265,Zeta电位为-37.1mV,表明构建的空白脂质体悬液分布均匀且稳定,能够有效包封入β-半乳糖苷酶。
2、脂质体模型筛选冻干保护剂,观察保护剂在冻干下对脂质体包封率的影响
(1)将β-半乳糖苷酶脂质体置于冷冻干燥过程中,通过加入海藻糖、透明质酸(HA)以及两者复配的保护剂溶液,以p-半乳糖苷酶渗透率为评价指标结果表明,不同保护剂对β-半乳糖苷酶脂质体的乳糖酶包封率有显著影响,保护效果依次为海藻糖和透明质酸复配溶液、透明质酸和海藻糖,表明复配保护剂能最好地降低脂质体膜的冻干损伤。
(2)研究了冻干过程中β-半乳糖苷酶脂质体包封率随三种保护剂不同浓度的变化情况。当海藻糖的浓度低于20mmg/100mL时,随着浓度的升高,包封率增加。当海藻糖浓度高于20mg/100mL时,包封率有一定的下降。说明海藻糖的浓度为20mg/100ml对脂质体的保护作用最佳。当透明质酸的浓度在0.1-0.8 mg/100mL之间,随着透明质酸浓度的增加,脂质体包封率逐步增加。说明透明质酸对脂质体的冻干保护作用随着浓度的增加而增强。当复配保护剂中海藻糖浓度为10mmg/100mL、透明质酸浓度为0.2 mg/100mL时,包封率最大。此时,使用的保护剂浓度为单独使用一种保护剂时的一半,表明海藻糖和HA有协同保护作用。
3、脂质体法筛选的保护剂在瑞士乳杆菌9中的应用
将脂质体法筛选获得的保护剂应用于瑞士乳杆菌9细胞冷冻干燥实践中,通过比较海藻糖、HA或海藻糖和HA复配三种保护剂存在下细胞膜的渗透率变化曲线可知,通过脂质体法筛选的透明质酸和复配保护剂,在应用于瑞士乳杆菌9时也能有效地减小细胞膜的泄露,降低冷冻干燥对乳杆菌细胞膜的损害。进一步地,比较了当使用脱脂乳作为保护剂时,应用脂质体法筛选的复配保护剂对菌株9冻干存活率和发酵力的影响。结果表明,与脱脂乳相比,选用复配保护剂能够使菌株在冷冻干燥下的存活率提高15%,凝乳时间缩短35%。这说明利用脂质体法筛选冻干保护剂是一种可行的尝试。
4、冻干保护剂的保护机理探索
(1)扫描电镜观察冻干乳杆菌菌体细胞的损伤表明,冻干过程破坏细胞膜完整性,导致部分细胞裂解、内容物泄漏。而采用海藻糖和HA复配作为保护剂时,能够有效地保持细胞膜的渗透屏障和结构完整,避免内容物泄漏。
(2)对菌株9分别采用从37℃降温至4℃C以及16℃进行冷应激诱导3h的代谢产物研究表明,当降温至4℃时,菌株9细胞代谢物中出现了一条分子量7kd的新蛋白质条带,说明经过低温诱导后菌株9合成了一种新的蛋白质,其分子量大小与目前确定的几种冷应激蛋白一致。
(3)对添加不同保护剂的瑞士乳杆菌9的冻干菌粉进行差式量热仪扫描发现,以海藻糖为保护体系的玻璃化温度(Tg)为125℃,透明质酸为保护剂体系的Tg为140℃,复配保护剂体系的Tg为132℃。经海藻糖和HA复配后,Tg高于海藻糖体系,低于HA体系,DSC曲线变化更平缓,表明复配体系有协同抗冻干逆境作用。
Lyoprotectants, as protective agent, prevent lactic acid bacteria cells subjected to harsh stresses like freezing or freeze-dry from damage or injury. The screening of lyoprotectants are one of key technology in the processing of food-grade starter cultures. However, the selection of lyoprotectants for starter cultures are time-using and labor-consuming. How to find a simple way to obtain lyoprotectants available for improving the survival of bacterial cells has received more attention. The objectives of this study were to:(1) use liposome to simulate bacterial cell membrane as a model and deal with the permeability of membrane-like according to the activity ofβ-galactosidase embedded; (2) investigate the influences of different lyoprotectants onβ-galactosidase activity contained on membrane-like liposomes subjected to freeze-dried environments; (3) rapidly obtain the protective agents which are available for the protection of lactic acid bacteria cells; and (4) apply these lyoprotectants which are screened form membrane-like liposomes technology to promote the survival of Lactobacillus helveticus 9 exposed to freeze-dried stresses. The following are main results.
