Enzyme cheese curd

Cathepsin D (EC3.4.23.5). It has been known for more than 20 years that milk also contains an acid proteinase, (optimum pH ss 4.0) which is now known to be cathepsin D, a lysozomal enzyme. It is relatively heat labile (inactivated by 70°C x 10 min). Its activity in milk has not been studied extensively and its significance is unknown. At least some of the indigenous acid proteinase is incorporated into cheese curd its specificity on asl- and / -caseins is quite similar to that of chymosin but it has very poor milk-clotting activity (McSweeney, Fox and Olson, 1995). It may contribute to proteolysis in cheese but its activity is probably normally overshadowed by chymosin, which is present at a much higher level. 

The other major casein in cheese is /3-casein, but it is generally not hydrolyzed by rennet in low-pH cheeses. Alkaline milk protease (plas-min) plays the major role in the hydrolysis of /3-casein (Richardson and Pearce 1981). The plasmin level in cheese is related to the pH of the curd at whey drainage, since plasmin dissociates from casein micelles as the pH is decreased. Richardson and Pearce (1981) found two or three times more plasmin activity in Swiss cheese than in Cheddar cheese. Swiss cheese curds are drained at pH 6.4 or higher, while Cheddar cheese curds are drained at pH 6.3 or lower. Proteolysis of /3-casein is significantly inhibited by 5% sodium chloride. The inhibitory influence of sodium chloride is most likely due to alteration of /3-casein or a reduction in the attractive forces between enzyme and substrate (Fox and Walley 1971). 

This liposome enzyme system is a combination of an endopeptidase and an exopeptidase. The enzymes are slowly released after the cheese is put into the aging chamber. Using these liposomes for cheddar cheese production showed that the liposomes were evenly distributed and 90% of the added enzyme was retained in the cheese curd. The resulting cheese ripened in half the normal time with excellent flavor and textural properties. 

Only about 7% of the coagulating enzyme activity is retained in the cheese curd (200) the remaining activity is expelled in the whey. Recently, Dulley (200) observed that cheeses containing normal and double normal amounts of rennet showed very little difference in peptides soluble in dilute trichloroacetic acid after 10 months maturation this suggests that the amount of rennet did not significantly affect proteolytic breakdown of the cheese proteins. Furthermore, Dulley (200) did not observe a significant difference in starch gel electrophoretic patterns of cheese proteins from cheese containing normal and double normal... 

On the assumption that proteolysis is the rate-limiting event in cheese ripening, there has been interest for several years in adding exogenous proteinases to cheese curd. The first problem encountered is the method of enzyme addition. Direct addition of the proteinase to the cheesemilk ensures its uniform distribution throughout the curd but since most proteinases are water-soluble, most of the added enzyme is lost in the whey, which is economically undesirable, and significant proteolysis may occur prior to coagulation with consequent loss of peptides in the whey and a reduction in cheese yield. 

Most of the lactose in milk is lost in the whey during cheese manufacture and hence most cheese contain only trace amounts of carbohydrate (Table XIII). Furthermore, the residual lactose in cheese curd is usually fermented to lactic acid by starter bacteria. Thus, cheeses are suitable dairy foods for lactose-malabsorbing individuals who are deficient in the intestinal enzyme, lactase. 

Holmes, D. G., Duersch, J. N., and Ernstrom, C. A. (1977). Distribution of milk clotting enzymes between curd and whey and their survival during Cheddar cheese making. J. Dairy Sci. 60, 862-869. 

Caseins are quantitatively the most important protein components in bovine milk. This protein complex, known as a micelle, comprises four different caseins (asi-, as2-, 3-, and /c-caseins), which are held together by non-covalent interactions and appear as a highly stabilized dispersion in mUk. " During the classical cheese making process, it is the casein fraction that constitutes the cheese curd after the enzyme-triggered milk coagulation step. 

Milk is a natural colloidal dispersion that contains casein micelles, self-assembled protein associates with a diameter of about 200 nm [20]. The casein micelles are protected against flocculation by an assembly of dense hairs (often called a brush ) at their surfaces. Polymer brushes can thus provide steric stabilization of colloids. For millennia, man used the fact that milk flocculates and gels when it is acidified, as in yogurt production. Below pH = 5 macroscopic flocculation of the casein micelles in milk is observed [21]. This means that the interactions between casein micelles change from repulsive to attractive. The explanation is that acidification leads to collapse of the casein brushes [22]. In cheese-making the steric stabilization is removed by enzymes, which induce gelation into cheese curd. 

As one would expect, microbial enzymes range in specificity. For example, lipases from Candida cylindracea and Staph aureus have little specificity while those from Geotrichum candidum are specific for long chain acids [57], which are of little value in this application. Fortunately, most microbial hpases show specificity for the one and three positions of the triglyceride where the short chain fatty acids are found. While substantial information exists in the literature characterizing lipase enzyme activity to help in enzyme choice [58], enzymes must be evaluated for function in a model system. This often is done in a curd slurry system, which is produced by blending two parts of fresh cheese curd plus one part sterile dilute salt solution (5.2%... 

