Fermentation-produced Chymosin Research Articles

Proteolytic activity of three variants of fermentation-produced chymosin on sodium caseinate and during Cheddar cheese ripening

Proteolytic activities and specificities of three types of fermentation-produced chymosin were investigated in cheese and in sodium caseinate (NaCN) digests made using these coagulants. The highest level of enzyme required to hydrolyse all intact αS1-CN in NaCN solution at pH 5.2 over 24 h was for a modified camel chymosin (mCC; 13.34 IMCU mL−1), followed by camel chymosin (4 IMCU mL−1) and bovine chymosin (0.4 IMCU mL−1). Many peptides were identified using liquid chromatography-mass spectrometry from both Cheddar cheese and NaCN digests produced using each chymosin. Besides previously reported casein-derived peptides produced by bovine and camel chymosins and corresponding cleavage sites, several new cleavage sites were identified. The proteolytic specificity of mCC on casein was determined. Most of the peptides observed in Cheddar cheese samples were also identified in NaCN digests. The overall proteolytic activity of mCC was the lowest, which may have implications for ripening and functionality of cheese.

Influence of different types of fermentation-produced chymosin on quality of soft cheeses

The disadvantage of soft cheeses is their short shelf life. In soft cheeses with a high moisture content, proteolysis occurs at a high rate, as a result of which the cheeses quickly overripe. The main proteolytic agent in soft cheeses is the milk-clotting enzyme (MCE). Increasing the shelf life of cheeses can be achieved by using MCE types having low proteolytic activity (PA). We have studied the effect of MCE based on different types of fermentation-produced chymosin: Chy-max® Extra (bovine chymosin), Chy-max® M (camel chymosin), Chy-max® Supreme (“modified” chymosin) on the dynamics of proteolysis in soft cheeses and related changes in the structure of cheeses during their storage. All 3 types of studied MCEs have different levels of nonspecific PA. The higher the level of nonspecific PA of the used MCE, the higher the rate of the proteolysis process in the resulting cheeses. Increasing the dose of MCE also increases the rate of proteolysis in cheeses. To increase the shelf life of soft cheeses, which depends on the period of preservation of a dense consistency, it is promising to use MCE with low PA based on camel chymosin (Chy-max® M) and “modified” chymosin (Chy-max® Supreme).

Assessing the authenticity of animal rennet using δ15N analysis of chymosin

Chymosin is a protease that curdles the milk casein. Animal rennet was the first discovered source of chymosin and its use is mandatory for the production of PDO cheeses such as Parmigiano Reggiano and Grana Padano. Of the alternatives, fermentation-produced chymosin is the most competitive because it functions in a similar way, but is much cheaper. Analytical tools are necessary in order to distinguish the 2 types of chymosin and verify the compulsory use of animal rennet in the production of PDO cheeses.In this work, a method to analyse 15N/14N in chymosin after extraction was developed. The δ15N values of animal rennet range from 5.7‰ to 8‰, whereas the δ15N values of fermentation-produced chymosin are significantly lower, ranging from −5.3‰ to 2.2‰. A threshold value of 5.7‰ was defined for authentic animal rennet.Addition of fermentation-produced chymosin to animal rennet, or its complete substitution, can be therefore detected.

Application of an Enzymatic Extract from Aspergillus niger as Coagulant for Cheddar Cheese Manufacture

Abstract The coagulation of milk by a serin protease from Aspergillus niger NRRL3 was studied by rheology. Cheddar-type cheese was manufactured using 3.5% (v/v) of fungal enzymatic extract and fermentation-produced chymosin was used as control coagulant. Full composition and ripening of both kinds of Cheddar cheese were studied. Differences in the proteolysis of caseins, not only during cheese manufacture but also during ripening, affected cheese composition, texture and peptide profile. Microbial development during ripening was not affected by the coagulant used.

