Bitter proteolysis

Milk from cows affected with mastitis alters the sensory quality of raw milk and cheese (Munro el al., 1984). Sensory defects are reported as increased rancidity and bitterness, factors which are consistent with higher levels of lipolysis and proteolysis (Ma et al., 2000). 

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). 

The earliest commercial milk protein enzymatic modification dates back to the 1940s, when the first formulas for allergenic infants were made. The aims of this process were to reduce allergenicity as well as to change the functional properties of proteins while preserving their nutritional value for clinical use. Unfortunately the hydrolysates thus obtained were characterized by bitter taste, and for mainly this reason proteolysis, as a technological process, enjoyed very little popularity. 

Depending on the nature of the protein and the protease used, progressive proteolysis can liberate bitter peptides from proteins, the bitterness of which is a function of amino acid composition and sequence as well as the peptide chain length (Adler-Nissen, 1986b). An excellent review of the chemistry of bitterness has been published (Roy, 1997) and the reader is directed to this for more details. 

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... 

Partial proteolysis has been used by several researchers to improve functional properties, i.e. foaming, solubility of proteins (7,8,9). The significant problems associated with enzyme hydrolysis of proteins are excessive hydrolysis occurring under batch conditions, the generation of bitter flavors during hydrolysis and the cost of enzymes. Extensive information on factors affecting proteolysis of proteins and the problem of bitterness has been reviewed by Fujimaki et al. (7) in conjunction with studies of the plastein reaction. 

An excess of proteolysis must be avoided as it can impair the sensory characteristics for the following reasons (Toldra 2002) (i) Development of a bitter and metallic taste as a consequence of the accumulation of an excess of peptides and free amino acids (ii) formation and random distribution of white crystals of tyrosine... 

D Suckau, J Kohl, G Karwath, K Schneider, M Casaretto, D Bitter-Sauermann, M Przybylski. Molecular epitope identification by limited proteolysis of a immobilized antigen-anitbody complex and mass spectrometric peptide mapping. Proc Natl Acad Sci USA 87 9848 9852, 1990. 

According to several authors, cheese taste is mainly due to the compounds found in the cheese water-soluble extract (WSE) (1, 2). Thus, to study cheese taste, the focus is usually on the cheese WSE which contains small polar molecules such as minerals, acids, sugars, amino acids, peptides and some volatile compounds produced by different processes such as lipolysis, proteolysis microbial metabolism (3). These compounds are responsible for the individual taste sensations like sourness, bitterness and saltiness which are the main taste descriptors for cheese. However, in a complex mixture they also exert otiier taste sensations due to taste / taste interactions (4). Peptides are generally considered to be the main bitter stimuli in cheese (5). However, it was shown that in goat cheese, bitterness resulted mainly from die bitterness of calcium and magnesium chlorides, partially masked by sodium chloride (6). 

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. 

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. 

Visser, F. M. W. (1977b). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 2. Development of bitterness and cheese flavour. Neth. Milk Dairy J. 31, 188-209. 

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],... 

Applications of liposomes in cheese ripening were developed by the 1980s (El Soda, 1986). Enhancement of proteolysis by encapsulated cyprosins was evident 24 h after manufacture of Manchego cheese. Addition of encapsulated cyprosins to milk perceptibly accelerated the development of flavor intensity in experimental cheese through 15 days of age without enhancing bitterness (Picon et al., 1996). The capability of neutral and charged liposomes to entrap the proteolytic enzyme neutrase, and the stability of the preparation, were evaluated in the ripening of Saint-Paulin cheese milk (Alkhalaf et al., 1989). 

Richardson, B.C., L.K. Creamer, Casein proteolysis and bitter peptides in cheddar cheese. New Zealand J. Dairy Sci. TechnoL, 8, p. 46, 1973. 

Precursors of bitter substances in milk and dairy product foods are quite often proteins and mineral salts. Bitter substances are some peptides produced by enzyme proteolysis (see Section 2.3.3.2.1). Bitterness is especially typical for certain dairy products such as cheeses, yoghurt and casein hydrolysates (see Section 2.3.3.2). For example, the bitterness of ripened Gouda cheese was found to be primarily induced by calcium (CaCl2) and magnesium (MgCl2)... 

In certain foods a bitter taste is definitely not desirable, therefore different debittering methods have been developed. Methods for removing the bitter taste of enzymatic protein hydrolysates (such as casein hydrolysates) are described in Section 2.3.2.2. These methods are mainly based on controlled proteolysis, plastein reaction, extraction with azeotropic mixtures of alcohols and masking of bitter substances. 

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