| Under the "macro view on food" the concept of "seeking heat and protein from cultivated land,grasslands,forests,oceans,plants,animals,and microorganisms,and developing food resources in a comprehensive and multi-channel manner".Food resources are facing a shift from primary grains to macro foods.The Northeast region,which is rich in natural forests,has unique advantages in plant resources.Corylus mandshurica Maxim with high protein content and abundant resources has strong development and application potential.The graded protein fractions(water-soluble protein fraction,salt-soluble protein fraction,alcohol-soluble protein fraction and alkali-soluble protein fraction)of Corylus mandshurica Maxim were divided into four groups(group A,group B,group C and group D)according to the different extraction orders by combining Osborne method,and then the solids and supernatant were sequential extractions.Differences in the effect of graded protein extraction were analyzed in combination with the Venn classification after obtaining the group with the maximum protein extraction rate,while the isolated proteins were extracted by alkaline solubilization and acid precipitation method to evaluate the nutritional value of the five proteins.The results showed that the extraction rate of Corylus mandshurica Maxim protein was the highest when extracted according to the order of group A,reaching 78.24%.Venn’s method analysis revealed dissolution crossover between the four graded proteins.The protein content of di-soluble protein with water-soluble protein,salt-soluble protein and alkali-soluble protein as the main components ranged from 11.56-21.16%,and the protein content of trisoluble protein reached about 55%,while the protein content of di-and tri-soluble protein with alcohol-soluble protein as the main components was only 3.44% and 2.42%.Amino acid analysis showed that the total amount of essential amino acids in the five proteins ranged from249.58-324.52 mg/g,with the first and second limiting amino acids being threonine and lysine,respectively.Moreover,except for the alcoholic soluble protein,the closeness of the other four proteins to egg protein was above 0.8,and the essential amino acid index and the relative ratio of essential amino acids were between 58.59-72.19 and 0.50-0.68.In addition,the amino acid ratio coefficient scores of isolated protein and water-soluble protein reached 68.49 and 62.46,and the nutritional indices reached 41.68 and 55.78,and the contents of presenting amino acids and medicinal amino acids were high,all around 70%.The solubility of isolated protein powder and water-soluble protein powder of Corylus mandshurica Maxim was improved and the mechanism of change was analyzed by ultrasonication,enzymatic hydrolysis and ultrasonic pretreatment followed by enzymatic modification.Determine the optimal solubility improvement conditions as follows: ultrasonic pretreatment power of 400 W and 600 W for isolated protein and water-soluble protein,respectively,with a treatment time of 20 min;the pH of neutral protease hydrolysis was 7.5,the amount of enzyme added was 2%,the hydrolysis temperature was 40 ℃,and the hydrolysis time was 60 minutes.Under these conditions,the solubility of isolated protein powder and water-soluble protein powder reached 51.68% and 66.51%,which increased by 31.72% and16.72% compared to untreated protein powder.Microstructure determination revealed that the main structure of both protein powders was sheet-like,and also contained some irregular rod-shaped and spherical structures,and different treatment methods would cause the protein powder sheet-like structure to break,the rod-shaped structure to break and the formation of smaller spherical structures.SDS–PAGE analysis showed that the molecular weights of the two proteins mainly consisted of 7 partial subunit bands,and some of the large molecular weight bands above 22 KD were degraded to small molecular weight bands below 14.4 KD after the treatment.Secondary structure calculations revealed that both α-helix and β-fold totals accounted for more than 70% of both protein powders,and about 10% of the α-helix structure was converted to β-turned structure in the enzymatically modified isolated protein powder after enzymatic digestion and ultrasonic pretreatment.The intrinsic fluorescence showed that the fluorescence intensity of the two proteins gradually increased and shifted to different degrees of blue with increasing solubility,indicating that the tertiary structures of both proteins changed to some extent.The modified isolated protein and water-soluble protein powder of Corylus mandshurica Maxim were subjected to heating and storage treatments and the changes in carbonyl content,sulfhydryl content,surface hydrophobicity and structure were measured to investigate the effects of heat treatment and storage on the quality of the two protein powders.The results showed that the carbonyl content of the isolated protein powder increased significantly(p<0.05)after the temperature reached 61 ℃,and the carbonyl content of the water-soluble protein powder increased significantly(p<0.05)in the temperature range of 73-85 ℃ and 109-121 ℃;the surface hydrophobicity of both protein powders decreased during the heat treatment;the total sulfhydryl and disulfide bond contents of the isolated protein and water-soluble protein powders showed a decreasing trend,but The content of total sulfhydryl groups and disulfide bonds of isolated protein and water-soluble protein powder showed a decreasing trend,but the content of both slightly increased with the increase of temperature in the range of 61-85 ℃,while the content of free sulfhydryl groups gradually increased with the increase of temperature.During storage,the carbonyl content of both protein powders increased more significantly(p<0.05)after 15 days of storage,and the carbonyl content changed significantly at 25 ℃ and 35 ℃ storage temperatures than at-20 ℃ and 4 ℃ storage temperatures,and the carbonyl content of isolated protein powder changed significantly than that of water-soluble protein powder;The surface hydrophobicity of protein powders showed a decreasing trend change and significantly decreased to 28.59 μg and 22.58 μg for isolated water-soluble protein powders at the 18 th day of storage at 35 ℃(p<0.05);The sulfhydryl content of both protein powders showed a decreasing trend during storage,but there was a small increase in the sulfhydryl content of both protein powders during the first 15 days of storage at 25 ℃ and35 ℃.SDS–PAGE analysis of the treated protein powder revealed that the subunit molecular weight of the protein did not change significantly between heat treatment and storage;The secondary structure of the protein powder changed relatively little,and only about 10% of theβ-turned of the secondary structure of the isolated protein powder was converted to α-helix when the heat treatment temperature was increased to 61 °C;The fluorescence spectra showed decreasing changes during both treatments,and the decrease in fluorescence intensity of isolated protein powder was more significant after the heat treatment up to 61 ℃ and under temperature storage at 25 ℃ and 35 ℃,and the tertiary structure changes were more obvious. |
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