Milk coagulating enzymes in cheese manufacture

Speaker: R.K. MALIK Principal Scientist DM Division

Coagulation
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

Chymosin attack, 1-st step of milk coagulation

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 (6.7), about 80% of -casein must be cleaved to permit aggregation of the micelles After loosing its water-soluble tail -casein can no longer keep the casein particles separated, the diameter of casein micelles is reduced 7-10 nm

Rennet
The active principle in rennet is an enzyme called chymosine, 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, acidity, and calcium content of the milk as well as other factors. The optimum temperature for rennet is in the region of 40C

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

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 35C
Rennet in powder form is normally 10x as strong as liquid rennet

Animal rennet
Traditionally manufactured by extracting the abomasum, the fourth stomach, 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 -casein which triggers the destabilisation of the casein micelles and, therefore, induces milk clotting Pepsin is much less specific and hydrolyses bonds with Phe, Tyr, Leu or Val residues

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, 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. There are three main types of substitute coagulants: Coagulating enzymes from plants, 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

Plant-derived coagulants

Vegetable enzymes, extracted by aqueous maceration from higher plant organs, have been extensively investigated as potential coagulants in cheese making For fig tree extracts, 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, however, not true for Cynara cardunculus L. extracts which have been used for centuries in traditional artisanal production of ewe milk cheeses such as Serra da Estrela, Manchego, La Serena or Serpa in Portugal and Spain

Cynara cardunculus L. 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. 2008; Pino et al. 2009) This leads to a soft and buttery cheese texture and, partly, to liquefaction and shape loss Bioactive peptides which were generated from casein by proteases of C. cardunculus L. were recently identified by Silva et al. (2006)

Microbial coagulants

Many extracellular proteases of microbial origin act similar as chymosin and are, partially, suitable for cheese production Such coagulants can be easily produced by fermentation and are, therefore, 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

The enzymes show, however, 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, which have been used in commercial cheese making since the 1960s, are of major importance. More than 100 fungal sources were reported by Garg and Johri (1994), 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)

Three species, namely Rhizomucor miehei, Rhizomucor pusillus and C. parasitica, have been established for large scale production The aspartic protease produced by R. miehei consists of a single polypeptide chain with a high similarity to chymosin in its three dimensional structure (Chitpinityol and Crabbe 1998) This protease (40.5 kDa, optimum milk clotting activity at pH 5.6, optimum proteolytic activity at pH 4.1, 50% loss of proteolytic activity after 30 min at 45C) is the most commonly used microbial coagulant for cheese production

Nomenclature and sources of major proteases in Rennets

Proteases Pepsin Gastricin

IUB Name and No. Pepsin A EC 3.4.23.1 Gastricin EC 3.4.23.3

Other Names Pepsin II Pepsin I Parapepsin II Pepsin B Pepsin C Rennin

Source Ruminants Pigs, Chicen Ruminants Pigs

Chymosin

Chymosin EC 3.4.23.6

Ruminants

Nomenclature and sources of major proteases in Rennets

Proteases IUB Name and No. Other Names Rennilase (Novo) Hannilase (Chr. Hansen) Fromase (Wallerstein) Marzyme (Miles) Emporase (Dairyland) Meito (Meito Sangyo) Noury (Vitex) Surecurd Suparen (Pfizer) Source Mucor Miehei

Mucor Miehei EC 3.4.23.6 Protease

M. pusillus Protease Endothia parasitica Protease

M. pusillus var. Lindt Endothia parasitica

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. (2006) Venera et al. (1997) Merheb et al. (2007)

Thermoascus aurantiacus Metschnikowia reukaufii Thermomucor indicae-seudaticae N31

Chi et al. (2009) Li et al. (2009) Merheb et al. (2010)

Micro-organisms Myxococcus xanthus

Properties Molecular mass: 40 kDa, highest clotting activity at pH 6 and 370C, acceptable yield and properties of the curd in cheese making experiments Successfully cloned into Escherichia coli Similar electrophoretic patterns of hydrolysed -casein as Rhizomucor miehei, effectively applied in Camembert cheese manufacture Milk clotting ability of extracellular extracts Optimisation of enzyme yield by fermentation conditions Ratio milk clotting to proteolytic activity comparable with commercial fungal proteases, but high thermostability Shows typical milk clotting kinetics

References Poza et al. (2003)

Poza et al. (2004) Sato et al. (2004)

Enterococcus faecalis

Nocardiopsis sp.

Cavalcanti et al. (2004) Cavalcanti et al. (2005) Dutt et al. (2008, 2009), Shieh et al. (2009) Ageitos et al. (2007)

Bacillus subtilis

Bacillus licheniformis

Genetically engineered chymosin

In 1990 the recombinant version of calf chymosin, usually denoted as fermentation produced chymosin (FPC), 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, but other parts of the world do also show increasing acceptance. There are no exact figures available but Johnson and Lucey (2006) estimated that FPC comprises 7080% of the global market for coagulants Recombinant Bos taurus chymosin is by far the most prominent genetically engineered clotting enzyme. After cloning pre prochymosin or pro chymosin cDNA, bacteria, yeasts or filamentous fungi served as hosts for recombinant enzyme expression (Mohanty et al. 1999)

Fermentation produced chymosin production comprises the isolation of mRNA from the hosts abomasum cells, 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, Bacillus subtilis, Lactococcus lactis, Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus niger, Aspergillus oryzae or Trichoderma reesii (Teuber 1990; Mohanty et al. 1999) Improved chymosin production by filamentous fungi, e.g. A. niger, was achieved by glycosylation of either the chymosin molecule itself, resulting in a more than 100% yield increase compared with the native enzyme (van den Brink et al. 2006)

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

Recombinant Chymosin Preparations

Source of DNA

Producing Microorganism

Producing company, Brand name Gist-brocades, Maxiren Genencor/Chr. Hansen, Chymogen Pfizer, Chy-Max

Calf abomasum

Kluyveromyces lactis

Calf abomasum

Aspergillus niger

Synthetic

Escherichia coli

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. (2001)

Caprine prochymosin

Escherichia coli

Vega-Hernandez et al. (2004) Kumar et al. (2007) Yang et al. (2007) Vallejo et al. (2008)

Water buffalo chymosin (Bubalus arnee bubalis) Camel chymosin (Camelus dromedarius)

Pichia pastoris

Higher affinity to casein compared with conventional buffalo chymosin Production in industrial scale

Aspergillus niger var. awawori

Kappeler et al. (2006)

Conclusions
Milk coagulants are essential for cheese making and one of the most important enzymes in the food industry. The understanding of the action of the enzymes during -casein cleavage and subsequent milk coagulation has increased substantially, but is still far from being complete. Although there is some significant competition on the market, 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, and which, when appropriately certified, can also be used in organic cheese making

Microbial coagulants, nowadays mainly originating from Rhizomucor miehei, are animal rennet substitutes used for almost 40 years

These enzymes do, however, differ in molecular structure and in proteolytic activity, and there are still reports that cheese yield and cheese quality is negatively affected Chymosin from recombinant micro-organisms has, because of its unvarying composition and its specific action, some advantages in industrial cheese making but, in some regions, lacks acceptance by the consumer