| Beer foam as one important factor can directly reflect the quality of beer. High quality of beer foam can not only bring the consumers the feeling of freash, but also prevent the oxidation reaction on the materials in beer. There are many factors influencing the quality of beer foam, protein as the most important factor has effects on beer foam. As one of the detected protein in beer, protein Z has been proved to owing high surface viscosity and elasticity. Protein Z can resist the high temperature and enzymolysis during the brewing process, and it has the ability to maintain beer foam stability eventually. At present, few researches on the structural and physicochemical properties of protein Z during the brewing process. The mechanism of influences on beer foam from protein Z is still unknown. Therefore, it is significant to revell the relationship between beer foam and protein Z. In this paper, a new method on foam stability detection was established. The degree of correlation between beer foam and protein Z was studied. The secondary structural transforamtion of protein Z during the brewing process was studied. The expression of protein Z in Pichia pastoris was conduted. The glycosylations and glycations of protein Z were also analyzed. The results presented the structural and modified characteristics of protein Z which were beneficial to beer foam stability. The main results were showed as follows:(1) A new method on foam stability detection by ultrasonic vibration was established, and the method made use of the dissolved CO2 to foaming. By analyzing the foam stability value, the effect degree on the stability of method was ranked. The effect degree: pretreatment temperature> ultrasonic vibration frequency>ultrasonic vibration frequency. Meanwhile, the detection conditions were determined. The pretreatment temperature of beer sample was 20 oC, the ultrasonic vibration frequency was 28 k Hz and ultrasonic vibration frequency was 15 s. The response surface analysis presented the same detection condition, and there were no influences on each other of the three detection condition.(2) The traits of proteins in beer were analyzed, such as the contents of protein in beer, relative contents of hydrolyzed amino acids and the hydrophobicity of protein. After analysis, the concentration of protein in beer foam was higher than which in beer, meanwhile, the opposite tendency were found in the hydrophobicity analysis of proteins in beer foam. After analyzing the relative amino acids contents of hydrolyzed proteins in beer and foam, the results presented that proteins in beer contained high relative contents of Met, His, Phe, Cys, Lys could improve the foam stability. On the contrary, the proteins in beer contained high relative contents of Glu, Pro had the negative effects on the foam stability. The relative contents and concentrations of protein Z in beer and foam were also detected by densitometric method and enzyme-linked immuno sorbent assay. The results showed that the relative contents of protein Z in beer and foam were respectively in the range of 3.52%-27.46% and 8.16%-35.90%, and the concentrations of protein Z in beer and foam were respectively in the range of 7.14 ?g·m L-1-55.53 ?g·m L-1, 10.41 ?g·m L-1-86.95 ?g·m L-1. The results showed that protein Z gathered in foam after foaming. Protein Z as one high content of proten in beer had high relationship with beer foam stability.(3) Based on the correlation between foam stability and protein Z, the secondary structural transformation of protein Z during the brewing process was monitored. The purification method for protein Z by ammonium sulfate precipitation(40%-60%), anion exchange chromatography and gel filtration chromatography was eatshlished firstly. From the results of Circular Dichroism spectrum, the ?-sheets and ?-turns were the main structure of protein Z from malt.Except that, the ?-hlices and random coils were also founded in protein Z. The ?-helices and ?-sheets were transformed into ?-turns and random coils during mashing and boiling processes. The relative contents of ?-hlices and ?-sheets were from 13.25% and 35.03% decreased to 10.09% and 25.05%, respectively. On the contrary, the relative contents of ?-turns and random coils were from 37.15% and 14.57% increased to 45.62% and 19.24%, respectively. There were few random coils altering to ?-hlices during fermentation process. Except that, protein Z could hold the structure during the fermentation process. After the brewing process, the whole structure of protein Z became loose which was beneficial to foam stability. From the structural stability analysis of protein Z, the temperature, p H and ethonal concentration were not the reason for the structural transformations of protein Z during mashing and boiling processes. However, the glycations of protein Z should be the main reason for structural alterations.(4) Protein Z was successfully expressed in Pichia pastoris. For the influences from glycaosylations on recombinant protein Z, the molecular weight increased to 45 k Da. The relative contents of ?-helices, ?-sheets, ?-turns and random coils in recombinant protein Z were 14.99%?24.54%?46.31% and 14.15%. Compared with the protein Z from malt, the more content of ?-helices in reombinat protein Z was detected. Meanwhile, the results of influences on recombinant protein Z from temperature, p H and ethonal concentration were also presented that recombinant protein Z had higher structural stability.(5) From the results of contrastive analysis between protein Z from malt and recombinant protein Z, protein Z from malt had lower hydrophobicity. And the relative contents of hydrolysed Lys, His which had positive effects on beer foam were higher in both of protein Z from malt and recombinant protein Z. The typical mannose structure was the mian glycosylation type of protein Z from malt and recombinant protein Z. However, the hyperglycosylations occurred to recombinant protein Z. After analyzing the glycation reations of protein Z, the results showed that glycaion reation was easier occurred to protein Z from malt and the high temperature was one advantage condition. Meanwhile, protein Z had the selectivity to the reacted saccharides. The experiments of the influences on foam stability from protein Z showed that the glycated protein Z could improve the foam stability, and just glycosylated protein Z had no positive effects on foam stability. Glycations on protein Z was not only the main reason which caused the structural transformations of protein Z during brewing process, but also the reason which protein Z maintained the foam stability. |
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