Milk coagulating enzymes in cheese manufacture

Speaker: R.K. MALIK Principal Scientist DM Division

It happens when the casein micelles stick together Casein micelles are hydrophobic and their natural tendency is to aggregate In normal milk this process is prevented by glucomacropeptide and negative charge on the micelles

Enzymatic coagulation The primary phase of rennet coagulation involves the specific enzymatic modification of casein micelles Aggregation of the rennet.altered micelles is the secondary phase of coagulation .

1-st step of milk coagulation .Chymosin attack.

the diameter of casein micelles is reduced 7-10 nm .7).Start of aggregation Rennet coagulation follows the specific hydrolyses of micelle stabilizing surface layer during this step glucomacropeptide is lost At the natural pH of milk (6. about 80% of κ-casein must be cleaved to permit aggregation of the micelles After loosing its water-soluble tail κ-casein can no longer keep the casein particles separated.

acidity.Rennet The active principle in rennet is an enzyme called chymosine. and calcium content of the milk as well as other factors. The optimum temperature for rennet is in the region of 40°C . and coagulation takes place shortly after the rennet is added to the milk Two stages: • Transformation of casein to paracasein under the influence of rennet • Precipitation of paracasein in the presence of calcium ions The whole process is governed by the temperature.

for example Chy-Max (Chr. Hansen) Bovine pepsin mixed with chymosin is also used (Stabo) .Several proteinases will coagulate milk under suitable conditions. most of it is produced by microorganisms today. but most of them are too proteolytic Chymosin is the best.

Rennet is extracted from the stomachs of young calves and marketed in form of a solution with a strength of… 1:10 000 to 1:15 000 …which means that one part of rennet can coagulate 10 000-15 000 parts of milk in… 40 minutes at 35°C Rennet in powder form is normally 10x as strong as liquid rennet .

of young ruminants. mainly of calves Rennet contains chymosin and pepsin in fractions Chymosin is known for its high specificity for cleaving the caseinomacropeptide from Ƙ-casein which triggers the destabilisation of the casein micelles and. therefore. the fourth stomach. induces milk clotting Pepsin is much less specific and hydrolyses bonds with Phe.Animal rennet Traditionally manufactured by extracting the abomasum. Leu or Val residues . Tyr.

There are three main types of substitute coagulants: • Coagulating enzymes from plants.Substitute of animal rennet Vegetarians' do not accept cheese made with animal rennet In the Muslim world. the use of porcine rennet is out of the question. • Coagulating enzymes from microorganisms • Recombinant Coagulating enzymes Investigations have shown that coagulation ability is generally good with preparations made from plant enzymes A disadvantage is that the cheese very often develops a bitter taste during storage . which is a further important reason to find adequate substitutes Interest in substitute products has grown more widespread in recent years due to a shortage of animal rennet of good quality.

papain from papaya leaves. bromelain from pineapple or other enzymes the ratio of milk clotting activity to proteolytic activity is not high enough for commercial cheese making This is. have been extensively investigated as potential coagulants in cheese making For fig tree extracts.Plant-derived coagulants Vegetable enzymes. La Serena or Serpa in Portugal and Spain . extracts which have been used for centuries in traditional artisanal production of ewe milk cheeses such as Serra da Estrela. however. extracted by aqueous maceration from higher plant organs. Manchego. not true for Cynara cardunculus L.

were recently identified by Silva et al. to liquefaction and shape loss Bioactive peptides which were generated from casein by proteases of C. Pino et al.Cynara cardunculus L. (2006) . is a thistle variety which mainly grows in dry and stony areas of Portugal and some other parts of the Iberian Peninsula (Sales-Gomes and Lima-Costa 2008) It is a special feature of cheeses processed with plant coagulants that proteolysis is more pronounced (Pereira et al. 2009) This leads to a soft and buttery cheese texture and. partly. 2008. cardunculus L.

