Hydrolysis of bitter peptides

Hydrolysis of Bitter Peptides from Soy Protein by CPase Top. Three bitter peptides were incubated with cmde CPase Top at 30 C, pH 4.0, with the bitterness and the amount of liberated amino acids being evaluated throughout incubation. When a 1 % bitter peptide solution from soy protein was incubated with crude CPase Top (enzyme/substrate ratio = 1 /tkat/g), the bitterness diminished as reaction time increased. The bitterness was almost completely eliminated after 15 hr (Fig. 1). The amount of amino acids liberated increased with time of incubation. 

Hydrolysis of Bitter Peptides from Casein by CPase Top. When a 0.5% solution of bitter peptides from casein was incubated with crude CPase Top (enzyme/substrate ratio = 2 /xkat/g), most of the bitterness was eliminated after 2 hr, but some bitterness remained even after 15 hr (Fig. 2). The liberated amino acids increased with time similarly as with the soy bitter peptides. 

Figure 1. Hydrolysis of bitter peptides from soy protein by CPase Top. Reaction conditions 3(rc, pH 4.0.

Extensive proteolysis of a protein often results in the formation of bitter peptides ( ). Therefore, a compromise between high protein yield and low bitterness has to be found when choosing the DH-value at which the hydrolysis reaction should be terminated. For the present process a DH-value of about 10% seems to be a reasonable value. The termination is performed by acid inactivation of the enzyme and the acid used should be chosen in accordance with the desired organoleptic characteristics of the final hydrolysate. A totally non-bitter product can be produced by use of an organic acid like malic or citric acid. Due to the masking effects of such acids, absolutely no bitterness can be detected even when the taste evaluation is performed at neutral pH. Such products are found most suitable for soft drinks. However, when inorganic acids, e.g. hydrochloric or phosphoric acids are used, a slight bitterness may be detected in the pure hydrolysate. However, when evaluated in for instance a meat product, no bitterness at all can be tasted even when the hydrolysate is added up to a proportion of 1 3 of hydrolyzed protein to meat protein. 

Preparation of Bitter Peptides. The bitter peptides were prepared by hydrolysis of soy protein or com gluten with pepsin and hydrolysis of casein with trypsin. In the preparation of bitter peptides from soy protein, a 5% suspension of soy protein was hydrolyzed by pepsin [enzyme/substrate ratio = 1/100 (w/w)] at 37 C at pH... 

The three available industrial methods for the production of HVPs are (i) enzymatic a slow process that may result in the formation of bitter peptides and typically lacks the desired aroma/taste profile [20,21,39] (ii) alkaline hydrolysis which typically results in unacceptable flavor profile and an unbalanced amino acid content, and (iii) acid hydrolysis the most preferred method that is cost effective and yields a range of good flavors (see Chapter 11, section 11.4.1.2 Hydrolyzed Vegetable Proteins). 

Some potential uses of the plastein reaction are given in Table V. The plastein reaction has been proposed for removing bitter peptides formed through previous hydrolysis of proteins by facilitating the resyn-... 

Bitterness occurs as a defect in dairy products as a result of casein proteolysis by enzymes that produce bitter peptides. Bitter peptides are produced in cheese because of an undesirable pattern of hydrolysis of milk casein (Habibi-Najafi and Lee 1996). According to Ney (1979), bitterness in amino acids and peptides is related to hydrophobic-ity. Each amino acid has a hydrophobicity value (Af), which is defined as the free energy of transfer of the side chains and is based on solubility properties (Table 7-6). The average hydrophobicity of a peptide, Q, is obtained as the sum of the Af of component amino acids divided by the number of amino acid residues. Ney (1979) reported that bitterness is found only in peptides with molecular weights... 

The use of other acid proteases as substitutes for rennin in cheese making is determined by whether bitter peptides are formed during ripening of the cheese and by whether initial rapid hydrolysis causes excessive protein losses in the whey. Some of the acid proteases used in cheese making include preparations obtained from the organisms Endo-thia parasitica, Mucor miehei, and Mucor pusillus. Rennin contains the enzyme chy-mosin, and the scarcity of this natural enzyme preparation for cheese making resulted in the use of pepsin for this purpose. Pepsin and chymosin have primary structures that have about 50 percent homology... 

