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Send EmailChymosin, Rennin, Rennet, Milk-Clotting Enzyme, Aspartic Protease, 9001-98-3
Chymosin / Rennin / Rennet / Milk-Clotting Enzyme / Aspartic Protease
CAS Number: 9001-98-3
EC Number: 232-645-0
Enzyme Class: EC 3.4.23.4
| Parameter | Information |
|---|---|
| Product Name | Chymosin |
| Other Names | Rennin, Rennet, Milk-Clotting Enzyme, Aspartic Protease, Calf Rennet, Microbial Rennet |
| CAS Number | 9001-98-3 |
| EC Number (EINECS) | 232-645-0 |
| Enzyme Class | Hydrolase (EC 3.4.23.4) |
| Systematic Name | Aspartic protease (chymosin) |
| Chemical Structure | Protein (polypeptide chain), globular structure, aspartic protease |
| Source | Animal (calf stomach mucosa), Microbial (Aspergillus niger, Aspergillus oryzae, Rhizomucor miehei, Rhizomucor pusillus), Recombinant (genetically modified microorganisms) |
| Appearance | Light brown to off-white powder or liquid formulation |
| Solubility | Readily soluble in water |
| Molecular Weight | 30,000 - 40,000 Da (chymosin); 30,000 - 45,000 Da (microbial rennet) |
| Protease Class | Aspartic protease (acidic protease) |
Description:
Chymosin (also known as rennin or rennet) is an aspartic protease enzyme that clots milk proteins (especially casein). Chymosin initiates clotting by hydrolyzing soluble κ-casein in milk. This reaction separates milk into curds and whey. In cheese production, it is used to clot milk, forming the structure and texture of cheese. Traditionally obtained from animal sources (calf stomach mucosa), microbial and recombinant chymosins are now widely used. Recombinant chymosin is chemically identical to animal chymosin and is preferred for vegetarian cheese production.
| Type | Name |
|---|---|
| Common Names | Chymosin, Rennin, Rennet, Milk-Clotting Enzyme, Calf Rennet |
| Enzymatic Names | Aspartic protease, Milk-clotting enzyme, Coagulase |
| EC Number | 3.4.23.4 |
| CAS Number | 9001-98-3 |
| HS Code | 3507.10.00 |
| WGK Germany | 1 (low hazard to water) |
3.1. Molecular Structure
Chymosin is an aspartic protease consisting of two main domains. It contains two lobes: an N-terminal and a C-terminal lobe. The active site is located between two aspartic acid residues (Asp-32 and Asp-215) in both lobes. The enzyme catalyzes the coagulation of milk proteins (especially κ-casein). Animal chymosin contains approximately 323 amino acids and has a molecular weight of 30,000-40,000 Da. Microbial rennets are aspartic proteases similar to chymosin but with different amino acid sequences.
3.2. Catalytic Mechanism
Chymosin initiates coagulation by hydrolyzing the Phe105-Met106 bond of κ-casein in milk:
Substrate Binding: The κ-casein molecule binds to the active site of the enzyme.
Catalytic Attack: Two aspartic acid residues (Asp-32 and Asp-215) in the active site activate a water molecule, which attacks the Phe105-Met106 bond of κ-casein.
Bond Cleavage: The Phe105-Met106 bond is hydrolyzed, splitting κ-casein into two parts: para-casein and glycomacropeptide (GMP).
Coagulation: Para-casein interacts with other casein molecules in the presence of calcium ions, forming a clot. GMP passes into the whey.
