Bitter from /3-casein

It has been demonstrated that CPase Top enzyme is useful for eliminating bitterness originating from soy protein and com gluten. With respect to the bitterness from casein, although the bitterness was not completely eliminated, it was effective in reducing most of the bitterness. It is postulated that the bitterness of peptides is due to the hydrophobic amino acids in the C-terminus position (4). This assumption is necessary for the elimination of bitterness by liberating a hydrophobic amino acid at the C-terminus by the substrate specificity of CPase Top. 

The bitter peptide BPI a 114, isolated by Okai et al.239 > from casein hydrolysates, and delicious tasting peptides from fish proteins, will undoubtedly achieve practical importance in the food industry. 

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 2. Hydrolysis of bitter peptides from casein by CPase Top. Reaction conditions , 30°C, pH 4.0.

Gallagher et al. [138] investigated the future application of two enzymes, bromelain and a bacillus protease (Bacillus subtilis) in the production of peptides from casein in point of view of the functional properties of the products. Bromelain action resulted in a hydrolysate with a great number of high-molecular-mass peptides this may have improved the functional properties of a food product. The bacillus protease seemed to be more suitable for producing bitter peptides for future research and/or for future food. 

PVPP is useful for minimizing a tendency to browning in white wines. Alone, or combined with casein, it inhibits maderization by eliminating tannins, oxidizable cinnamic acids and the quinones formed when they oxidize. It acts differently from casein, as it eliminates the oxidizable phenols, whereas casein inhibits the oxidative phenomena (Table 10.9). The doses of 20-30 g/hl required to prevent browning do not produce any negative organoleptic changes. On the contrary, PVPP attenuates the bitterness of certain wines. 

Clegg, K.M., C.L. Lim, W. Manson, The structure of a bitter peptide derived from casein by digestion with papain, J. Dairy Res., 41, p. 283, 1974. 

Cyclo(Leu-Trp), a bitter compound isolated from the fermentation of milk casein by Bacillus subtilis, opened up the field to flavor and fragrance properties. It was further noted that dipeptides became more bitter when blockage of both the amino and carboxyl groups occurred or the dipeptide was converted into a DKP. This phenomenon opened the field of taste exhibition. ... 

The gross proteolysis of casein is probably due solely to rennet and plasmin activity (O Keeffe et al. 1978). Bacterial proteases and peptides are responsible for subsequent breakdown of the large peptides produced by rennet and plasmin into successively smaller peptides and finally amino acids (O Keeffe et al. 1978). If the relative rate of proteinase activity by rennet, plasmin, and bacterial proteases exceeds that of the bacterial peptidase system, bitterness in the cheese could result. Bitter peptides can be produced from a,-,- or /3-casein by the action of rennet or the activity of bacterial proteinase on /3-casein (Visser et al. 1983). The proteolytic breakdown of /3-casein and the subsequent development of bitterness are strongly retarded by the presence of salt (Fox and Walley 1971 Stadhouders et al. 1983). The principal source of bitter peptides in Gouda cheese is 3-casein, and more particularly the C-terminal region, i.e., 3(193-209) and 3(193-207) (Visser et al. 1983). In model systems, bitter peptides are completely debittered by a peptidases system of S. cremoris (Visser et al. 1983). 

Extraction of a portion of whey proteins, peptides, and amino acids Suitable for extraction of caseins and peptides from young cheeses. Not as effect as water Extraction of bitter and astringent peptides... 

Enzymatic hydrolysates of various proteins have a bitter taste, which may be one of the main drawbacks to their use in food. Arai el al. [90] showed that the bitterness of peptides from soybean protein hydrolysates was reduced by treatment of Aspergillus acid carboxypeptidase from A. saitoi. Significant amounts of free leucine and phenylalanine were liberated by Aspergillus carboxypeptidase from the tetracosapeptide of the peptic hydrolysate of soybean as a compound having a bitter taste. Furthermore, the bitter peptide fractions obtained from peptic hydrolysates of casein, fish protein, and soybean protein were treated with wheat carboxypeptidase W [91], The bitterness of the peptides lessened with an increase in free amino acids. Carboxypeptidase W can eliminate bitter tastes in enzymatic proteins and is commercially available for food processing. 