The extent of hydrolysis of protein hydrolysates is measured by the ratio of the amount of amino nitrogen to the total amount of nitrogen present in the raw material (AN/ TN ratio). Highly hydrolyzed materials have AN/TN ratios of 0.50 to 0.60. To obtain the desired level of hydrolysis in a protein, a combination of proteases is selected. Serine protease prepared from Bacillus lichenifor-mis has broad specificity and some preference for terminal hydrophobic amino acids. Peptides containing terminal hydrophobic amino acids cause bitterness. Usually a mixture of different proteases is employed. The hydrolysis reaction is terminated by adjust-... 

Hydrolysis of proteins without taste by proteases often produces bitter peptides. Hydrophobic amino acid residues located in the interior of protein molecules in aqueous solution are exposed by fragmentation of the protein molecules treated with proteases, and the peptides containing a number of hydrophobic amino acid residues occur in the solution (13). Many bitter peptides as shovm in Table 4-have been isolated from protein digests with proteinases (14-22). 

Molecular and enzymatic properties of serine carboxypeptidase (EC.3.4.1.6.1, CPase Top), isolated and refined from the common squid (Todarodes pacificus) liver, were studied. It was found that this enzyme reacts well at the C-terminal position of peptides having hydrophobic amino acids. Because of this property, it was anticipated that this enzyme would have the effect of eliminating bitterness of some peptides. This enzyme was used on bitter peptides prepared by hydrolysis of proteins with pepsin and trypsin. It was found that this CPase Top can eliminate bitter peptides prepared from soy protein and com gluten. 

Solubilization of Protein. Fish protein concentrate has high nutritional quality as determined both from its essential amino acid composition and from animal feeding experiments. Unfortunately, the concentrate is quite insoluble in water because of its denaturation by the solvent extraction method used in processing thus it contributes no functional properties to a food and must be used in bakery products primarily. A potentially useful method of solubilizing the protein is by proteolysis (9-12). As is the case with protein hydrolysates of casein and soybean protein, bitter peptides are formed during the hydrolysis. Papain and ficin produce more of these bitter peptides than does Pronase, for example (12). Pronase was found to produce a more brothy taste (13). A possible method of removing the bitter peptides is to convert the concentrated protein hydrolysate to plastein by further proteolytic enzyme action (14) to remove the bitter peptides. 

Food proteins, especially those of plant origin, often require modification to achieve desirable functional properties for use as food ingredients. For instance, soy protein has limited water solubility at acid pH, which restricts its use in acidic foods such as coffee whitener and acidic beverages. Improved solubility at acid pH for commercial soy protein isolate can generally be achieved by hydrolysis. However, the hydrolysis has to be carefully controlled, because excessive peptide bond hydrolysis may release bitter peptides, resulting in undesirable off-flavors. Scientists are constantly looking for better and safer methods to improve the functional properties of protein to meet the needs of the food industry. 

As mentioned earlier, deamidation by excessive protein hydrolysis is undesirable for food use because it could result in reduced protein functionality and the release of bitter-tasting peptides [19]. Alkali-catalyzed deamidation is also undesirable because, in addition to indiscriminate hydrolysis, alkali treatment results in products such as lysinoalanine that have been implicated in kidney damage in rats [20,21],... 

Shipe, W.F., G.F. Senyk, R.A. Ledford, D.K. Handler, E.T. Wolff, Elavor and chemical evaluations of fresh and aged market milk, J. Dairy Sci., 63(Suppl. 1), p. 43, 1980. Chandran, R.C., K.M. Shahani, Milk lipases a review, J. Dairy Sci., 47, p. 471, 1964. Kwak, H., I.J. Jeon, S.K. Pemg, Statistical patterns of lipase activities on the release of short-chain fatty acids in Cheddar cheese slurries, J. Food Sci., 54, p. 1559, 1989. Murry, T.K., B.E. Baker, Studies on protein hydrolysis I — preliminary observations on the taste of enzymic protein hydrolysates, J. Sci. Food Agric., 3, p. 470, 1952. Fujimaki, M., M. Yamashita, Y. Okazawa, S. Aral, Diffusible bitter peptides in peptic hydrolyzate of soybean protein, Agric. Biol. Chem., 32, p. 794, 1968. 

In the course of an investigation into the role of hydrophobicity in the bitter taste of certain peptides, a series of 2,3-di-0-aminoacylated derivatives (28) have been synthesised from methyl 4,6-0-benzylidene-Q -D-glucopyranoside and BcK-protected di-, tri-, or tetra-peptides by DCC-CMAP promoted condensation and subsequent acetal hydrolysis, and a similar condensation method was used in the synthesis of the Lipid A subunit analogues (30) from the known precursor (29) to acylate the free hydroxyl group of the ester substituent at C -3. ... 

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