Intervention Behaviors On In Vitro Digestion Of Nutrient And Safety Evaluation Of Carboxymethylated CNF

Cellulose nanofibril（CNF）,as a typical cellulose nanomaterial,has been widely used in the field of food due to its outstanding physical and chemical properties such as large Young’s modulus,high aspect ratio and sustainable source of raw materials（plant source）.For example,they are used as fillers in food packaging to extend the shelf life of food,or as texture modifiers to enhance the sensory appeal of food.In addition,the addition of CNF has been found to affect the rheological properties of digestive juices in the gastrointestinal tract,or to interact with digestive enzymes,thereby interfering with the digestion and absorption of nutrients in food（including protein,fat,and starch）.At the same time,the particle surface charge is a key characteristic that determines the physicochemical properties of nanoparticles.Based on the above findings,four kinds of CNFs with different surface charges were prepared by carboxymethyl modification,and their safety was evaluated by cell models in vitro and animal experiments in vivo.Through in vitro digestion model,the intervention behavior of surface charge difference of nanoparticles on the digestibility of protein,fat and starch in different food models was explored.The main research results are as follows:（1）Preparation and safety evaluation of carboxymethylated CNFFour carboxymethylated CNF（0,0.36,0.72 and 1.24 mmol/g）with different carboxymethylation degrees were prepared from cotton wool by carboxymethylation pretreatment and high-pressure homogenization.The surface charge of particles increases significantly with the increase of carboxyl substitution（p<0.05）,12.4±1.7,21.8±1.2,26.7±1.0 and 34.2±2.2 m V,respectively.By constructing Caco-2 cell model,it was found that CNFs with four different charges had no cytotoxicity.The mice model of repeated gavage was further established（8 weeks）,and the experimental results showed that there was no lethal situation in the experimental group.Compared with the control group,the experimental group showed no significant differences in the blood analysis and serum biochemical analysis.The results showed that the four kinds of CNF with different surface charges had no toxicity.（2）Effects of carboxymethylated CNF on in vitro digestion of myofibrillar protein gelThe results showed that the addition of CNF could help the protein gel form a dense and uniform network structure,and improve the gel hardness up to 205.10 g,with an increase of65%.The water holding capacity of the gel was significantly increased from 64%to 80%,while the water phase mobility was reduced.These changes all depend on the strengthening effect of CNF on the gel microstructure.With the increase of the surface charge of the particles,the effect is first strengthened and then weakened.Among them,C0.72 gel has the highest hardness,the most compact and uniform gel network structure,and the lowest water fluidity,indicating that excessive surface charge of C1.24 weakens its own strengthening effect.In addition,fluorescence bleaching restoration experiments showed that the dense network structure and limited water mobility reduced the diffusion rate of pepsin within the gel by 45%.Thus,the structural strengthening of CNF slows down the digestibility of the protein gel in the simulated stomach phase.（3）Effects of carboxymethylated CNF on in vitro digestion of lipid emulsionThe experimental results show that in Pickering emulsion system prepared by CNF emulsified corn oil,the particle size of emulsion decreases and the surface potential of droplets increases with the increase of particle surface charge.In the gastric phase environment with strong acid and high salt ions,the laser confocal results showed that CNF could promote the formation of gel structure of emulsion and promote the accumulation of oil droplets.In addition,the surface charge difference of CNF will affect the compactness of emulsion gel structure,among which,C₀can induce the emulsion to form a firmer and denser gel structure.The free fatty acid release rate in blank group was 80.2%,while C₀ could effectively reduce the release rate by 38%.The results show that the dense emulsion gel structure can slow down the hydrolysis of fat,mainly because the gel structure reduces the contact surface area of pancreatic lipase and bile salts in the intestinal phase.（4）Effects of carboxymethylated CNF on in vitro digestion of starchInhibition ofα-amylase and amyloglucosidase by CNF with different surface charges was studied by enzyme activity inhibition assay,fluorescence spectrum analysis and secondary structure change.The results showed that the four kinds of CNFs with different charges could effectively delay the hydrolysis of starch,and the effect was negatively correlated with the particle surface charge.Among them,C0 with the smallest particle surface charge had the strongest inhibitory effect onα-amylase（9.81 mg/m L）and amyloglucosidase（13.16 mg/m L）activities.The inhibitory effect is mainly through the combination of CNF withα-amylase or amyloglucosidase to form new complexes,and the microenvironment of starch hydrolase is changed by static quenching mode.According to thermodynamic parameters calculation,CNF and starch hydrolase spontaneously form complexes through hydrophobic interaction.In addition,the formation of new complexes can change the secondary structure of starch hydrolase.（5）Effects of pea fiber on in vitro digestion of plant-based protein composite gelsThe pea fiber was refined by using nanotechnology and prepared into two sizes of micron and nano.The pea protein,corn oil and pea starch were then filled with pea fiber as reinforcing material to prepare plant-based protein meat as a food model for in vitro digestion.The results showed that the nano-pea fiber was more favorable to the establishment of protein complex gel network structure,and the dense gel structure delayed the digestion of pea protein in the stomach phase,but did not affect the intestinal phase digestion.In addition,compared with micron-sized fibers,nano-sized pea fibers can better inhibit the activity of pancreatic amylase（up to 17.1%）,which can better inhibit the hydrolysis of starch.However,different from the experimental results of nanocellulose delaying fat digestion in the previous chapters,micro and nano sized pea fibers in this chapter did not have significant inhibition on fat digestion,which may be closely related to the form of fat and the type of nanoparticles.