Separation And Purification Of Bitter Peptides And Debittering Methods In Hydrolysates Of Cod Fish

Cod is a deep-sea fish with is rich in nutrient elements. It is the largest species of aquatic products imported in china to the processing capacity, the enzymatic hydrolysis of the cod has varity biological activities. Cod is a common market in fish, but it is only used as a low-value product, but cod would be used as high value products such as dry powder of cod and cod oral high value products. It will greatly enhance its market value. But cod fish hydrolyzate after hydrolysis produced has a bitter taste, wich affecting their applications in food markets. Currently the bitterness of the protein hydrolyzate research is still a global problem, so the study of bitter taste of hydrolysates has important significance. Therefore, this study investigates the bitter peptides from codfish. It combines enzymatic hydrolysis, cation exchange chromatography, HPLC to separate and purify 2 bitter peptides, and the peptides structures were mesured by LC-Q-TOF-Ms/Ms. This study also researched a variety of factors which influence the bitterness intensity of the separation bitter and non-bitter group. At last the study researched three different debittering methods and optimized the debitterizing process.The main results of this paper are as follows:1. Cod fish was used as research materials and the main compositions were measured. Cod fish is a kind of fish with hige level of protein content (20.20%), low level of fat content(2.54%). The essential amino acids content in adults reached 36.59% and the content of essential amino acids for children up to 45.13%, thus the protein of cod fish is a high quality protein.2. Using different protease to hydrolyze the cod fish, choose the hydrolysate with a high content for polypeptide(4.90mg/mL), high level of DH(15.10%) and high bitterness intensity(7.25±0.96) as sample to the next study, so the alkaline protease hydrolysis was used as sample to separate bitter peptides and remove the bitter taste.3. An approach of separation and identification of cod fish derived bitter peptides is established. Within the 2 identified peptides, only 1 peptide is synthesized. By using SP Sephadex C-25 cation chromatography and RP-HPLC, the bitter peptides were separated, by LC-Q-TOF-Ms/Ms, the bitter peptides were identified. The bitter peptides were HWPWMK and AVVL?, with the bitterness intensity were 9.40±0.82 and 8.90±0.90.4. A variety of factors to the bitterness intensity were explored in the hydrolysis of cod fish:cod fish hydrolysis bitterness is due to small hydrophobic molecules polypeptides produced by enzymes of cod protein. The DH, hydrophobicity of polypeptides, molecular weight, amino acid composition etc. are all important factors to the bitterness. The results showed that the intensity of bitterness increased and then declined with the increase of DH. When the degree of hydrolysis reached to 15.95%, the bitterness intensity reached to 5.00±0.71. For bitterness peptides generally hydrophobic peptides with a molecular weight of lower than 6kDa, mainly in the polypeptide 3kDa-6kDa in the hydrolysis of cod fish, but lower than 3kDa peptides also have bitterness. The reach of comparing with different components in the terminal amino acid shows that hydrophobic amino acids and basic amino acid are important to the bitterness intensity.5. The study of debilitating methods with activated charcoal, ?-cyclodextrin and yeast found that:the method by using activated carbon, the ablity of removal of bitterness is better, but for the solution of nutrients heavy losses; the method by using ?-cyclodextrin, although the sample nutrients are retained, the effect is not obvious removed from the hydrolysis, while increasing ?-CD dosage, the flavor also have other adverse taste; the method by using yeast, retain the nutrition on the samples with the degree of bitterness removal is higher. So the method by using yeast is the best method to remove the bitter in hydrolysis of cod fish. Compard the polypeptide content and So, the yeast debittering method shows that the sample after debittering, the polypeptide content was almost unchanged (decreased 2.98%), but the hydrophobic index So significantly reduced from the original 466.56 down to 278.31. By studying the enzyme activity of protease and aminopeptidase after debittering, the results show that protease and aminopeptidase have an important role in debittering process. It is generally select protease (2.38U/mL) and aminopeptidase (4.98U/ mL) higher activity as debittering conditions.