Major Technological Advances and Trends in Cheese

Over the last 25 yr, cheese production in the United States has more than doubled with most of the increase due to production in the western states. Processing large volumes of milk into cheese has necessitated changes in vat size and design, reliance on computer software, and milk standardization, including use of membrane concentration of milk either at the cheese plant or on the farm. There has been increased interest in specialty cheeses including cheese made from sheep, goat, and organic milks. In addition, membrane processing of whey into various value-added components has become routine. Changes in cheese manufacturing protocols have resulted in a reduction of the manufacturing time and the necessity for consistent and reliable starter activity. Major advances in the genetics of microorganisms have not only resulted in widespread use of fermentation-produced chymosin but also in starter bacteria with improved resistance to bacteriophage infection. Genomics and proteomics have increased the likelihood of the development of nonstarter adjuncts with specific enzymatic activity. Indeed, the use of adjunct microorganisms to produce cheese with a unique flavor profile or to produce cheese with more consistent or better quality flavor has gained almost universal acceptance.

Arylsulphatase activity in milk and rennet from different sources

Abstract Arylsulphatase activity on two substrates, p -nitrocatechol sulphate ( p NCS) and p -nitrophenyl sulphate ( p NPS), in milk and commercial rennet from different sources was measured. Cows’ and sheep's milk contained arylsulphatase active on p NCS, and batch pasteurization reduced the activity. Rennets from animal sources, including calf, kid and lamb, exhibited arylsulphatase activity with different substrate specificities. A fermentation-produced chymosin from a genetically engineered microorganism did not exhibit detectable arylsulphatase activity. A commercial rennet paste showed arylsulphatase activity after dialysis.

Effect of gelation temperature on the properties of skim milk gels made from plant coagulants and chymosin

Reconstituted skim milk was gelled at 25–40°C with the plant-origin coagulants from Cynara cardunculus L. or Cynara humilis L. or with fermentation-produced chymosin. Gel formation and ageing were monitored by low amplitude oscillatory rheology and confocal scanning laser microscopy. Arrhenius plots for the rate of milk gelation were also determined. Plant coagulants had shorter gelation time (tg) at 25°C, 35°C and 40°C, and higher initial rate of increase in G′ values at all temperatures tested. The firmest gels at long ageing times were produced by chymosin at 30°C and 32°C. At a gelation temperature of 25°C, the differences in rheological and microstructural characteristics between plant coagulants and chymosin were considerable; plant coagulants had shorter tg and higher G′ values. For the lowest gelation temperatures, plant coagulants had smaller activation energy values for gelation. Most of the gelation results were similar between plant coagulants, but some differences were found in the values of tg, the rate of increase in G′ and loss tangent parameter. The characteristics of gels produced with plant coagulants were influenced less by the changes in temperature compared with chymosin-produced gels, which may be an important consideration in using plant-origin coagulants in the production of cheeses with a wider range of gelation temperatures.

Influence of milk‐clotting enzymes on acidification rate of natural whey starter culture

The aim of this work was to study the influence of milk‐clotting enzymes on whey starter culture and hard cheesemaking. Four cheeses were prepared simultaneously per cheesemaking day, and the experiment repeated on eight consecutive days. Adult bovine rennet was used in the control cheeses, and three formulations of fermentation‐produced chymosin (FPC) in the experimental cheeses. Whey cultures were obtained from the whey of the preceding cheeses, incubated individually for 24 h at 42°C. pH values of the control and experimental whey did not differ significantly before incubation, but after 24 h incubation the pH of the FPC whey was significantly higher than that of the control. Soluble nitrogen content in trichloroacetic acid 2% and 12% and in phosphotungstic acid 2.5% was significantly lower for all experimental whey samples than for control whey. This probably explains the decrease in the acidification rate of the whey cultures when bovine coagulant is replaced by FPC in hard cheesemaking.

Rheological properties of milk gels made with coagulants of plant origin and chymosin

The rheological properties of milk gels made using coagulants obtained from the plants Cynara cardunculus L. and Cynara humilis L. were compared with those of fermentation-produced chymosin, using dynamic low amplitude oscillation as well as large strain (yield) testing. Gelation experiments were performed at 321C using skim milk powder that had been reconstituted for 2 or 16 h at 321C. The storage modulus (G 0 ), loss tangent (tand) at low frequency (0.002 Hz) andyieldstress were higher for chymosin-induced gels than those of plant coagulants, when tested B6 h after coagulant addition. Plant coagulants were slightly more proteolytic than chymosin, andcasein hydrolysis may have resultedin lower gel firmness. Most of the rheological properties were similar for the two plant coagulants, in agreement with their similar enzyme contents. Gelation properties were different in milk reconstituted for 2 or 16 h. This behaviour was probably due to casein hydrolysis by plasmin, as milk reconstituted for 16 h at 321C hadsignificant levels of degradation of both as- and b-caseins. The addition of soybean trypsin inhibitor which inhibits plasmin activity resulted in similar gelation profiles for gels made from milk reconstituted for 16 and 2 h. r 2002 Publishedby Elsevier Science Ltd .

