| 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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