3.3. Molecular Weight
30,000 - 40,000 Da (chymosin); 30,000 - 45,000 Da (microbial rennet)
3.4. Basic Structural Properties
| Property | Description |
|---|---|
| Catalytic Domain | Aspartic protease (two aspartic acid residues) |
| Active Site | Asp-32 and Asp-215 |
| Substrate Specificity | κ-Casein (Phe105-Met106 bond) |
| Solubility | Readily soluble in water |
| Isoelectric Point (pI) | ~4.5 - 5.0 |
| Thermal Stability | Stable at 37°C; inactivated above 45°C |
| Property | Value |
|---|---|
| Appearance | Light brown to off-white powder, granule, or liquid |
| Odor | Slight fermented or characteristic (enzymatic) odor |
| Color (Powder) | Light brown to off-white |
| Molecular Weight | 30,000 - 40,000 Da (chymosin); 30,000 - 45,000 Da (microbial) |
| Isoelectric Point (pI) | ~4.5 - 5.0 |
| Solubility in Water | Readily soluble |
| Solubility in Ethanol | Insoluble (precipitated) |
| Solubility in Acetone | Insoluble (precipitated) |
| Density (Powder) | ~0.5 - 0.7 g/cm³ (uncompacted) |
| Loss on Drying (Powder) | ≤ 8.0 % |
| Optimum pH | 5.5 – 6.5 |
| pH Stability | 5.0 – 7.0 |
| Optimum Temperature | 30 – 37°C |
| Temperature Stability | Stable at 37°C; activity decreases above 42°C; rapidly inactivated at 45°C |
| Calcium Requirement | Calcium (Ca²⁺) ions are required for coagulation |
| Activity Inhibitors | Heavy metals (Hg²⁺, Cu²⁺), SDS, Pepstatin A (aspartic protease inhibitor), EDTA |
| Activity Activators | Calcium (Ca²⁺) ions, Sodium (Na⁺) ions |
| Parameter | Information |
|---|---|
| EC Number | 3.4.23.4 |
| Systematic Name | Aspartic protease (chymosin) |
| Reaction | κ-Casein + H₂O → Para-casein + Glycomacropeptide (GMP) |
| Substrate Specificity | κ-Casein (Phe105-Met106 bond), α-casein (partially), β-casein (partially) |
| Cleavage Type | Endopeptidase |
| Bond Specificity | Phenylalanine-Methionine bond (Phe-Met) |
| Product Type | Para-casein (forms clot), Glycomacropeptide (GMP) |
| Clotting Unit (U) | 1 U = amount of enzyme that clots a specific amount of milk in 1 minute at pH 6.3 and 35°C (IMCU - International Milk Clotting Unit) |
| Specific Activity | 50 - 500 IMCU/mg (depending on purity and source) |
| Activity Assay | Milk clotting test (Berridge method), Spectrophotometric method (casein substrate) |
| Clotting Time | Typically 5-30 minutes under standard conditions (35°C, pH 6.3); varies with enzyme dosage, temperature, and pH |
| Milk Type | Cow milk, goat milk, sheep milk, buffalo milk |
Chymosin catalyzes milk coagulation through a mechanism involving the following steps:
6.1. Substrate Binding
The κ-casein molecule (the soluble casein fraction in milk) binds to the active site of the enzyme. κ-Casein acts as a "protector" that stabilizes other casein molecules in milk and prevents coagulation.
6.2. Enzymatic Hydrolysis
Chymosin hydrolyzes the Phe105-Met106 bond of the κ-casein molecule. This splits κ-casein into two parts:
Para-casein (κ-casein amino acids 1-105): Participates in coagulation in the presence of calcium.
Glycomacropeptide (GMP, κ-casein amino acids 106-169): Soluble and passes into the whey.
6.3. Coagulation (Primary and Secondary Stages)
Primary Stage (Enzymatic): Hydrolysis of κ-casein removes the protective effect.
Secondary Stage (Chemical): Para-casein molecules interact with each other in the presence of calcium ions (Ca²⁺), forming a three-dimensional network. This network causes milk coagulation and curd formation.
6.4. Curd Syneresis
The formed curd contracts over time, expelling whey. This process determines the moisture content and texture of the cheese. Cutting and processing the curd affects the final product properties.
