Design Of A High-efficiency Delivery System For Lactase And Study On Its Stability

Lactose intolerance is a common chronic disease,with 70%of the world’s population suffering from this disease.The prevalence and high incidence of lactose intolerance make it an essential issue in global public health.Insufficientβ-galactosidase（β-Gal）activity in the small intestine is the leading cause of lactose intolerance.Compared to dietary lactose elimination,β-Gal supplementation does not cause changes in food quality or nutritional status,nor does it negatively affect human health.However,β-Gal preparations face many adverse environments during processing,transportation,storage,and ingestion,such as lyophilization,temperature changes,acidic pH in the gastrointestinal tract environment,and various digestive enzymes,which will significantly reduce the activity ofβ-Gal.The development and application of oral delivery systems forβ-Gal are expected to break this predicament.The problems withβ-Gal formulations are the lack of nutritionally efficient excipients,poor oral stability,lack of sustained-release formulations,and poor activity.This study used two strategies（excipient strategy and oral delivery system strategy）by constructing oral delivery systems with different interaction mechanisms;the effects of different mechanisms of action on improving the performance ofβ-Gal preparations are systematically explored.Moreover,we try to build a synergistic system ofβ-Gal and probiotics to relieve lactose intolerance more efficiently.This study is expected to provide a more explicit theoretical reference for the development ofβ-Gal formulations and accelerate the commercial use of food-gradeβ-Gal formulations.The main findings are as follows:1.The effect of oligosaccharides on the stability ofβ-Gal and its mechanism of actionThis study investigated the ability of oligosaccharides to protectβ-Gal against heat stress.Four kinds of oligosaccharides,including Isomalto-oligosaccharides（IMO）,Xylo-oligosaccharides（XOS）,Konjac-oligosaccharides（KOS）,and Mycose significantly increased the activity retention ofβ-Gal under heat treatment.The results of three assays,including circular dichroism,fluorescence,and fourier transform infrared spectroscopy（FTIR）,illustrated that these oligosaccharides could stabilize the secondary and tertiary structure ofβ-Gal under thermal conditions through hydrogen bond interaction.Unlike these four oligosaccharides,Chito-oligosaccharides（COS）changed the secondary and tertiary structure ofβ-Gal,thus decreasing its activity retention rate.Under heat treatment,the activity retention rate ofβ-Gal with optimal composition（30%IMO,w/v and 40%XOS,w/v）reached 82.12±1.13%,significantly higher than that of the nativeβ-Gal（the activity retention rate of 20.00±0.96%）.This study provides an insight into the mechanism by which sugar stabilizes protein under heat stress and offers guidance for applying liquid lactase to the food industry.2.Study on the protective effect of plant-based microcapsules from different sources onβ-GalThis study extracted sporopollenin exine capsules（SECs）from pollen or spores of different plant sources,and thenβ-Gal was encapsulated into SECs from different plant sources to fabricate an oral-controlled release system.The extraction process of SECs from different plant sources was optimized.L.clavatum spores were treated with 85%（w/v）H₃PO₄ for 10 h;sunflower pollen grains（SPGs）were treated with 85%（w/v）H₃PO₄ for 5 h;pine pollen grains（PPGs）were treated with 40%（w/v）H₃PO₄ for 2 h.Five different initial ratios of SECs:β-Gal and the loading time were optimized with the maximum enzyme retention rate reaching 98.96±1.06%（PPGs）,82.75±2.16%（SPGs）,and 79.40±1.96%（L.clavatum）.Furthermore,β-Gal-loaded SECs entrapped in carboxymethylpachymaran（CMP）could control the release ofβ-Gal under simulated gastrointestinal conditions（SGC）.Within 24 h in SGC,the optimal enzyme retention rate reached 68.66±1.02%（SPGs）,65.33±1.46%（L.clavatum）,and 61.20±1.34%（PPGs）.More importantly,the microstructure of different plant-extracted SECs remained unchanged throughout theβ-Gal encapsulation and simulated digestion process,showing excellent stability.Collectively,these results indicated that the entrapped SECs could be used as an effective oral delivery vehicle ofβ-Gal to improve its performance as a dietary supplement in the digestion of lactose.3.Comparative study on the protective effect and mechanism of polysaccharide physical and ionic crosslinking modified plant-based microcapsules onβ-GalThe SECs were structured to be pH-responsive to improve the stability of theβ-Gal