suitable for cheese production Such coagulants can be easily produced by fermentation and are. partially. therefore.Microbial coagulants Many extracellular proteases of microbial origin act similar as chymosin and are. almost unlimited available As the enzymes are not derived from ruminant tissue there are no constraints as regards bovine spongiform encephalopathy or scrapie. and cheeses made with microbial clotting enzymes are accepted by lacto-vegetarians .

which have been used in commercial cheese making since the 1960s. which reflects the high scientific interest in alternative coagulants for cheese production Fungi producing milk clotting proteases are ubiquitary and may easily be isolated from various environments (Tubesha and Al-Delaimy 2003) . higher proteolytic activity during cheese making. which may lead to a loss of protein degradation products into the whey and thus negatively affect cheese yield At present microbial coagulants of fungal origin. are of major importance. More than 100 fungal sources were reported by Garg and Johri (1994).The enzymes show. however.

parasitica. Rhizomucor pusillus and C.1. 50% loss of proteolytic activity after 30 min at 45C) is the most commonly used microbial coagulant for cheese production .5 kDa.Three species. miehei consists of a single polypeptide chain with a high similarity to chymosin in its three dimensional structure (Chitpinityol and Crabbe 1998) This protease (40. optimum milk clotting activity at pH 5. optimum proteolytic activity at pH 4. namely Rhizomucor miehei. have been established for large scale production The aspartic protease produced by R.6.

Chicen Ruminants Pigs Chymosin Chymosin EC 3.23.Nomenclature and sources of major proteases in Rennets Proteases Pepsin Gastricin IUB Name and No. Gastricin EC 3. Pepsin A EC Ruminants .3 Other Names Pepsin II Pepsin I Parapepsin II Pepsin B Pepsin C Rennin Source Ruminants Pigs.

Hansen) Fromase (Wallerstein) Marzyme (Miles) Emporase (Dairyland) Meito (Meito Sangyo) Noury (Vitex) Source Mucor Miehei Mucor Miehei EC 3.23.6 Protease M. pusillus var.4. Other Names Rennilase (Novo) Hannilase (Chr. Lindt Endothia parasitica Protease Surecurd Suparen (Pfizer) Endothia parasitica . pusillus Protease M.Nomenclature and sources of major proteases in Rennets Proteases IUB Name and No.

(2007) Thermoascus aurantiacus Metschnikowia reukaufii Thermomucor indicae-seudaticae N31 Chi et al. (2006) Venera et al. (2009) Li et al. (2009) Merheb et al. (1997) Merheb et al. (2010) .Recent research on new microbial proteases Micro-organisms Pleurotus sajorcaju (white rot fungus) Mucor bacilliformis Properties Clotting activity under cheese making conditions High structural similarity to bovine chymosin Lower thermostability than Rhizomucor miehei protease Enzymatic hydrolysis of bovine casein differed largely from proteolysis patterns generated by bovine chymosin Milk clotting activity Successfully cloned into Escherichia coli Crude enzymatic extract showed high milk clotting and low proteolytic activity and low thermostability References Moharib (2007) Machalinski et al.

2009). Cavalcanti et al. (2007) Bacillus licheniformis . (2004) Cavalcanti et al. (2008. (2004) Enterococcus faecalis Nocardiopsis sp. (2003) Poza et al. Shieh et al. effectively applied in Camembert cheese manufacture Milk clotting ability of extracellular extracts Optimisation of enzyme yield by fermentation conditions References Poza et al.Micro-organisms Myxococcus xanthus Properties Molecular mass: 40 kDa. acceptable yield and properties of the curd in cheese making experiments Successfully cloned into Escherichia coli Similar electrophoretic patterns of hydrolysed Ƙ-casein as Rhizomucor miehei. but high thermostability Shows typical milk clotting kinetics Dutt et al. (2009) Ageitos et al. (2005) Bacillus subtilis Ratio milk clotting to proteolytic activity comparable with commercial fungal proteases. highest clotting activity at pH 6 and 370C. (2004) Sato et al.