7.1. κ-Casein Hydrolysis (Main Reaction)
κ-Casein + H₂O → Para-casein + Glycomacropeptide (GMP)
7.2. Coagulation Reaction
Para-casein + Ca²⁺ → Para-casein Calcium Complex (Clot)
7.3. Whey Formation
Milk + Chymosin → Curd (Para-casein + Ca²⁺) + Whey (GMP + Lactose + Minerals)
7.4. Enzyme Inactivation (Thermal)
Chymosin (active) → Chymosin (inactive) (≥45°C, 10-15 minutes)
7.5. Enzyme Inhibition (Pepstatin A)
Chymosin + Pepstatin A → Chymosin-Pepstatin A Complex (inactive)
| Type | Source | Optimum pH | Optimum Temp. | Clotting Activity | Proteolytic Activity | Application Area |
|---|---|---|---|---|---|---|
| Animal Chymosin | Calf stomach | 5.5-6.0 | 35-37°C | High | Low | Traditional cheese |
| Microbial Rennet | Rhizomucor miehei | 5.5-6.5 | 35-40°C | Medium-High | Medium | Cheese production |
| Microbial Rennet | Aspergillus niger | 4.5-5.5 | 30-35°C | Medium | High | Cheese production |
| Recombinant Chymosin | Genetically modified microorganisms (E. coli, Pichia pastoris) | 5.5-6.5 | 30-37°C | High | Low | Vegetarian cheese |
| Animal Rennet (Mixture) | Calf, lamb, goat stomach | 5.5-6.5 | 30-37°C | Medium-High | Medium | Traditional cheese |
| Plant Rennet | Fig, papaya (papain) | 5.0-6.0 | 30-35°C | Low-Medium | High | Specialty cheeses |
9.1. Cheese Production
| Application | Function | Typical Usage Condition |
|---|---|---|
| Milk Clotting | Clots milk, forming cheese curd and whey. | 0.01-0.05% (milk weight); 30-37°C; pH 6.2-6.6; 30-60 minutes |
| Cheese Texture | Determines the structure, texture, and elasticity of cheese. | Depends on milk type and cheese variety |
| Cheese Yield | Increases cheese yield by minimizing casein loss. | Optimum clotting conditions |
| Cheese Ripening | Contributes to flavor development through proteolytic activity during cheese ripening. | Depends on cheese variety |
Example Recipe – Cheese Clotting (Cow Milk):
Cow milk: 1000 L
Chymosin (Recombinant): 10-50 g (depending on activity)
Calcium chloride (CaCl₂): 10-20 g (optional)
Application temperature: 30-35°C
Clotting time: 30-60 minutes
Heat milk to 30-35°C. Optionally add calcium chloride (to optimize milk's calcium content). Dissolve chymosin in a small amount of cold water and add to milk. Stir and leave to clot for the specified time. Check clot formation and cut the curd.
9.2. Cheese Varieties
| Cheese Variety | Chymosin Type | Clotting Conditions | Characteristics |
|---|---|---|---|
| Hard Cheese (Cheddar, Parmesan) | Recombinant / Animal | 30-32°C; 45-60 minutes | Hard texture, long ripening |
| Soft Cheese (Camembert, Brie) | Microbial / Recombinant | 30-35°C; 30-45 minutes | Soft texture, short ripening |
| Semi-Hard Cheese (Gouda, Edam) | Recombinant / Animal | 30-32°C; 40-50 minutes | Medium-hard texture |
| Fresh Cheese (Mozzarella, Ricotta) | Microbial / Recombinant | 35-37°C; 20-30 minutes | Elastic, fresh texture |
| Goat Cheese | Animal / Recombinant | 30-35°C; 30-60 minutes | Soft, creamy texture |
9.3. Dairy Products and Protein Coagulation
| Application | Function | Typical Usage Condition |
|---|---|---|
| Casein Production | Coagulation for casein isolation from milk. | 30-35°C; pH 6.2-6.6 |
| Milk Protein Concentrates | Concentration of milk proteins through coagulation. | 30-35°C; pH 6.2-6.6 |
| Yogurt (Texture Improvement) | Casein modification to improve yogurt consistency. | 30-35°C; pH 5.5-6.0 |
| Whey Protein | Coagulation of whey proteins. | 30-35°C; pH 6.0-6.5 |
9.4. Pharmaceutical and Biotechnology
| Application | Function | Typical Usage Condition |
|---|---|---|
| Protein Modification | Enzymatic modification of specific proteins. | Optimized conditions |
| Biocatalysis | Biocatalytic reactions as aspartic protease. | 30-37°C; pH 5.5-6.5 |
