Mechanistic Study Of The Structural Features And Digestion Properties Of Starches In Intact Pulse Cells

Pulses are dry edible seeds in legumes.They are high in carbohydrates,high in protein,low in fat,rich in vitamins and minerals,and are excellent food sources.Starch is the main carbohydrate in pulses,and an important energy source for pulse food.Pulse starches are natural slowly digestible starch with a lower glycemic index compared with other kinds of starches.Currently,the whole food diet is recognized as a healthy,nutritious diet because the whole food system has a complete tissue structure that limits the accessibility of starch with digestive enzymes and thus reduces the starch digestibility.The whole pulse food can resist the digestive enzymes due to the entrapped starch inside cell walls to reduce the digestion,and prevent the risk of type 2 diabetes,obesity and cardiovascular disease.In this paper,the isolated pulse cells are used as a model to investigate the whole pulse food,and the research is carried out to investigate the starch structure and digestive kinetics changes in different types of pulses by using modern advanced techniques such as laser confocal microscopy,X-ray diffraction,scanning electron microscopy,differential scanning calorimetry,etc.The structural and digestion kinetic changes of starches in pulse cells were compared with methods of limiting starch gelatinization in different pulse cells,different pulse cells isolation methods,dry heat treatment on pulse cells with different cell breakage degree.The main research contents include the following aspects:1.Eight kinds of pulses were chosen as the research objects.Purified starches were isolated from eight kinds of pulses,and the starch structure（from nanometer to micrometer）,amylase binding ability of starch granules and in vitro digestion kinetics of starch granules were determined to clarify the relationship between the starch structural parameters and digestion kinetics parameters of pulses with Pearson matrix statistical analysis.The results showed that there was no significant correlation between the starch structural parameters(DP_AP1,DP_AP2,DP_AM1,DP_AM2,amylose content,1045/1022 cm^-1 and relative crystallinity)and the digestion kinetics parameters after mixing with the amylase.The starch damage was positively correlated with the starch digestion kinetics.Enzyme binding experiments showed that the residual enzyme activity of the supernatant bound to the starch granules under non-hydrolysis conditions was significantly negatively correlated with the digestibility of starch.Therefore,the influence of the surface structure characteristics of starch granules on the initial binding of enzymes to starch granules during enzyme-catalysed starch hydrolysis is a decisive factor for the in vitro starch digestibility of starch.2.Garbanzo bean and pinto bean were selected as examples,and the cotyledon cells were isolated as models to simulate the whole pulse food.Pulse cells were incubated at different hydrothermal temperatures to determine structure and digestion characteristics of starches in cells.The results of hot-stage polarized light microscopy,X-ray diffraction and thermal properties showed that the entrapped starches in pulse cells were not fully gelatinized,still remained certain amount of crystal structure.The digestion profiles of pulse cells and confocal microscopic images before and after digestion showed that the starch digestion rate and extent in pulse cells increased significantly with higher hydrothermal temperature.The digestion extent of garbanzo bean cell samples increased from 17.0%（60oC）to 41.9%（100oC）,while that of pinto bean counterparts increased from 8.1%（60oC）to 35.5%（100oC）,respectively,but still lower than that of pure starch counterparts treated at the same temperature.Pulse cell walls are key factors influencing the digestion kinetics of entrapped starches entrapped,which mainly affects the diffusion of digestive enzymes to starch substrates.Different hydrothermal temperatures may lead to different permeability of cells,thus affecting the starch digestion.Pulse cell wall acts as the primary physical barrier of the enzyme to starch substrate,which hinders the enzyme binding.At the same time,the cell structure also limits the starch gelatinization,which would further control the enzyme successipility.The inhibitory effect of soluble and insoluble components on the enzyme activity from pulse cells also helps to reduce the starch digestion,but the enzyme inhibition gradually decreases as a function of hydrothermal temperature.Therefore,the in vitro digestion characteristics of starches in pulse cells in this study are mainly affected by the enzyme binding to the starch substrate,which is controlled by two main factors:（i）the intactness of pulse cell walls;（ii）enzyme inhibitory of soluble and insoluble components from pulse cells.3.Garbanzo beans were chosen as samples.The intact cells were isolated with different methods（including pressure-heating,hydrothermal treatments and acid-alkali treatment alone or combination methods）.Advanced technologies of light microscopy,laser confocal microscopy,X-ray diffraction and DSC were used to investigate the starch structural and digestion kinetic changes,and the permeability of garbanzo bean cell walls was also evaluated.The results shows that starches in different garbanzo bean cells are not fully gelatinized,and retain certain amount of crystal structure,which is further,confirmed the limitation of cell walls on the starch gelatinization.The digestion kinetics of starch entrapped in garbanzo bean cells isolated with different methods,and the digestion kinetics of starch in intact and broken cells all showed obvious differences.Infiltration experiments of three representative intact cells（Pre-ISO-I,80-ISO-I,Al-ISO-I）by different molecular weight fluorescein isothiocyanate-dextran probes reveal that the cell wall of Pre-ISO-I samples with high digestion rate and extent(0.0013min^-1,28.9%)can pass three kinds of probes.However,the Al-ISO-I samples with the lowest digestion rate and extent(0.0002min^-1,6.6%)could not pass the three probes.The permeability of cell walls is also comfirmed by results of infrared spectroscopy that the Pre-ISO-I sample has a lower degree of methylation（1.68）,which parially demonstrates that the pectin in the cell wall of Pre-ISO-I samples is highly dissolved.After the broken treatment,the starch digestion rate and extent in the broken cells are significantly higher than that of intact cells,but still lower than that of pure starch counterparts.In this study,the cell wall barrier effect of the garbanzo bean cells are adjusted by different processing methods,which results in different cell wall permeability and thus affects the starch digestibility.Therefore,the changes in cell wall permeability induced by different treatments in this study is the key factor affecting the in vitro starch digestion in garbanzo bean cells.4.Garbanzo bean cells with different cell breakage degrees were prepared by cellulose degrading enzyme treatment at different times.The changes of starch structure and digestibility in garbanzo bean cells with different cell wall breakage before and after dry heat treatment were compared.After dry heat treatment,the initial gelatinization temperature（T_o）,enthalpy changes（ΔH）and crystallinity of garbanzo bean cells with different cell wall breakage and pure starch granules all decreased,but the（T_c-T_o）of garbanzo bean cells with different cell breakage degrees increased,whereas that of pure starches decreased.The results indicats that the crystal diversity of starches changes,and chains reorganization including long-chain cleavage（T_p decline）and crystallization disintegration may occur.The garbanzo bean cell walls can protect the starch granules from reducing the starch degradation degree.After dry heat treatment,the digestion rate and extent of garbanzo bean cells with different breakage and starches samples increase gradually,indicating higher susceptibility of starch granules to enzymes.The dry heat treatment also significantly reduces the enzyme inhibitory of the insoluble components,thereby promoting the starch hydrolysis.With higher cell wall breakage degree,the digestion rate and extent of garbanzo bean cells increased.The results which further confirme that the susceptibility of enzyme to starch substrates is the key factor for the in vitro digestion of entrapped starches in garbanzo bean cells.