适宜冷冻干燥保护剂提高植物乳杆菌LIP-1微胶囊性能

为了探讨添加冷冻干燥保护剂对Lactobacillus.plantarum（L.plantarum）LIP-1微胶囊性能的影响，该试验以植物乳杆菌（L.plantarum） LIP-1微胶囊的包埋率和冻干存活率为指标，通过单因素及正交试验，筛选出最佳冷冻干燥保护剂，在此基础上将其添加到微胶囊中，观察对L.plantarum LIP-1微胶囊形态、释放性等性能的影响。试验结果表明冷冻干燥保护剂的最佳配方为质量分数分别为甘油2%、麦芽糖1%、L-半胱氨酸2%、乳糖2%，此时微胶囊的包埋率为67.60%，冻干存活率为83.80%；与未添加保护剂的空白对照组相比，添加适宜保护剂的微胶囊在表观形态、肠液释放性、耐胃酸性及在不同温度（4、20、37℃）下的耐贮藏性能均显著提高（P<0.05）。添加适宜保护剂的微胶囊表面更加光滑致密，粒径更小，约100 μm（空白对照组约为150～200 μm）；在模拟肠液中，添加适宜保护剂的微胶囊完全释放仅需60 min，而空白对照组需要90 min才能释放完全；在耐胃酸性上，添加适宜保护剂的LIP-1微胶囊在120min后，活菌数才开始显著下降（P<0.05），150 min后，活菌数下降约30%；空白对照组在90 min后活菌数开始显著下降（P<0.05），150 min后，活菌数下降约44%；在4、20、37℃贮藏28 d后，加保护剂组的活菌数分别下降0.76、1.33、1.88 lg(cfu/g)，而空白对照组的活菌数分别下降0.96、 1.50、2.40 lg(cfu/g)。试验结果表明添加适宜的冷冻干燥保护剂可以提高L.plantarum LIP-1微胶囊的性能，为工业化生产中提高益生菌微胶囊的性能提供一定的理论和技术指导。

Proper cryoprotectants improving properties of L. plantarum LIP-1 microcapsules

Abstract: As all know, probiotics have many physiological functions, but the actual levels detected in probiotic products are often much lower due to adverse conditions during product storage, transportation, marketing and consumption. Many studies have shown that, microencapsulation techniques can provide protection against adverse conditions for probiotics. In order to enhance the storage stability of microcapsule, freeze drying is commonly used, but freeze-drying technique exposes the bacterial cells to additional stressful processing steps and the loss in viability of cells was observed. To prevent these adverse effects, cryoprotectants are commonly added to samples before freeze drying. In this study, we investigated the optimal ratio of cryoprotectants and the effect of cryoprotectants on the properties of L. plantarum LIP-1 microcapsules. Single factor and orthogonal experimental design were used to study the influence of adding different types of cryoprotectants on microencapsulation efficiency (ME) and survival rate of LIP-1 during freeze-drying process. Based on those data, the effect of optimal cryoprotectants on the properties of L. plantarum LIP-1 microcapsules was evaluated, such as surface morphology and microstructure by scanning electron microscopy (SEM), simulated gastric ?uid (SGF) resistance, release characteristic in simulated intestinal ?uid (SIF) and viability during four-week storage at different temperatures (4, 20 and 37°C). The results showed that the best cryoprotectant formulations were glycerol of 2%, maltose of 1%, L-cysteine of 2% and lactose of 2%, and based on this formulation, the ME was 67.60% and the survival rate was 83.80%, which increased the number of living bactera after freeze drying significantly (P<0.05) compared with the blank (microcapsules without cryoprotectants, the ME and the survival rate were 70.5% and 56.41%, respectively). The microcapsules with optimal cryoprotectants indicated a compact microcapsule structure and regular shape, and a finer and more dispersed substructure than the blank. The microcapsules with optimal cryoprotectants had significantly faster release properties in SIF than the blank, for the microcapsules with optimal cryoprotectants werecompletely released within 60 min, while the blank took 90 min to be fully released, which was maybe due to the difference in size and distribution status of those microcapsules. The size of microcapsules with optimal cryoprotectants was smaller and the distribution was better than the blank, resulting in a larger specific surface area for enzyme activity, and the increase of the speed of disintegration. The microcapsules with optimal cryoprotectants had a better SGF resistance than the blank. And in our study, free cells of LIP-1 were sensitive to low pH value, the viability began to decrease significantly (P<0.05) within 30 min, and they lost about 90% when exposed to acidic conditions for 120 min; the viability of microcapsules in blank began to decrease significantly (P<0.05) after 90 min, and lost about 44% when exposed to acidic conditions for 150 min; while the viability of microcapsules with optimal cryoprotectants began to decrease significantly (P<0.05) after 120 min, and they lost about 30% when exposed to acidic conditions for 150 min. So the microencapsulation provided a significant protection for L. plantarum under acidic conditions, and cryoprotectants could enhance the SGF resistance of LIP-1 significantly. Maybe because the surface of microcapsules with optimal cryoprotectants was denser, and had less aperture, it was likely to be more resistant to the penetration of SGF. The viability of microcapsules was significantly (P<0.05) higher than free cells during four- week storage at different temperatures (4, 20 and 37°C), while the viability of microcapsules with optimal cryoprotectants was significantly (P<0.05) higher than the blank. So the microencapsulation provided a significant protection for L. plantarum during storage at different temperature, and the microcapsules with optimal cryoprotectants could significantly (P<0.05) increase the viability of LIP-1 during storage at different temperature, for the dense membrane formed around the microcapsules may provide effective protection by separating LIP-1 from harmful factors, such as air, moisture, light. In addition, the oxidative stability of L-cysteine was in favor of the storage of microcapsules. Therefore, the results indicate that the adding of optimal cryoprotectants can improve the properties of L. plantarum LIP-1 microcapsules significantly, and also provide a theoretical basis and technical guidance for improving the properties of probiotic microcapsules during the commercial production.