| Protein Hydrolysates | Production of casein hydrolysates. | 30-37°C; pH 5.5-6.5 |
9.5. Other Applications
| Application | Function |
|---|---|
| Research and Development | Dairy product and protein research. |
| Quality Control | Testing clotting quality of milk and dairy products. |
| Food Analysis | Casein determination and protein analysis. |
| Enzyme Standardization | Standardization of clotting activity (IMCU). |
10.1. Animal Chymosin vs Recombinant Chymosin
| Property | Animal Chymosin | Recombinant Chymosin |
|---|---|---|
| Advantages | Traditional; natural source; well-characterized | Vegetarian; consistent quality; high purity; sustainable; no animal content |
| Disadvantages | Animal source; variable quality; limited availability; more expensive | Chemically identical; may not be considered "natural" by some consumers |
| Selection | Preferred for traditional cheese production and "natural" labeling | Preferred for vegetarian cheese, industrial production, and consistent quality |
10.2. Chymosin vs Microbial Rennet
| Property | Chymosin | Microbial Rennet |
|---|---|---|
| Advantages | High clotting/proteolytic activity ratio; specific; less bitter peptide formation | Lower cost; high availability; long shelf life |
| Disadvantages | Higher cost; animal source (traditional) | Higher proteolytic activity; risk of bitter peptide formation; less specific |
| Selection | Preferred for high-quality, long-ripened cheeses | Preferred for soft and semi-hard cheeses, low-cost production |
10.3. Chymosin vs Plant Rennet (Papain, Ficin, Bromelain)
| Property | Chymosin | Plant Rennet |
|---|---|---|
| Advantages | Specific; high clotting activity; low bitterness; consistent quality | Plant-based; vegetarian; natural; traditional |
| Disadvantages | Animal or recombinant source; high cost | Low clotting activity; high proteolytic activity; bitter peptide formation; variable quality |
| Selection | Preferred for industrial cheese production | Preferred for specialty cheeses, small-scale production, and traditional methods |
10.4. Chymosin vs Acid Coagulation
| Property | Chymosin | Acid Coagulation |
|---|---|---|
| Advantages | Specific; controlled coagulation; soft curd; high yield | Simple; low cost; fast |
| Disadvantages | High cost; temperature and pH sensitivity | Hard curd; acidic taste; low yield; less controlled |
| Selection | Preferred for cheese production | Preferred for fresh cheeses, casein production, and laboratory applications |
11.1. Acute Toxicity
| Parameter | Value |
|---|---|
| Acute Oral Toxicity (LD50, Rat) | Practically non-toxic (> 5,000 mg/kg) |
| Acute Dermal Toxicity | Low risk (> 2,000 mg/kg) |
| Acute Inhalation Toxicity | Powder form may cause respiratory sensitization |
| Eye Irritation | Mild to moderate irritant |
| Skin Irritation | Mild irritant; some forms may be sensitizing |
| Respiratory Sensitization | May cause occupational asthma upon repeated exposure (enzyme dust) |
| Metabolism | Enzyme protein is digested in the gastrointestinal tract and metabolized to amino acids |
| Allergenicity | Caution for individuals with milk allergy (risk of milk protein contamination) |
11.2. Regulatory Status
| Parameter | Information |
|---|---|
| FDA | GRAS (Generally Recognized As Safe) - 21 CFR 184.1685 |
| EFSA | Approved as a food enzyme; safety assessment completed |
| JECFA | Acceptable as a food additive; specifications established |
| Kosher Certification | Available for microbial and recombinant forms |
| Halal Certification | Available for microbial and recombinant forms |
| Vegetarian | Recombinant and microbial forms are vegetarian; animal form is not |