The most abundant milk protein is casein, of which there are several different kinds, usually designated a-, (1-, and K-casein. The different caseins relate to small differences in their amino acid sequences. Casein micelles in milk have diameters less than 300 nm. Disruption of the casein micelles occurs during the preparation of cheese. Lactic acid increases the acidity of the milk until the micelles crosslink and a curd develops. The liquid portion, known as whey, containing water, lactose and some protein, is removed. Addition of the enzyme rennet (chymosin) speeds up the process by hydrolysing a specific peptide bond in K-casein. This opens up the casein and encourages further cross-linking. 

Cheese is a concentrated dairy food produced from milk curds that are separated from whey. The curds may be partially degraded by natural milk or microbial enzymes during ripening, as in cured cheeses, or they may be consumed fresh, as in uncured cheeses like cottage cheese. Most commonly, a bacterial culture with the aid of a coagulating enzyme like rennin is responsible for producing the initial curd. The... 

Minerals found in milk which are insoluble remain in water in the curd and are more concentrated in the cheese than in milk. About two-thirds of the calcium and one-half of the phosphorus of milk remains in cheese. A major portion of the milk calcium is retained in the curd of cheese made with coagulating enzymes. Acid coagulation alone results in the loss of portions of both calcium and phosphorus salts in the acid whey, since these minerals are more soluble in the acidic medium. Most milk fat and fat-soluble vitamins are retained in the curd, but a considerable amount of water-soluble vitamins is lost during cheese manufacture. Retention of part of some B-complex vitamins in curd is due to their extended association with casein in the original milk. 

M. pusillus var. Lindt protease has given satisfactory results as a chymosin substitute in the manufacture of a number of cheese varieties, but not all varieties of M. pusillus var. Lindt are capable of producing acceptable cheese (Babel and Somkuti 1968). The clotting activity of M. pusillus var. Lindt protease is more sensitive to pH changes between 6.4 and 6.8 than chymosin, but is much less sensitive than that of porcine pepsin (Richardson et al 1967). The same authors reported that CaCL added to milk affected the clotting activity of M. pusillus var. Lindt rennet more than it did that of chymosin rennet. They also reported that this rennet was more stable than chymosin between pH 4.75 and 6.25. M. pusillus var. Lindt rennet is not destroyed during the manufacture of Cheddar cheese, although less than 2% of the enzyme added to the milk remains in the curd. Nearly all of it is found in the whey (Holmes et al. 1977). Mickelsen and Fish (1970) found M. pusillus var. Lindt rennet to be much less proteolytic than E. parasitica rennet but more proteolytic than chymosin rennet on whole casein, a8-casein and /3-casein at pH 6.65. 

Many enzymes extracted from higher plants have been tried for clotting cheese milk (Burnett 1976), however, attempts to use them have been unsuccessful. Most plant proteases are strongly proteolytic and cause extensive digestion of the curd, which has resulted in reduced yields, bitter flavors, and pasty-bodied cheese. 

Originally enzymes were given nondescriptive names such as rennin curding of milk to start cheese-making processcr pepsin hydrolyzes proteins at acidic pH trypsin hydrolyzes proteins at mild alkaline pH... 

Part of the process to make cheese involves the flocculation of an electrostatically stabilized colloidal O/W emulsion of oil droplets coated with milk casein. The flocculation is caused by the addition of a salt, leading to the formation of networks which eventually gel. The other part of the process involves reaction with an enzyme (such as rennet), an acid (such as lactic acid), and possibly heat, pressure and microorganisms, to help with the ripening [811]. The final aggregates (curd) trap much of the fat and some of the water and lactose. The remaining liquid is the whey, much of which readily separates out from the curd. Adding heat to the curd (-38 °C) helps to further separate out the whey and convert the curd from a suspension to an elastic solid. There are about 20 different basic kinds of cheese, with nearly 1000 types and regional names. Potter provides some classification [811]. 

Cheese is made by coagulating milk by the addition of rennet to produce curds. The curds are separated from the liquid whey and then processed and matured to produce a wide variety of cheeses. The active ingredient of rennet is the enzyme chymosin. Until 1990, most rennet was produced from the stomach of slaughtered newly born calves. These days, at a cost one tenth of that before 1990, chymosin is produced by genetically engineered bacteria into which the gene for this enzyme has been inserted, and is used for making cheese in the United States, Europe, and other parts of the world. 

Acid proteases probably also play a role in the breakdown of cheese proteins by species of Fenicillia used to produce blue cheeses (Roquefort, Stilton, Danish Blue) and soft cheeses (Camembert, Brie, etc.). The curds are inoculated with spore preparations of the appropriate mold. The growing mold then converts the curd into the desired cheese through the action of different enzymes. 

The characteristic flavor of various cheeses is primarily due to the enzymatic action of microbial flora contained in the curd. Enzymes extracted from these microorganisms and reacted with corresponding substrates may also produce a specific cheese flavor. The flavor produced may be economical and could be classified as "natural". 

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