Rhizomucor miehei aspartic proteinases having improved properties.

Aspartic proteinases are widely spread in most organisms, where they carry out many different functions. The oldest and most well–known use of aspartic proteinases is as coagulants in cheese–making. The coagulants are probably still the only commercial products containing aspartic proteinases as the active substance. The main commercial interest in coagulants is focused on how to enhance the enzyme activity and its performance during cheese–making. In contrast to this, most commercial interests in other aspartic proteinases deal with how to inhibit their activity in order to control their function. The main commercial coagulants can be divided into three groups according to their origin, animal rennet, fermentation produced chymosin (FPC) and microbial coagulants.1 Of animal rennets, calf rennet is the traditional and the most desired coagulant for cheese–making, but FPC products made by recombinant organisms and microbial coagulants, the natural aspartic proteinases of the fungi, make up a considerable part of the coagulant market. Fermentation produced chymosin is the product of the future, and the great interest in microbial coagulants is due to the fact that they constitute a significant part of the market because of their lower prices. Rhizomucor miehei aspartic proteinase (EC 3.4.23.23) constitutes the active component of the most widely used microbial coagulant. The molecule consists of 361 amino acids, has a molecular weight of 38701 and contains in addition approx. 6% carbohydrate.2 The enzyme is commercially available in its natural form, in various heat labile forms made by post-treatment of the natural form (oxidation) and as recombinant coagulant produced in Aspergillus oryzae.

Detection of recombinant chymosins in calf rennet by enzyme-linked immunosorbent assay

Summary - The presence and the origin of recombinant chymosin can be detected in milk-c1otting enzyme solutions by antigen coat plate-enzyme-Iinked immunosorbent assay (ACP-ELISA) or sandwich ELISA. The method is based on the detection of contaminant proteins derived from microorganisms or from the culture medium, which are always present in fermentation-produced chymosin solutions. The specifie polyclonal antibodies obtained from the sera of rab bit and hen permitted identification of the contaminant proteins in the commercial solutions. A simple dilution of the sample is sufficient to detect the addition of 0.5% Chymogen (Hansen) or Maxiren (Gist-Brocades) and 2.5% Chymax (Pfizer) in calf rennet.

Cheese Yield Performance of Fermentation-Produced Chymosin and Other Milk Coagulants

Fat recovery, protein recovery, and cheese yield performance of a fermentation-produced chymosin was compared with other commonly used milk coagulants. In trial 1, performance of fermentation-produced chymosin was compared with proteases from Mucor miehei and Mucor pusillus. In trial 2, fermentation-produced chymosin was compared with calf rennet and adult bovine pepsin. In each trial, three vats of Cheddar cheese were made simultaneously from the same milk, using the same starter culture, with the three different coagulants. This was replicated 12 times in trial 1 and 9 times in trial 2. Generally, higher fat and protein losses in the whey were observed for proteases from Mucor miehei and Mucor pusillus than for fermentation-produced chymosin or calf rennet. Adult bovine pepsin had higher fat losses in the whey, but not higher protein losses in the whey, than fermentation-produced chymosin or calf rennet. In trial 1, fermentation-produced chymosin had a higher cheese yield efficiency than proteases from Mucor miehei and Mucor pusillus (.54 and .74%, respectively) with a protected least significant difference of .34%. In trial 2, fermentation-produced chymosin (100% chymosin) and calf rennet (94% chymosin) had virtually identical cheese yield efficiencies, but adult bovine pepsin had a lower (.39%) cheese yield efficiency with a protected least significant difference of .27%.