| Vegan | Recombinant and microbial forms are vegan; animal form is not |
| Hazard Class | Category | H-Statement |
|---|---|---|
| Respiratory Sensitization | Category 1 | H334: May cause allergy or asthma symptoms if inhaled |
| Eye Irritation | Category 2 | H319: Causes serious eye irritation |
| Specific Target Organ Toxicity (STOT SE) | Category 3 | H335: May cause respiratory irritation |
Signal Word: Danger
Hazard Pictograms: GHS07 (Exclamation mark), GHS08 (Health Hazard)
Hazard Statements (H-Codes):
H334: May cause allergy or asthma symptoms if inhaled
H319: Causes serious eye irritation
H335: May cause respiratory irritation
Safety Statements (Historical):
| Code | Statement |
|---|---|
| S22 | Do not breathe dust. |
| S24/25 | Avoid contact with skin and eyes. |
| S26 | In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. |
| S36/37/39 | Wear suitable protective clothing, gloves, and eye/face protection. |
| Code | Statement |
|---|---|
| P260 | Do not breathe dust. |
| P264 | Wash hands thoroughly after handling. |
| P271 | Use only outdoors or in a well-ventilated area. |
| P280 | Wear protective gloves/protective clothing/eye protection/face protection. |
| P302+P352 | IF ON SKIN: Wash with plenty of soap and water. |
| P304+P340 | IF INHALED: Remove person to fresh air and keep comfortable for breathing. |
| P305+P351+P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present. |
| P337+P313 | If eye irritation persists: Get medical advice/attention. |
| P342+P311 | If experiencing respiratory symptoms: Call a POISON CENTER/doctor. |
| P403+P233 | Store in a well-ventilated place. Keep container tightly closed. |
| P501 | Dispose of contents/container in accordance with local regulations. |
| Exposure Route | Action to Take |
|---|---|
| Inhalation | Remove from dusty environment. Move to fresh air. If breathing difficulty occurs, seek medical attention. If symptoms persist, consult a doctor. |
| Skin Contact | Wash with plenty of soap and water. Remove contaminated clothing. If redness or irritation occurs, seek medical attention. |
| Eye Contact | Rinse cautiously with water for at least 15 minutes. Remove contact lenses if present. If irritation persists, seek medical attention. |
| Ingestion | Rinse mouth. Drink plenty of water. Do NOT induce vomiting. If unconscious, do not give anything by mouth. Seek medical attention. |
| Note | In case of allergic reaction (respiratory difficulty, skin rash), seek immediate medical attention. Caution for individuals with milk allergy. |
| Parameter | Information |
|---|---|
| Fire Hazard | Organic dust may pose a fire hazard; dust clouds may be explosive in air |
| Suitable Extinguishing Media | Water spray, CO₂, dry chemical powder, alcohol-resistant foam |
| Specific Hazards | Thermal decomposition produces toxic gases (CO, CO₂, nitrogen oxides, sulfur oxides) |
| Protective Equipment | Self-contained breathing apparatus (SCBA), full protective clothing |
| Special Precautions | Avoid dust cloud formation. Cool containers with water spray. |
| Explosion Risks | Dust/air mixtures may be explosive. Provide ventilation and ignition source control. |
| Parameter | Information |
|---|---|
| Personal Protection | Protective goggles, chemical-resistant gloves, dust mask (P2/P3), coverall |
| Ventilation | Increase ventilation; use local exhaust; prevent dust from dispersing into air |
| Containment | Absorb with inert material (sand, vermiculite); prevent dust spread |
| Cleaning Methods | Collect with absorbent material. Use vacuum cleaner to prevent dust formation. Avoid sweeping as it may raise dust; wet cleaning is preferred. |
| Environmental Precautions | Prevent entry into sewers, drains, and water bodies. Prevent contamination of soil and surface water. |