1. Firstly liposome suspension was successfully prepared. Thenβ-galactosidase was encapsulated to liposome suspension by film dispersion method. Several factors affected the preparation of liposome containing the enzyme. Soy lecithin and octadecylamine as raw materials in molar ratio 10:1, equal to 144μmol, were used to make the liposome. In the preparation of liposome containing the enzyme, ether was selected as the organic solvent, and ultrasonic disrupter with a 240W ultrasonic power and 5minx2 ultrasonic time used to homogenize the particle size.5ml commercialβ-galactosidase, after diluted five times, was encapsulated to form the final membrane-like liposomes. Theβ-galactosidase liposomes obtained had an average particle size of 110nm, with a dispersion coefficient of 0.265 and -37.1mV Zeta potential. Next, trehalose, hyaluronic acid (HA) or their mixture used as 3 protective agents were added to theβ-galactosidase liposomes subjected to the process of freeze- dried. The penetration ofβ-galactosidase was used to evaluate the roles of 3 protective agents in protecting lactase encapsulated in liposomes from inactivity. Among 3 lyoprotectants, the mixture of trehalose and HA had the best protective effect in preventing lactase from damage under harsh stresses, followed by hyaluronic acid and trehalose. Perhaps the mixed protective agent was able to reduce the damage of freeze-dried to membrane-like liposomes.
2. Three lyoprotectants with various concentrations were added to the membrane-like liposomes containingβ-galactosidase exposed to freeze-dried enviroments. Lactase penetration observed varied according to the concentrations of 3 protective agents. Trehalose less than 20mg/100ml as lyoprotectant improved the efficiency of encapsulatedβ-galactosidase. However, if the concentration of trehalose was higher than 20mg/100ml, the efficiency of lactase encapsulated in membrane-like liposomes declined. Trehalose of 20mg/100m] was suggested to protect membrane-like liposomes from the destroy of freeze-dired. Hyaluronic acid with a concentration of 0.1 to 0.8mg/100ml promoted membrane-like liposomes to encapsulate more lactase. The mixture of trehalose (10mg/100ml) and hyaluronic acid (0.2mg/100ml) as protective agent was observed to have the highest encapsulation efficiency, because only 50% the mixture produced a protection which corresponds to that of trehalose or HA applied alone. Clearly, the combination of trehalose and HA formed a synergistic protection of membrane-like liposomes aginst harsh streeses.
3. The mixture of trehalose and HA, screened by membrane-like liposomes, was observed to improve the survival of L. helveticus 9 during freeze-dried stresses.12% skim-milk, trehalose and HA as the lyoprotectants of strain 9 were also used as the control, respectively. HA and the mixture protected strain 9 better from the freeze-drying damage due to less leak of bacterial cell membrane. The mixture "lyoprotectant" produced the same protection of lactobacilli strain 9 cells as thatβ-galactosidase encapsulated in membrane-like liposomes. Two lyophilized preparations of strain 9 cells were applied to ferment milk. One was made with skim-milk as lyoprotectant, in short SML preparation. The another was made with the mixture of trehalose and HA as lyoprotectant, in short THAL preparation. Compared to the cells from SML, the survival of strain 9 in THAL was improved by 15%. Involvement of THAL in milk produced a faster coagulation than that of SML did. Time that milk coagulated was shortened by 35% due to the use of THAL preparation.
