Study On The Microencapsulation Of Lactobacillus Rhamnosus GG And The Mechanism Behind The Viability Enhancement

Probiotics are live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host. There is accumulating clinical data supporting the role of probiotics in human health. However, the ability to maintain high probiotic viability during drying and storage presents a significant challenge. Two microencapsulation methods were tried in this study. The viabilitis of Lactobacillus rhamnosus GG (LGG) during storage were significantly enhanced when samples were encapsulated by those two methods. The mechenisam of this viability enhancing phenomenon was also investigated.A multi-shell microencapsulated (LGG) were developed. The LGG was encapsulated with skim milk powder then extruded into small pieces with diameter of0.5-1mm. The sample then was coated with shellac and wax via fluid bed. The viability of this multi-shell microencapsulated LGG was enhanced by23times compare to the blank control, which was only encapsulated by skim milk powder, after storage at a relative humidity of70%and25℃for56days.A composite microcapsule matrix, which was formulated with protein: maltodextrin:D-glucose (1:1:1), were developed. A significant enhancement of LGG viability, which was about5011times better than blank control, was also observed, when those samples were stored at a relative humidity of33%RH and25℃over5weeks.The protective effect of a metabolizable and a non-metabolizable stereoisomer of glucose on encapsulated Lactobacillus rhamnosus GG (LGG) bacteria in the dry state was investigated. Separate experiments confirmed that the probiotic strain metabolises only D-glucose. Encapsulated LGG formulations (2wt%LGG, dry basis) were prepared by freeze drying a mixture of the freshly cultured bacteria within various whey protein-carbohydrate matrices. The encapsulant matrices used were (i) a1:2mixture of protein:maltodextrin,(ii) a1:1:1mixture of protein:maltodextrin: D-glucose and (iii) a1:1:1mixture of protein:maltodextrin:L-glucose. The partial substitution of maltodextrin with either D-glucose or L-glucose in the encapsulant matrix significantly enhanced the survival of LGG powders stored at a relative humidity of33%RH and25℃over5weeks. In comparison, there was little protective effect afforded by partial substitution of maltodextrin with either stereoisomer of glucose during storage of LGG powders at relative humidity of70%RH and25℃over2weeks and a negative effect on longer term storage (5weeks). These experiments allowed discrimination between the physical and nutritional roles of glucose on LGG viability. The results suggest that the protection afforded to LGG by the incorporation of D-or L-glucose in the encapsulant matrix was primarily due to the physico-chemical effect of the glucose molecules rather than D-glucose being a metabolizable sugar. This suggests that the incorporation of a metabolizable substrate alone does not enhance the long term survival of probiotics in dried LGG formulations.The cell integrity and the viability of freeze dried LGG encapsulated in matrices containing whey protein isolate (WPI)-maltodextrin (ratio1:1), WPI-maltodextrin-glucose (ratios4:3:1,2:1:1or4:1:3) or WPI-glucose (ratio1:1) were assessed after freeze drying and during storage at25℃and33%and70%relative humidity (RH) over5weeks. Cell integrity was assessed by measurement of the absorbance of extracellular cell contents after treatment of cells with DNase. Confocal laser scanning microscopy (CLSM) and a plate counting method were used to assess probiotic viability. The inclusion of glucose in the matrix formulation enhanced the cell integrity during drying and storage at25℃and33%RH but not during storage at25℃and70%RH. Cell integrity was linearly correlated (p<0.01) to probiotic viability.This work also tried to gain an understanding of the mechanism of the encapsulant matrix (e.g. sugar/glucose) protects the microorganisms (e.g. probiotic cells) during non-refrigerated storage. Time domain1H nuclear magnetic resonance (NMR) was used to measure the proton mobility of freeze-dried microencapsulated LGG and the whey protein isolate (WPI) and carbohydrate (maltodextrin DE5and glucose) encapsulant matrices(1WPI:2maltodextrin;1WPI:1maltodextrin: ID-glucose; and1WPI:1maltodextrin:1L-glucose) during storage at25℃and33%and70%RH environment. It was found that when LGG was encapsulated in matrices containing either D-or L-glucose, the rate of LGG viability loss was slower at25℃and33%RH; whereas the opposite trend was observed at25℃and70%RH. There was a strong correlation between the rate of LGG viability loss and the molecular mobility (T2(long) or%T2(long)) and the moisture uptake of the encapsulant matrices (p<0.05). The results from this study indicated that a less mobile encapsulant matrix and a less moisture uptake would contribute to the enhanced survival of microencapsulated probiotic cells during storage. This finding has the potential to provide some practical guiding rules for the encapsulant materials selection and formulation design for probiotics.In summary, two kinds of microencapsulation methods were developed. Viability of LGG was increased by23and5011times when sample were stored at certain conditions. The mechanism behind the viability enhancement was also investigated: the nutition effect of glucose; the protective effect of glucose on LGG cell membrane, as well as the proton mobility of microcapsule matrix. Three rules were found:a. L-glucose (non-metabolic sugar), similar to D-glucose, can significantly improve the viability of LGG during dehydration and storage.b. Glucose has a protective effect on the cell membrane. The inclusion of glucose in the matrix formulation enhanced the cell integrity during drying and storage at25℃and33%RH; c. The proton mobility of microcapsule closely related to the inaction rate of LGG. A matrix with less mobility and less moisture uptake would contribute to the enhanced survival of microencapsulated probiotic cells during storage.