| Waste Disposal | Dispose of in accordance with local regulations. Collect contaminated materials in closed containers. |
| Parameter | Information |
|---|---|
| Storage Conditions | Store in a cool, dry, well-ventilated area. Protect from direct sunlight. Protect from moisture. |
| Temperature | Recommended: 4-25°C. For long-term storage, 4°C (refrigeration) is recommended. Avoid freezing. |
| Container Requirements | Tightly closed, moisture-resistant containers. Use HDPE, PP, or aluminum containers. |
| Materials to Avoid | High temperature (>40°C), high humidity, direct sunlight, strong oxidizers, heavy metals (Hg²⁺, Cu²⁺). |
| Material Compatibility | HDPE, PP, aluminum, stainless steel. Avoid: Copper, zinc, iron (may cause activity loss). |
| Shelf Life | 12-24 months (in unopened original packaging, under recommended storage conditions) |
| Stability Note | Hygroscopic; absorbs moisture. Store in tightly closed containers to prevent activity loss. Enzyme activity declines at elevated temperatures. Calcium presence may improve stability. |
| Packaging Type | Quantity | Material |
|---|---|---|
| Powder Form | 1 kg, 5 kg, 10 kg, 20 kg, 25 kg | Aluminum foil bag / HDPE drum / Multi-layer kraft bag |
| Liquid Form | 1 L, 5 L, 10 L, 20 L, 200 L | HDPE bottle / HDPE drum / IBC tank |
| Bulk (Powder) | 500 – 1000 kg | Big bag / FIBC |
| Bulk (Liquid) | 1000 L | IBC Tank |
| Small Containers | 50 g, 100 g, 500 g | Aluminum foil bag / HDPE bottle |
| Parameter | Information |
|---|---|
| UN Number | Not applicable (not classified as dangerous goods) |
| Hazard Class | Not classified as dangerous for transport |
| Packing Group | Not applicable |
| ADR/RID | Not regulated |
| IMDG Code | Not regulated |
| IATA (Air) | Not regulated (consult airline for powder form) |
| Marine Pollutant | No |
| Transport Temperature | Ambient temperature; protect from moisture and direct sunlight. |
| Special Transport Conditions | Prevent dust dispersion from powder form. Prevent leakage from liquid form. |
| Parameter | Information |
|---|---|
| Aquatic Toxicity | Low toxicity to aquatic organisms (EC50 > 100 mg/L) |
| Biodegradability | Readily biodegradable (due to protein structure) |
| Bioaccumulation | Low potential (proteins generally do not bioaccumulate) |
| Mobility in Soil | Moderate; protein structure degrades in soil |
| Vapor Behavior | Non-volatile (powder form) / Low vapor pressure (liquid form) |
| WGK Germany | 1 (low hazard to water) |
| Ecotoxicology | Low toxicity to soil organisms; does not affect beneficial microorganisms |
| Waste Disposal | Incineration or disposal in accordance with local regulations; do not discharge into sewers |
| Region / Authority | Status |
|---|---|
| European Union (EFSA) | Approved as a food enzyme; safety assessment completed |
| European Union (REACH) | Registered; approved for use as a food enzyme |
| USA (FDA) | GRAS (21 CFR 184.1685) |
| Turkey | Approved as a food enzyme; complies with Turkish Food Codex |
| JECFA | Acceptable as a food additive; specifications established |
| Kosher Certification | Available for microbial and recombinant forms |
| Halal Certification | Available for microbial and recombinant forms |
| Vegetarian | Recombinant and microbial forms are vegetarian |
| Vegan | Recombinant and microbial forms are vegan |
Q1: Is chymosin safe?
A1: Yes. Chymosin is classified as GRAS (Generally Recognized As Safe) by the FDA and approved as a food enzyme by EFSA. It has been safely used in cheese production for many years. However, the powder form may cause allergic reactions when inhaled, so appropriate personal protective equipment should be used.