4. Scanning electron microscopy was applied to observe the damage of L. helveticus 9 cells subjected to freeze-dried-rehydrated stresses. The process of freeze-dried-rehydration destroy the integrity of cell membrane, causing bacterial cell lysis and leakage of cellular materials. Instead, the cells of strain 9 encapsulated by the mixture of trehalose and HA as a protective agent, although exposed to freeze-dried-rehydrated stresses, maintained cell membrane permeability barrier and structural integrity. Strain 9 cells were cold-induced from 37℃down to 16℃, and then to 4℃for 3h. A new protein band appeared on SDS-PAGE electrophoresis, compared to the control. The molecular weight of the new protein was about 7kd, close to those of Csp proteins found in the cold treatment of bacterial cell. Low temperature from freeze-dried treatment might induce strain 9 to synthesize a new protein that help cells to challenge harsh stress. The lyophilized preparations of strain 9 cells made with 3 protective agents were scanned for their glass transition temperature (Tg). Tg was 125℃for trehalose as lyoprotectant,140℃for HA, and 132℃for the mixture of trehalose and HA, respectively. High Tg showed lyoprotectant to have good protection of bacterial cells. The mixture of trehalose and HA provided a protection of L. helveticus 9 cells against the destroy of lyophilized treatment.
In conclusions, the present data proved that use of membrane-like liposomes to screen lyoprotectants should be a good attempt in the manufacture of starter cultures.
1、β-半乳糖苷酶脂质体模型的构建与相关影响因素研究
对影响酶脂质体制备的相关因素的研究表明,逆向蒸发法适宜制备β-半乳糖苷酶脂质体,具体操作方法如下：即原材料选用大豆卵磷脂和十八胺,摩尔比10：1,共144μmol；选用乙醚为有机溶剂；超声破碎仪匀化粒径,超声功率240W,超声时间5minx2：包封β-半乳糖苷酶量为5ml稀释5倍后的市售酶液。制得β-半乳糖苷酶脂质体平均粒径为110nm,分散系数为0.265,Zeta电位为-37.1mV,表明构建的空白脂质体悬液分布均匀且稳定,能够有效包封入β-半乳糖苷酶。
2、脂质体模型筛选冻干保护剂,观察保护剂在冻干下对脂质体包封率的影响
(1)将β-半乳糖苷酶脂质体置于冷冻干燥过程中,通过加入海藻糖、透明质酸(HA)以及两者复配的保护剂溶液,以p-半乳糖苷酶渗透率为评价指标结果表明,不同保护剂对β-半乳糖苷酶脂质体的乳糖酶包封率有显著影响,保护效果依次为海藻糖和透明质酸复配溶液、透明质酸和海藻糖,表明复配保护剂能最好地降低脂质体膜的冻干损伤。
(2)研究了冻干过程中β-半乳糖苷酶脂质体包封率随三种保护剂不同浓度的变化情况。当海藻糖的浓度低于20mmg/100mL时,随着浓度的升高,包封率增加。当海藻糖浓度高于20mg/100mL时,包封率有一定的下降。说明海藻糖的浓度为20mg/100ml对脂质体的保护作用最佳。当透明质酸的浓度在0.1-0.8 mg/100mL之间,随着透明质酸浓度的增加,脂质体包封率逐步增加。说明透明质酸对脂质体的冻干保护作用随着浓度的增加而增强。当复配保护剂中海藻糖浓度为10mmg/100mL、透明质酸浓度为0.2 mg/100mL时,包封率最大。此时,使用的保护剂浓度为单独使用一种保护剂时的一半,表明海藻糖和HA有协同保护作用。
3、脂质体法筛选的保护剂在瑞士乳杆菌9中的应用
将脂质体法筛选获得的保护剂应用于瑞士乳杆菌9细胞冷冻干燥实践中,通过比较海藻糖、HA或海藻糖和HA复配三种保护剂存在下细胞膜的渗透率变化曲线可知,通过脂质体法筛选的透明质酸和复配保护剂,在应用于瑞士乳杆菌9时也能有效地减小细胞膜的泄露,降低冷冻干燥对乳杆菌细胞膜的损害。进一步地,比较了当使用脱脂乳作为保护剂时,应用脂质体法筛选的复配保护剂对菌株9冻干存活率和发酵力的影响。结果表明,与脱脂乳相比,选用复配保护剂能够使菌株在冷冻干燥下的存活率提高15%,凝乳时间缩短35%。这说明利用脂质体法筛选冻干保护剂是一种可行的尝试。
4、冻干保护剂的保护机理探索
(1)扫描电镜观察冻干乳杆菌菌体细胞的损伤表明,冻干过程破坏细胞膜完整性,导致部分细胞裂解、内容物泄漏。而采用海藻糖和HA复配作为保护剂时,能够有效地保持细胞膜的渗透屏障和结构完整,避免内容物泄漏。
(2)对菌株9分别采用从37℃降温至4℃C以及16℃进行冷应激诱导3h的代谢产物研究表明,当降温至4℃时,菌株9细胞代谢物中出现了一条分子量7kd的新蛋白质条带,说明经过低温诱导后菌株9合成了一种新的蛋白质,其分子量大小与目前确定的几种冷应激蛋白一致。