delivery system in the gastrointestinal tract（Gastrointestinal tract,GIT）.This study compared the effect of two structured design forms of SECs（physical entrapment of polysaccharide and entrapment of polysaccharide-metal ion cross-linking）on the release behavior ofβ-Gal in SGC.Then,the interaction and binding mechanisms between CMP and metal ions in the vehicle were investigated.Notably,the system based on CMP and metal ion crosslinking embedding（CMP/metal ion system）not only markedly decreased the CMP dosage but also achieved a valid long-term release compared with the system based on CMP physical embedding（CMP system）.Among all the systems,the CMP/3%Al Cl₃ system showed the best ability to control the release with the maximum residual activity ofβ-Gal at nearly 70.55±1.03%after 24 h of treatment.Subsequently,the interaction mechanism between CMP and metal ions was characterized by microstructure,rheological properties,and spectroscopy characteristics.The results indicated that the low pH conditions lead to increased carboxyl protonation in CMP,decreased intermolecular electrostatic repulsion,and increased intermolecular hydrogen bond strength,resulting in high gel strength and thus a dense structure.Since the CMP/metal ion system has excellent pH responsiveness,which can impact the controlled release ofβ-Gal in GIT.4.Comparative study on the efficient delivery and protection mechanism of hydrophobin non-covalently and covalently modified plant-based microcapsules toβ-GalBased on previous studies,the SECs were modified to improve their sustained-release performance and storage stability as aβ-Gal delivery system in GIT,and reduce manufacturing costs.This study compared the effects of non-covalent and covalently modified SECs based on hydrophobic proteins on the release behavior ofβ-Gal in SGC and the system’s stability.At the same time,we provided insights into the sustained-release mechanism by exploring the interaction between zein and tannic acid（TA）.Notably,compared with the system based on zein physical modification（zein system）and the system based on zein non-covalent modification（zein/CMP system）,the system based on zein covalent modification（zein/TA system）not only significantly decreased the zein dosage but also achieved a sustained release,and had good storage stability.The residual activity ofβ-Gal in the zein₃/TA₂ system was about 70.26±1.33%after 36 h in SGC and about 90.03±1.61%for 28 d at 25℃.Additionally,FTIR analysis demonstrated that the stability of the zein/TA system depends on both hydrogen bonding and specific covalent bonding through the Schiff-base reaction,and the bonding strength regulates the sustained release.5.Construction and protection mechanism ofβ-Gal/probiotic co-encapsulation system based on plant-based microcapsulesTo explore the possibility of SECs co-encapsulatingβ-Gal and probiotics and prepare aβ-Gal/probiotic oral delivery system to relieve lactose intolerance synergistically.This study developed a new core-shell structure（SECs as the core and Ca-alginate（Alg）/CMP gel as the shell）for the co-encapsulation of L.plantarum andβ-Gal.We investigated the effects of this core-shell structure on gastrointestinal stability,freeze-drying stability,storage stability,and thermal stability ofβ-Gal and probiotics.The results showed that the maximum probiotic loading capacity and the residual activity ofβ-Gal reached 9.63±0.11×10⁹ CFU/g and 80.72±2.33%,respectively.In the Ca-Alg/CMP shell,the introduction of CMP can change the water-binding capacity of the gel and promote the gel to form a denser spatial network structure;the mass ratio of CMP:Alg could affect the microstructure and swelling behavior of the shell by influencing the hydrogen bonds between CMP and Alg.Therefore,the introduction of CMP can enhance the pH responsiveness of the shell gel,thereby regulating the release behavior ofβ-Gal and probiotics in SGC.After 600 min under simulated gastrointestinal conditions,the number of viable cells exceeded 10⁷ CFU/m L,and the residual activity ofβ-Gal was around 62.02±1.79%.Compared with the control group（pure L.plantarum andβ-Gal）,this system can significantly reduce the loss rate of probiotic count and enzyme activity after lyophilization and during storage（p<0.05）.Finally,using methylcellulose（MC）to coat the core-shell structure can significantly improve（p<0.05）the thermal stability of L.plantarum andβ-Gal.Overall,this core-shell for the protection ofβ-Gal and probiotics could not only enhance the storage,lyophilization,and thermal stability ofβ-Gal and probiotics but also achieve sustained release in the gastrointestinal tract.