Q2: What is the difference between chymosin and rennet?
A2: Chymosin is the main enzyme that clots milk. Rennet is traditionally obtained from calf stomach and contains chymosin and other proteases (pepsin). Today, the term "rennet" is used as a general term for all milk-clotting enzymes. Recombinant chymosin is the active component of animal rennet.
Q3: Is chymosin suitable for vegetarian products?
A3: Recombinant chymosin (produced by microbial fermentation) and microbial rennet are vegetarian and can be halal/kosher certified. Animal-derived chymosin (from calf stomach) is not vegetarian. Relevant certificates can be obtained from manufacturers.
Q4: Why is chymosin used in cheese production?
A4: Chymosin hydrolyzes κ-casein in milk, causing milk coagulation. This initiates the formation of cheese curd and whey. Chymosin is the most important enzyme that determines the structure, texture, and ripening properties of cheese. Different chymosin types and dosages are used for different cheese varieties.
Q5: Is chymosin suitable for vegan products?
A5: Recombinant chymosin (produced by microbial fermentation) is vegan. Animal-derived chymosin (from calf stomach) is not vegan. Recombinant chymosin is produced using genetically modified microorganisms and contains no animal ingredients. It is preferred for vegetarian and vegan cheese production.
Q6: How should chymosin be stored?
A6: Store in a cool (preferably 4-25°C), dry, well-ventilated area in tightly closed containers. Moisture, high temperature (>40°C), and direct sunlight cause activity loss. Contact with heavy metals (copper, mercury) should be avoided. Long-term storage at 4°C is recommended.
Q7: What are the optimum working conditions for chymosin?
A7: The optimum pH of chymosin is between 5.5-6.5, and the optimum temperature is between 30-37°C. Calcium ions (Ca²⁺) are required for coagulation. The enzyme is stable up to 37°C, activity decreases above 42°C, and it is rapidly inactivated at 45°C. Milk pH, temperature, and calcium content directly affect clotting time.
Q8: How does chymosin affect cheese yield?
A8: Chymosin increases cheese yield by minimizing casein loss. Under optimum clotting conditions (correct temperature, pH, calcium, and enzyme dosage), all casein is converted to curd. Excessive enzyme use or incorrect conditions can cause casein loss and low yield. Recombinant chymosin provides yield increase due to its high purity.
Q9: What is the difference between recombinant chymosin and animal chymosin?
A9: Recombinant chymosin is produced using genetically modified microorganisms (E. coli, Pichia pastoris, Aspergillus niger) and is chemically identical to animal chymosin. Animal chymosin is obtained from calf stomach. Recombinant chymosin is vegetarian and vegan as it is not animal-derived, has more consistent quality, and is sustainable.
Q10: What are the effects of inhaling chymosin powder?
A10: Chymosin powder, when inhaled, can cause allergic reactions, asthma-like symptoms, and respiratory tract irritation. Occupational asthma can develop upon repeated exposure to enzyme dust in cheese production facilities. Therefore, a dust mask (P2/P3) must always be used when handling the powder form, and local exhaust ventilation should be provided. Caution for individuals with milk allergy.
| Property | Value |
|---|---|
| CAS Number | 9001-98-3 |
| EC Number | 232-645-0 |
| EC Class | 3.4.23.4 |
| Systematic Name | Aspartic protease (chymosin) |
| Appearance | Light brown powder or liquid |
| Odor | Slight fermented / characteristic |
| Molecular Weight | 30,000 - 40,000 Da |
| Optimum pH | 5.5 – 6.5 |
| Optimum Temperature | 30 – 37°C |
| Solubility in Water | Readily soluble |
| Substrate | κ-Casein |
| Product | Para-casein + Glycomacropeptide (GMP) |
| Calcium Requirement | Yes (Ca²⁺) |
| Activity | ≥ 100,000 IMCU/g (powder) / ≥ 5,000 IMCU/mL (liquid) |
| Shelf Life | 12-24 months |
| Primary Uses | Cheese production, milk clotting |
| UN Number | Not applicable |
| WGK Germany | 1 |
CRITICAL WARNINGS:
Dust Control and Respiratory Sensitization: Chymosin powder can cause occupational asthma upon repeated exposure. Use local exhaust ventilation to prevent dust dispersion and always wear a dust mask (P2/P3). Liquid formulations are safer than powder forms.