(3)对添加不同保护剂的瑞士乳杆菌9的冻干菌粉进行差式量热仪扫描发现,以海藻糖为保护体系的玻璃化温度(Tg)为125℃,透明质酸为保护剂体系的Tg为140℃,复配保护剂体系的Tg为132℃。经海藻糖和HA复配后,Tg高于海藻糖体系,低于HA体系,DSC曲线变化更平缓,表明复配体系有协同抗冻干逆境作用。
Lyoprotectants, as protective agent, prevent lactic acid bacteria cells subjected to harsh stresses like freezing or freeze-dry from damage or injury. The screening of lyoprotectants are one of key technology in the processing of food-grade starter cultures. However, the selection of lyoprotectants for starter cultures are time-using and labor-consuming. How to find a simple way to obtain lyoprotectants available for improving the survival of bacterial cells has received more attention. The objectives of this study were to:(1) use liposome to simulate bacterial cell membrane as a model and deal with the permeability of membrane-like according to the activity ofβ-galactosidase embedded; (2) investigate the influences of different lyoprotectants onβ-galactosidase activity contained on membrane-like liposomes subjected to freeze-dried environments; (3) rapidly obtain the protective agents which are available for the protection of lactic acid bacteria cells; and (4) apply these lyoprotectants which are screened form membrane-like liposomes technology to promote the survival of Lactobacillus helveticus 9 exposed to freeze-dried stresses. The following are main results.
1. Firstly liposome suspension was successfully prepared. Thenβ-galactosidase was encapsulated to liposome suspension by film dispersion method. Several factors affected the preparation of liposome containing the enzyme. Soy lecithin and octadecylamine as raw materials in molar ratio 10:1, equal to 144μmol, were used to make the liposome. In the preparation of liposome containing the enzyme, ether was selected as the organic solvent, and ultrasonic disrupter with a 240W ultrasonic power and 5minx2 ultrasonic time used to homogenize the particle size.5ml commercialβ-galactosidase, after diluted five times, was encapsulated to form the final membrane-like liposomes. Theβ-galactosidase liposomes obtained had an average particle size of 110nm, with a dispersion coefficient of 0.265 and -37.1mV Zeta potential. Next, trehalose, hyaluronic acid (HA) or their mixture used as 3 protective agents were added to theβ-galactosidase liposomes subjected to the process of freeze- dried. The penetration ofβ-galactosidase was used to evaluate the roles of 3 protective agents in protecting lactase encapsulated in liposomes from inactivity. Among 3 lyoprotectants, the mixture of trehalose and HA had the best protective effect in preventing lactase from damage under harsh stresses, followed by hyaluronic acid and trehalose. Perhaps the mixed protective agent was able to reduce the damage of freeze-dried to membrane-like liposomes.
