Microencapsulation And Storage Stability Of Probiotics Based On Glass Theory

Lactic acid bacteria（LAB）are the oldest type of probiotics used by human and are considered as the safest probiotics.Many studies have shown that LAB have good effects on maintaining the integrity of intestinal function,increasing intestinal motility,treating colitis,lowering blood sugar and enhancing immune activity,which are beneficial to body health.In recent years,the processing and application of LAB have ushered in a new climax with the continuous advancement of the activity research of lactic acid bacteria and the continuous advancement of food processing technology.However,there are few reports on the physical state and stability mechanism of Lactobacillus plantarum（L_p）microcapsules,which restricts the further processing and application of L_pin the food industry.In this study,the freeze-drying method was used to encapsulated Lactobacillus plantarum(L_p)under the protect of dextran and its derivatives and small molecular sugars based on the glass state theory.In order to provide theoretical guidance for microbial microencapsulation technology and explain the relationship between microcapsules stability and wall materials from the glassy theory,the effects of water migration,glass state formation,and physical and chemical properties of wall materials on the stability of microcapsules were discussed.（1）In this paper,the GAB moisture adsorption isotherm and Gordon-Taylor model of the mixtures of dextran with trehalose,lactose,sucrose were established firstly.The thermal stability,morphological changes and crystallinity of the mixtures of dextran and small molecular sugars were analyzed by TGA,SEM and XRD analysis.In the dynamic water adsorption experiment,pure trehalose did not lose water when the relative humidity（RH）was 11%to 76%,while sucrose and lactose appeared water loss at the 196th and 48th hours when the relative humidity was 33%and 43%,respectively.Thereafter,as the relative humidity increased,the time of water loss for lactose and sucrose occurred earlier.In the GAB model,the K value<1,indicating that GAB model is suitable to establish the water adsorption isotherm of the mixtures of dextran and small molecular sugars.At the same proportion,the m₀of the mixtures are:dextran&sucrose>dextran&trehalose>dextran&lactose.The higher proportion of dextran in mixtures,the higher C value,indicating the interaction between water and the monolayer water increased.In anhydrous state,dextran&trehalose and dextran&lactose had one T_g,while dextran&sucrose had two T_g.At the same time,dextran&trehalose mixtures have one degradation peak after 200°C in the thermogravimetric analysis,while dextran&lactose and dextran&sucrose mixtures have two,indicating the compatibility between dextran and small molecular sugar is:trehalose>lactose>sucrose.Mixtures has the best inhibitory effect on water plasticization（k=3.50）,when the mixing ratio of dextran&trehalose is 2:1.L_pwas microencapsulated by vacuum freeze-drying technology.Moreover,dextran was used as macromolecular protective agent,and trehalose,lactose and sucrose in different proportions from 2%to 9%were added as small molecule protective agents and the storage stability of different microcapsules stored at room temperature for 4 weeks and gastrointestinal tolerance of L_pwith the optimal ratio of microcapsules were analyzed.The results showed that the microcapsules had the best storage stability,when the addition of trehalose,lactose and sucrose were 6%,7%and4%,respectively,and the K values were 0.158,0.161 and 0.156,respectively.L_phad good gastrointestinal tolerance in the gastrointestinal environment after microcapsule.（2）Carboxymethylated dextran with different degrees of substitution was prepared by using monochloroacetic acid.The physicochemical properties of carboxymethylated dextran were studied by the sugar content,degree of substitution,UV and IR spectra.Futhermore,the thermal stability,T_g,microstructure and crystallinity of carboxymethylated dextran were analyzed by TGA,DSC,SEM and XRD techniques.The sugar content of carboxymethylated dextran were 97.95%±1.54%,84.08%±2.54%,86.63%±0.29%and 76.56%±3.07%,respectively.The degree of substitution were 0.57±0.03,0.78±0.02,1.13±0.01 and 1.25±0.01,respectively.With the increase of substitution degree,the thermal stability of carboxymethylated dextran increased.Furthermore,CD.dex₃had the highest T_g（215.96℃）.L_pwas microencapsulated by freeze-drying technology while carboxymethylated dextran was used as the wall materials.Moreover,the viability after encapsuled,gastrointestinal tolerance and storage stability of L_pwere studied.The viability of L_pafter encapsulated by dextran,CM.dex₁,CM.dex₂,CM.dex₃and CM.dex₄were48.98±1.36,70.87±4.82,58.55±2.98,15.62±2.68 and 7.48±0.66,respectively.The K values were 0.416,0.216,0.276,0.495 and 0.529,respectively.CD.dex₁had the highest viability and the best storage stability.L_phad good gastrointestinal tolerance in the gastrointestinal environment after microcapsule.