There are no exact figures available but Johnson and Lucey (2006) estimated that FPC comprises 70–80% of the global market for coagulants Recombinant Bos taurus chymosin is by far the most prominent genetically engineered clotting enzyme. yeasts or filamentous fungi served as hosts for recombinant enzyme expression (Mohanty et al. usually denoted as fermentation produced chymosin (FPC). bacteria. After cloning pre prochymosin or pro chymosin cDNA. was the first processing aid for food processing produced with recombinant DNA technology which has been registered by the FDA Recombinant chymosin is primarily used in the United States.Genetically engineered chymosin In 1990 the recombinant version of calf chymosin. 1999) . but other parts of the world do also show increasing acceptance.

Mohanty et al.g. Kluyveromyces lactis. 1999) Improved chymosin production by filamentous fungi. Saccharomyces cerevisiae. e. resulting in a more than 100% yield increase compared with the native enzyme (van den Brink et al. Lactococcus lactis. was achieved by glycosylation of either the chymosin molecule itself. Aspergillus oryzae or Trichoderma reesii (Teuber 1990. 2006) . and reverse transcriptase transfers the information to cDNA After incorporation into a vector DNA it can be transferred into suitable GRAS microorganisms such as Escherichia coli. Aspergillus niger. Bacillus subtilis.Fermentation produced chymosin production comprises the isolation of mRNA from the host’s abomasum cells. niger. A.

awamori. giraffe. This enzyme. ovine. buffalo. is now produced in industrial scale by Chr. (2006) described appropriate pilot scale production and purification. Kappeler et al. a 70% higher specific activity towards bovine Ƙ-casein than bovine FPC and a lower general proteolytic activity. . 2007) Most recent work on nonruminants focused on camel (Camelus dromedarius) chymosin expressed in A. using affinity chromatography. Camelidae and Equidae species (Kappeler et al. Denmark) and commercially available since the end of 2009.Reports on recombinant chymosin cloned from other animals include deer. antelope. niger var. caprine. porcine. Hansen A⁄S (Hoersholm. which has a molecular mass of about 40 kDa.

Chy-Max . Chymogen Calf abomasum Kluyveromyces lactis Calf abomasum Aspergillus niger Synthetic Escherichia coli Pfizer. Hansen. Brand name Gist-brocades. Maxiren Genencor/Chr.Recombinant Chymosin Preparations Source of DNA Producing Microorganism Producing company.

(2008) Water buffalo chymosin (Bubalus arnee bubalis) Camel chymosin (Camelus dromedarius) Pichia pastoris Higher affinity to Ƙcasein compared with conventional buffalo chymosin Production in industrial scale Aspergillus niger var. (2006) . (2007) Vallejo et al. (2007) Yang et al. (2001) Caprine prochymosin Escherichia coli Vega-Herna´ndez et al. (2004) Kumar et al. awawori Kappeler et al.Fermentation produced chymosin (FPC) from animals other than calf FPC Lamb prochymosin Host Escherichia coli Results Clotting and proteolytic activity similar to calf chymosin Proposed as alternative enzyme References Rogelj et al.

when appropriately certified. each of the coagulant types has its specific use Advances in separation and purification technology are responsible for a significant improvement of these enzymes which are accepted by vegetarians.Conclusions Milk coagulants are essential for cheese making and one of the most important enzymes in the food industry. and which. Although there is some significant competition on the market. The understanding of the action of the enzymes during Ƙ-casein cleavage and subsequent milk coagulation has increased substantially. but is still far from being complete. can also be used in organic cheese making .

some advantages in industrial cheese making but. because of its unvarying composition and its specific action. in some regions. and there are still reports that cheese yield and cheese quality is negatively affected Chymosin from recombinant micro-organisms has.Microbial coagulants. lacks acceptance by the consumer . nowadays mainly originating from Rhizomucor miehei. however. differ in molecular structure and in proteolytic activity. are animal rennet substitutes used for almost 40 years These enzymes do.

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