2. Three lyoprotectants with various concentrations were added to the membrane-like liposomes containingβ-galactosidase exposed to freeze-dried enviroments. Lactase penetration observed varied according to the concentrations of 3 protective agents. Trehalose less than 20mg/100ml as lyoprotectant improved the efficiency of encapsulatedβ-galactosidase. However, if the concentration of trehalose was higher than 20mg/100ml, the efficiency of lactase encapsulated in membrane-like liposomes declined. Trehalose of 20mg/100m] was suggested to protect membrane-like liposomes from the destroy of freeze-dired. Hyaluronic acid with a concentration of 0.1 to 0.8mg/100ml promoted membrane-like liposomes to encapsulate more lactase. The mixture of trehalose (10mg/100ml) and hyaluronic acid (0.2mg/100ml) as protective agent was observed to have the highest encapsulation efficiency, because only 50% the mixture produced a protection which corresponds to that of trehalose or HA applied alone. Clearly, the combination of trehalose and HA formed a synergistic protection of membrane-like liposomes aginst harsh streeses.
3. The mixture of trehalose and HA, screened by membrane-like liposomes, was observed to improve the survival of L. helveticus 9 during freeze-dried stresses.12% skim-milk, trehalose and HA as the lyoprotectants of strain 9 were also used as the control, respectively. HA and the mixture protected strain 9 better from the freeze-drying damage due to less leak of bacterial cell membrane. The mixture "lyoprotectant" produced the same protection of lactobacilli strain 9 cells as thatβ-galactosidase encapsulated in membrane-like liposomes. Two lyophilized preparations of strain 9 cells were applied to ferment milk. One was made with skim-milk as lyoprotectant, in short SML preparation. The another was made with the mixture of trehalose and HA as lyoprotectant, in short THAL preparation. Compared to the cells from SML, the survival of strain 9 in THAL was improved by 15%. Involvement of THAL in milk produced a faster coagulation than that of SML did. Time that milk coagulated was shortened by 35% due to the use of THAL preparation.
4. Scanning electron microscopy was applied to observe the damage of L. helveticus 9 cells subjected to freeze-dried-rehydrated stresses. The process of freeze-dried-rehydration destroy the integrity of cell membrane, causing bacterial cell lysis and leakage of cellular materials. Instead, the cells of strain 9 encapsulated by the mixture of trehalose and HA as a protective agent, although exposed to freeze-dried-rehydrated stresses, maintained cell membrane permeability barrier and structural integrity. Strain 9 cells were cold-induced from 37℃down to 16℃, and then to 4℃for 3h. A new protein band appeared on SDS-PAGE electrophoresis, compared to the control. The molecular weight of the new protein was about 7kd, close to those of Csp proteins found in the cold treatment of bacterial cell. Low temperature from freeze-dried treatment might induce strain 9 to synthesize a new protein that help cells to challenge harsh stress. The lyophilized preparations of strain 9 cells made with 3 protective agents were scanned for their glass transition temperature (Tg). Tg was 125℃for trehalose as lyoprotectant,140℃for HA, and 132℃for the mixture of trehalose and HA, respectively. High Tg showed lyoprotectant to have good protection of bacterial cells. The mixture of trehalose and HA provided a protection of L. helveticus 9 cells against the destroy of lyophilized treatment.
In conclusions, the present data proved that use of membrane-like liposomes to screen lyoprotectants should be a good attempt in the manufacture of starter cultures.
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