（3）Three Maillard reaction products with different grafting degrees were prepared under the conditions of 79%RH and 60℃for 1 day,3 days and 5 days(WP-CD_1d,WP-CD_3dand WP-CD_5d).The physical and chemical properties of Maillard reaction products were studied by SDS-PAGE electrophoresis,infrared spectroscopy,fluorescence spectroscopy and free amino group experiments,and the T_gof Maillard reaction products was determined by DSC experiment.The degree of Maillard reaction products increased with the increase of reaction time,and the grafting degrees of Maillard reaction products were 53.87%±1.78%、57.60%±0.85%and 61.21%±1.02%,respectively.The Tg of Maillard reaction products increased with the degree of reaction,which were 115.53℃,137.21℃and 156.31℃,respectively.L_pwas microencapsulated by freeze-drying technology while Maillard reaction products was used as the wall materials.Moreover,the viability after encapsuled,gastrointestinal tolerance and storage stability of L_pwere studied.The viability of L_punder the protection of WP-CD_1d,WP-CD_3d,WP-CD_5d,CM.dex₁and whey protein were 69.75±1.39,79.23±3.58,87.37±1.80,70.87±4.82 and 59.80±2.42,respectively.Furthermore,L_phad the highest viability and the best storage stability,while WP-CD_5das the wall material.L_phad good gastrointestinal tolerance in the gastrointestinal environment after microcapsule.（4）L_pwas microencapsulated by vacuum freeze-drying technology,while WP-CD_5dwas used as macromolecular protective agent,and trehalose,lactose and sucrose were added as small molecule protective agents.The moisture adsorption isotherms and T_gof microcapsules were established by GAB model and Gordon-Taylor equation.The molecular mobility of microcapsules were studied by solid-state NMR technique.Moreover,the gastrointestinal tolerability of microcapsules and the storage stability of the microcapsules were investigated.The viability of L_pafter freeze-drying under the protective of WP-CD_5d,WP-CD_5dH,WP-CD_5dR,and WP-CD_5dZ were 87.37±1.80,97.84%±0.88%,98.49%±0.51%,81.73%±0.41%,respectively.The water adsorption experiment showed that when the water activity was 0.76,WP-CD_5dmicrocapsule occurred water loss after stored14 days indicating that the physical state of the WP-CD_5dmicrocapsule have changed,and the amorphous state had begun to change to the crystalline state.However,WP-CD_5dH,WP-CD_5dR,and WP-CD_5dZ did not occurred water loss,which indicated that the physical state of microcapsules increased after the small molecular sugars added.In the GAB model,the K value<1,indicating that GAB model is suitable to establish the water adsorption isotherm of WP-CD_5d,WP-CD_5dH,WP-CD_5dR,and WP-CD_5dmicrocapsules.The m₀value of microcapsules is WP-CD_5d>WP-CD_5dH>WP-CD_5dZ>WP-CD_5dR,indicating that the addition of small molecular sugar protective agents can reduce the m₀of the microcapsules.The C value of the microcapsules is:WP-CD_5dR>WP-CD_5dH>WP-CD_5d>WP-CD_5dZ,indicating that the addition of trehalose and lactose to the microcapsules can increase the interaction between the wall material and the monolayer water.However,the addition of sucrose decreased the intermolecular forces.In the Gordon-Taylor equation,the k values of microcapsules were:WP-CD_5d>WP-CD_5dZ>WP-CD_5dR>WP-CD_5dH,which indicated that the addition of small molecular sugars can reduce the influence of water plasticization on microcapsules.Furthermore,WP-CD_5dH had the best inhibitory effect on water plasticization with a k value was 4.03.The ¹³C NMR spectrum analysis indicated that the microcapsules were maintain amorphous state when stored 11%,33%,and 53%relative humidifies for 14 d at 4°C.The ¹H NMR spectrum showed that,compared with WP-CD_5dand WP-CD_5dR,WP-CD_5dH had the smallest chemical shift when stored at 53%relative humidity for 14 d,indicating that WP-CD_5dH had the lowest molecular mobility.WP-CD_5d,WP-CD_5dH,and WP-CD_5dZ had the best storage stability at 11%RH,which K were 0.041,0.021,and 0.026,respectively;WP-CD_5dR had the best storage stability at 23%RH,which K was 0.027.L_phad good gastrointestinal tolerance in the gastrointestinal environment after microcapsule.