Preparation Of Narpodon Nehereus Protein And The Isolation, Purification And Characterization Of Low-Molecular-Weight Bitter Peptides

This paper studied on the hydrolysis technology of Harpodon Nehereus protein with protease, the isolation, purification and characterization of low-molecular weight bitter peptides in the fish protein hydrolysates, as well as the chromatographic distribution patterns of bitter peptides and the identification of bitterness intensity using chromatographic methodology.By the screening of five kinds of proteases, exopeptidase was recognized as hydrolysase for Harpodon Nehereus hydrolysis technology. On the basis of enzyme screening, this paper made a deeper research on the detailed technological conditions using 4-factors orthogonal rotation regression design and response surface methodology. Data from the analysis of the regression mathematical model and its 3-dimension response surface plot, and 2-dimension contour plot showed that the impact of enzyme dosage added, temperature and time on improving degree of hydrolysis possessed optimum zone of action; Within the experimental conditions, pH had insignificant effect on the hydrolysis technology; Temperature and time possessed obvious interaction. By choosing the amount of enzyme added as 233IU/g (fish paste), temperature as 45℃, and time as 4hr., a relatively high degree of hydrolysis ( 43.7%) could be achieved, efficiently and energy-savingly.Under the guidance of technology prioritization scheme previously studied, this paper performed a scale-up experiment on Harpodon Nehereus hydrolysis technology. Data from the measurement of the content of free amino acid nitrogen and the content of protein in the hydrolysates, as well as the analysis of amino acid composition suggested that what we had acquired was a low-molecular-weight peptide product which holded the properties of non-bitterness, joyful umami taste, and high nutrition value. The gain rate of product was 66.3%, and the protein transformation rate was 61.6%.The molecular weight distribution of peptides in Harpodon Nehereus hydrolysates and their bitter peptide fractions were determined by gel permeation chromatography on a Sephadex G-25 super fine column. Results indicated that the molecular weight range of the fraction enriched in bitter peptides was 470-1370 Da. Subsequently, the bitter fractions recognized were separated by reversed phase HPLC on LUNA C18 column and confirmed by sensory testing, three peptides with the most bitter flavor(p2, p4, p8) were obtained. Data from Tricine-SDS-PAGE electrophoresisshowed that the molecular weight of the peptides were l,460Da, 1,100 Da, 930 Da, respectively. The homogeneity of p2, p8 were identified by cLC capillary liquid chromatography.An attempt on the process analysis of miscellaneous components in Harpodon Nehereus hydrolysates was made using SDS-PAGE and Tricine-SDS-PAGE electrophoresis. Results showed that in comparision with SDS-PAGE, Tricine-SDS-PAGE manifested an improved performance on the separation of compositions in low molecular and complex system. The transformation detail of the compositions in Harpodon Nehereus hydrolysates during the process of hydrolysis could be observed with the electrophoregram, clearly.A series of Harpodon Nephereus hydrolysates with different intensity of bitterness were analysed by gel permeation HPLC on HW40S column. From the perspective of chromatography, the process of fish protein hydrolysis, as well as the formation and evolution of bitterness were elaborated. By the fitting of linear relativity between chromatographic peak area and bitter taste, the distribution pattern and transformation rule of bitter peptides were found and a preliminary, chromatographic testing system for the obscure sensory index-bitterness was advanced. Researches in this aspect provided a new method for obscure organoleptic testing.