Preparation Of Chlorophyll Microcapsules Based On Multi-Carrier Agents And Encapsulation Techniques And Their Kinetics,Thermodynamics And Bio-Accessibility Evaluation

The burgeoning awareness of consumers on the negative health implications of synthetic food colorants has stimulated the need to seek for healthier alternatives.Chlorophyll,a natural pigment found in many green vegetables is highly valued due to its chemo-preventive effects.Despite the huge health benefits,the exposure of natural chlorophyll to external factors such as heat,light,oxygen,and acid during processing and storage limit the gains associated with this pigment.To find more novel approaches to mitigate natural chlorophyll degradation,researchers must explore a variety of encapsulation methods.In this study,natural chlorophyll was extracted from spinach leaves（Spinacia oleracea）and then optimization of the different encapsulation techniques using different carrier agents was performed.The related physico-chemical and morphological properties of the fabricated microcapsules was carried out.Furthermore,simulated gastrointestinal digestion was performed to evaluate chlorophyll bioaccessibility and the impact of digestion condition on the fabricated microcapsules.The main findings of this study are as follow:（1）Soy protein isolate（SPI）and chitosan were used as carriers to encapsulate chlorophyll.Optimization conditions utilizing the Box-Behnken design used 1:7（wet basis）mass ratio of chitosan to SPI,1:14.8（wet basis）mass ratio of chlorophyll to SPI,and p H6.91.Under this condition,the predicted and experimental encapsulation efficiency was97.95%and 96.21%respectively.Using complex coacervation and vibration nozzle technology,different chlorophyll-loaded microparticles were obtained with acceptable green colour,satisfactory encapsulation efficiency（94.9-96.1%）and loading capacity（3.32-4.22%）.Z-average diameter,SEM,and TEM showed nanoparticle sizes were nano-sized（273-373 nm）,porous and largely spherical with good dispersibility.FTIR,Raman,and DSC analyses confirmed significant interaction between chlorophyll molecules and carriers.Furthermore,chlorophyll retention of microparticles after 21 days of exposure to sunlight at room temperature ranged from 58.3-62.3%.At p H 2.5,chlorophyll retention ranged from 59.4-69.5%.In weak acid condition（p H 4.5 and 6.5）,nearly all（97.7-100%）chlorophylls remained.（2）Chlorophyll-loaded nano/microparticles were fabricated using zein,casein,and whey protein isolate as carrier agents.Chlorophyll was adequately loaded in these wall materials with encapsulation efficiency,loading capacity,and particle size ranging from83.6 to 96.3%,0.8 to 5.5%,and 483 to 1020 nm respectively.As unveiled by differential scanning calorimetry（DSC）,chlorophyll existed in a non-crystalline state inside the different wall materials.The encapsulation techniques and different carrier agents were effective in protecting chlorophyll from acid p H（p H 2 to 6,chlorophyll retention;39.3 to97.8%）and light（chlorophyll retention;41.6 to 65.5%）conditions.（3）Box-behnken design was used to optimize spray drying conditions for chlorophyll.Optimum conditions were given as inlet temperature,132℃;inulin-to-whey protein isolate ratio,61%:39%;pump rate,25%,resulting in 92.3%encapsulation efficiency,69.4%solubility,and-13.5 m V zeta potential at a desirability level of 0.901.The particle size,Carr index,bulk and tapped density,polydispersity index,and colour showed satisfactory results.Crystallinity,endothermic peak melting temperature,and enthalpy of chlorophyll-loaded microcapsules increased when compared to the blank microcapsules suggesting decreased hygroscopicity and enhanced thermal stability.In addition,the suitability of fabricated microcapsules using yogurt as a food model was assessed.Yogurt incorporated with chlorophyll-loaded microcapsules showed no significant p H modification with better apparent viscosity than control and sodium copper chlorophyllin（SCC）yogurt after 9 days of refrigerated storage.（4）The thermal degradation of chlorophyll in the different fabricated chlorophyll microcapsules was investigated.The degradation kinetics of chlorophyll,which was well fitted into the first-order model was evaluated at temperatures ranging from 70–90℃.The rate constant ranged from 0.0022/hr to 0.0851/hr with I-W-Chl and S-C-Chl^V having the lowest and highest rate constant respectively.Activation energy and z-value ranged from 39.7 to 70.1 KJ/mol and 32 to 56.4℃.Reaction rate,activation energy,and z-value result indicated that I-W-Chl was generally more stable at the studied temperatures.The D-value and t_1/2 ranged from 27.02 to 885.61 hours and 8.13 to 282.92 hours for all temperature conditions.Gibbs free energy,enthalpy,and entropy change ranges from 92.2to 100.0 k J mol^-1,36.7 to 67.4 k J mol^-1,and-172.0 to-86.4 J mol^-1 K^-1,respectively.（5）Finally,in vitro digestion was performed,and the bioaccessibility of chlorophyll in the different encapsulation systems was carried out.The zeta potential,particle size,and PDI were significantly modified after the micellarization of digested microcapsules.I-W-Chl presented with the highest total chlorophyll recovery and micellarization rate of 54%and 43%,respectively.In the aqueous micellar fraction,the different encapsulation systems had total chlorophylls,pheophytins,and pheophorbides ranging from 13–49%,42–77%,and 3–22%respectively.The bioaccessibility of total chlorophyll pigment ranging from7%to 20%is given in the following order:I-W-Chl>WPI-Chl>Z-Chl>Ca-Chl>S-C-Chl^V>S-C-Chl^C.Results of in vitro digestion study was verified in the experiment of gavage in mice.The results of this study show that the type of carrier and different encapsulation techniques can significantly affect the bioaccessibility of chlorophyll,which can provide assistance in the development of lipid-soluble phytochemical delivery systems.The optimization process developed in this study for complex coacervation and spray drying can be applied in the food industry to prepare microcapsules of chlorophyll─a natural food coloring agent with higher resistance,wider application range and enhanced functionality.The prepared chlorophyll microcapsules can minimize chlorophyll degradation and provide a reference scheme for functional food design.