Umetsu, H., Matsuoka, H., and Ichishima, E. (1983). Debittering mechanism of bitter peptides from milk casein by wheat arboxypeptidase. J. Agric. Food Chem., 31, 50-53. 

Degradation of a bitter peptide obtained from the action of Bacillus subtilis on Casein... 

As given in Table XII the Q-method was also successfully applied in the case of bitter peptides from the rennet-sensitive sequence of K-casein (36) ... 

Series of bitter peptides have been isolated from enzymatic hydrolysates of proteins, esp. casein and soybean protein. 

Figure 3 gives the sequence of p-casein - which represents JO % of casein - and the bitter peptides derived from it and isolated by the groups of Clegg (49), Kloster-meyer (46), Gordon (6k). Here also the Q-values of the bitter peptides are above 1400. Please note, that no special single amino acid or sequence is needed to impart the bitter taste. 

Figure 2, Bitter peptides from aa,-casein ( ) = hydrophilic regions...

It is interesting to see that proteins with high Q-values above 1400 as e.g. soybean protein, casein wheat gluten, potato protein, Zein are the "parents" of bitter peptides, whereas no bitter peptides have been isolated from hydrolysates prepared from collagen or gelatin, proteins with Q-values below 1300. 

They also observed that the bitter taste of the peptides depended on many parameters DH being one of them. It seemed clear from their work that non-bitter hydrolysates could be obtained at high DH-values (above 20%) an observation which is in accordance with the results of Clegg and McMillan (4) who removed the bitter taste of casein hydrolysates by applying an exopeptidase. 

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 water soluble portion was dialyzed and then freeze dried to yield bitter peptides. Bitter peptides from com gluten and casein were prepared in a similar manner. 

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. 

A bitter principle, isolated from bacterial proteinase casein hydrolysate, and originally thought to be a cyclic tetrapeptide, has been now shown to have the... 

Bitter peptides that have been isolated from cheese are summarized in Table VIII. As expected, bitter peptides originate principally from hydro-phobic regions of the caseins, e.g., sequences 14 to 34, 91 to 101, and 143 to 151 of asi-casein, and 46 to 90 or 190 to 209 of /8-casein. As discussed by McSweeney et al. (1996) the majority of these peptides show evidence of some degradation by lactococcal proteinases and/or peptidases. 

Lemieux, L., and Simard, R. E. (1991). Bitter flavour in dairy products. I. A review of the factors likely to influence its development, mainly in cheese manufacture. Lait 71,599-636. Lemieux, L., and Simard, R. E. (1992). Bitter flavour in dairy products. II. A review of bitter peptides from the caseins Their formation, isolation and identification, structure masking and inhibition. Lait 72, 335-382. 

Traditionally fermented dairy products have been used as beverages, meal components, and ingredients for many new products [60], The formation of flavor in fermented dairy products is a result of reactions of milk components lactose, fat, and casein. Particularly, the enzymatic degradation of proteins leads to the formation of key-flavor components that contribute to the sensory perception of the products [55], Methyl ketones are responsible for the fruity, musty, and blue cheese flavors of cheese and other dairy products. Aromatic amino acids, branched-chain amino acids, and methionine are the most relevant substrates for cheese flavor development [55]. Volatile sulfur compounds derived from methionine, such as methanethiol, dimethylsulflde, and dimethyltrisul-fide, are regarded as essential components in many cheese varieties [61], Conversion of tryptophan or phenylalanine can also lead to benzaldehyde formation. This compound, which is found in various hard- and soft-type cheeses, contributes positively to the overall flavor [57,62]. The conversion of caseins is undoubtedly the most important biochemical pathway for flavor formation in several cheese types [62,63]. A good balance between proteolysis and peptidolysis prevents the formation of bitterness in cheese [64,65],... 

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