Isolation And Purification Of Rice Protein Bodies And Their Structure Characterization

Rice,as the highest yielding grain in the world,is the staple food for about two-thirds of the population and is rich in nutrients.Rice is rich in starch and is the main source of energy for people’s diet.Protein is the second largest component in rice and has high nutritional value.The storage protein in rice mainly exists in the form of protein bodies,while starch exists in the form of starch granules.Although studies have shown that rice with high protein content has lower eating quality,it is not clear for the effect of protein on starch gelatinization and eating quality in rice.This paper aims to separate and purify protein bodies from rice,and characterize their structure,providing a methodological basis for studying the impact of proteins in rice on eating quality,and also providing a new pathway for studying the effect of proteins on starch gelatinization.A new method for separating protein bodies dilute alkali method was established,using brown rice with high protein content as raw material.The obtained protein bodies were purified and characterized.The main conclusions obtained are as follows:（1）Isolation and purification of protein bodies from brown rice.A method was established to separate protein bodies from brown rice using dilute alkali（sodium hydroxide）.On the basis of single factor experiments on alkali concentration,liquid material ratio,stirring time,ultrasonic power,ultrasonic temperature,and ultrasonic time,the Box-Behnken response surface optimization was used for separating protein bodies from brown rice.The optimal conditions for separating protein bodies were obtained as follows:alkali concentration 14 mmol/L,liquid to material ratio 12 m L/g,stirring time 90 minutes,ultrasound power 90 W,ultrasound temperature 35℃,ultrasound time 40 minutes.Under these conditions,the volume fraction of protein bodies in the separated mixture was 20.10%.After centrifuging the separated mixture and fitrating through 0.10μm and 5μm microporous membranes,the residual starch is removed using calcium chloride to obtain the purified protein bodies.（2）Morphological observation and structural characterization of brown rice protein bodies.Fluorescence microscopy and scanning electron microscopy observation showed that the obtained protein bodies were microspheres with fluorescence,with a size range of 0.5μm-3.0μm.After filtration with a microporous filter membrane,residual starch is removed by calcium chloride,and the resulting protein bodies are all discrete individuals that do not aggregate with each other.There are several micropores on the surface of the protein body,with a few large pits visible on the surface of the protein body.The protein body is white and cannot be stained by Coomassie Brilliant Blue.But after microporous filtration,if sodium carbonate is used to remove the residual starch,a small portion of the obtained protein bodies will be in an aggregated state,and the number of discrete protein bodies will be relatively small.It indicates that sodium carbonate has a poor effect on removing residual starch.According to SDS-PAGE electrophoresis,the protein body has protein subunits of 16k Da,22 k Da,33 k Da,and 57 k Da.The diffraction peak at 19.9°in the XRD spectrum belongs to proteinβ-folding peak.The absorption peaks appearing in the FT-IR spectrum at 1464 cm^-1and 1534 cm^-1belong to the Amide II band of the protein.The scattering peaks of the Raman spectrum appear at 1303 cm^-1and 1651 cm^-1,respectively,belonging to the Amide III and Amide I bands,which are characteristic peaks of proteins.Therefore,it is feasible to obtain protein bodies from rice using dilute alkali separation method and microporous filtration membrane and calcium chloride purification methods,which lays a methodological foundation for studying the structure and properties of protein bodies in rice endosperm and their impact on the formation of rice eating quality.（3）Isolation,purification and structural characterization of protein bodies in polished rice.The protein content in polished rice is lower than that in brown rice.A method for separating and purifying protein bodies from polished rice was studied,and its structure were preliminarily characterized to lay the foundation for studying the relationship between protein changes and the rice eating quality.Under the conditions of alkaline solution concentration of 14 mmol/L,ultrasound power of 90W,ultrasound temperature of 35℃,and ultrasound time of 40 minutes,the results showed that the size of the protein bodies separated from polished rice was also 0.5μm-3.0μm,the number of protein bodies separated from polished rice after storage is relatively reduced.XRD showed that the protein bodies of polished rice also appeared at 19.9°C,belonging to the proteinβ-folding diffraction peak,and the stronger diffraction peak of the protein body separated from the stored polished rice indicate thatβ-folding increases.Meanwhile,the protein bodies isolated from stored polished rice exhibited a infrared protein amide-I band absorption peak near 1642cm^-1,which was weaker than that isolated from polished rice before storage.The above research results indicate that although protein bodies are enriched in the cortex of brown rice,thus a lower protein content in polished rice,it is also feasible to separate protein bodies from polished rice.But the number of protein bodies separated from polished rice after storage is smaller,β-folding increases,and the infrared absorption peak of protein amide-I band weakens.The research results provide feasibility for studying the effect of protein body changes in polished rice on the formation of rice eating quality.In summary,it is feasible to separate and purify protein bodies from both brown and polished rice,and protein body samples for structural characterization can be obtained.This lays a methodological foundation for further research on the influence of protein bodies in rice on the formation of eating quality,and also provides new ideas for studying the effect of protein on starch gelatinization.