Calcium Requirement: Chymosin requires calcium ions (Ca²⁺) for coagulation. Milk's natural calcium content may be insufficient; therefore, calcium chloride (CaCl₂) may need to be added. Optimum calcium concentration is ~1.5-2.5 mM.
Activity Loss - Temperature: Chymosin is rapidly and irreversibly inactivated above 45°C. Strictly maintain application temperatures within the optimum range (30-37°C). Temperature control is critical in cheese production.
Activity Loss - pH: Chymosin loses stability below pH 5.0 or above pH 7.0. Maintain application pH within the optimum range (5.5-6.5). Excessively acidic or basic conditions cause permanent enzyme inactivation.
Milk Type and Quality: Different milk types (cow, goat, sheep, buffalo) show different clotting properties. Milk pH, calcium content, and protein composition affect clotting time. Optimum conditions should be determined for each milk type.
Activity Loss - Heavy Metals: Mercury (Hg²⁺), copper (Cu²⁺), and other heavy metals inhibit chymosin. Avoid these metals in equipment that comes into contact with the enzyme. Stainless steel, HDPE, or glass materials are preferred.
Food Grade: Only FDA/EFSA-approved food-grade chymosin meeting specifications should be used in food applications. Industrial grade enzymes may contain impurities and are not suitable for human consumption.
Proteolytic Activity: Chymosin has low proteolytic activity (compared to pepsin). This reduces bitter peptide formation in cheese. Microbial rennets may have higher proteolytic activity. The correct enzyme should be selected according to cheese variety.
BEST PRACTICE RECOMMENDATIONS:
Storage: Store in a cool (4-25°C), dry, well-ventilated area in tightly closed containers. For long-term storage, +4°C (refrigeration) is recommended. Use within a short period after opening.
Handling: Use protective gloves, dust mask (P2/P3), and protective goggles. To prevent dust dispersion, preferably use liquid formulations. Prepare working solutions fresh for each application.
Milk Preparation: Heat milk to optimum temperature (30-37°C). If necessary, add calcium chloride (CaCl₂). Maintain milk pH between 6.2-6.6. Post-pasteurization milk may have altered clotting properties.
Dosage: Calculate the correct dosage according to enzyme activity (IMCU units). Check enzyme activity for each batch. Start with low dosage and adjust based on results. Typical dosage ranges are 0.01-0.05% of milk weight.
Clotting Control: Regularly check clotting time. Optimum clotting time is 30-60 minutes. Evaluate curd consistency and firmness. Continue processing after cutting the curd.
Enzyme Inactivation: Inactivate the enzyme by heat treatment (≥45°C, 10-15 minutes) after clotting is complete. Heat treatments applied during cheese processing will inactivate the enzyme.
Quality Control: Perform activity testing for each batch. Maintain production and handling hygiene to prevent microbiological contamination. Monitor clotting time, curd firmness, and cheese yield for consistent product quality.
Waste Management: Dispose of enzyme and contaminated materials in accordance with local regulations. Do not discharge into sewers. If released into wastewater, inactivate the enzyme by heat treatment before disposal.
This Technical Data Sheet (TDS) is for informational purposes only and is prepared based on available technical data. The user is responsible for determining the suitability of the product for their specific application and for complying with all local, national, and international regulations. For complete safety, storage, handling, transport, waste, and regulatory compliance information, refer to the official Safety Data Sheet (SDS/MSDS) provided by the manufacturer/supplier. Chymosin powder may cause sensitization when inhaled; appropriate protective equipment should be used. Caution for individuals with milk allergy. This document does not replace professional or medical advice.