Introduction
Rennet is an aspartate protease first discovered in the stomach of weaned calves, which can specifically cleave milk κ- The peptide bond between Phe105 Met106 of casein breaks the casein micelles, causing milk to coagulate. The coagulation and protein hydrolysis abilities of rennet make it a key enzyme in cheese production, forming texture and special flavors. It is widely used in the production of cheese and yogurt.
Mammalian gastric juice contains rennet, which can cause proteins in milk to condense into cheese, which is easily digested by various protein enzymes. Chymosin only improves the efficiency of enzymes and is not actually counted as an enzyme. Animals other than mammals rarely have rennet because they do not consume milk.
Chymosin is an essential preparation for cheese production, accounting for 15.5% of the total enzyme production value. The main source is the gastric mucosa of weaned calves.
1. Source
Animal derived rennet: rennet originated from the fourth gastric mucosa of calves and was subsequently extracted from the stomachs of other animals. However, due to the instability of animal derived rennet sources and considerations of race, beliefs, and other factors, researchers have shifted their research focus to microbial and plant derived rennet.
Plant derived rennet: Many plants contain a wide range of proteases that can coagulate milk. At present, there are several types of proteases that have been extensively studied, including papain, ficin, bromelain, gingeroprotease, acacia protease, artichoke protease, etc.
Microbial derived rennet: Microorganisms have the advantages of short growth cycle, high yield, limited time and space, low production cost, convenient extraction, and high economic benefits, making it the most promising development direction for rennet sources at present. At present, more than 40 types of microorganisms have been found to produce certain vitality of rennet, mainly fungi, actinomycetes, bacteria, etc.
2. Charactics
The solubility of rennet is influenced by pH value, temperature, and solution ion strength. Soluble in a NaCl solution with a pH of 5.5 and a concentration of 1mol/L, the crystal structure of rennet is relatively stable at 25 ℃, and its solubility increases with increasing ion strength. The isoelectric point of rennet is pH 4.5, and it is most stable at pH 5.3-6.3. However, its activity decreases due to self degradation at pH 3-4. Under alkaline conditions, irreversible conformational changes occur, leading to a decrease in activity.
3. Type
There are three states of rennet: liquid, powder, and tablet. Traditionally, rennet was extracted from the fourth stomach of calves to produce rennet. With the progress of rennet research, its source is constantly expanding, and now it can be classified into three categories: animal rennet, which comes from the stomach of cows, pigs, and sheep; Plant derived rennet, derived from fig tree sap and pineapple fruit; Microbial rennet, derived from fungi and yeast. The coagulase derived from mold has been widely used in the production of Danish cheese.
4.Coagulation mechanism
The coagulation effect of rennet is divided into two steps: the first step is the enzyme specific hydrolysis of milk κ- The peptide bond between Phe105 Met106 of casein polypeptide chain forms a stable accessory κ- Casein and hydrophilic glycopeptides; The second step is to take the overall κ- When about 80% of casein is hydrolyzed, it forms clumps or solidified milk through chemical bonds formed between casein particles in the presence of calcium ions. This is due to κ- Casein molecules are located on the surface of casein micelles, and subunits are connected together through hydrophobic bonds and colloidal calcium phosphate interactions.
5.Function
Promote the coagulation of raw milk and provide conditions for the discharge of whey.
6. Vitality
The number of milliliters that can coagulate raw milk in 40 minutes with 1 milliliter of rennet solution or 1 gram of dry powder under 35 ℃ conditions. The activity of standard liquid rennet is usually between 12000 and 15000, while powdered rennet is 10 times higher than the former, at 120000 to 150000.
7. Influencing factor
(1) PH: In acidic environments, the activity of rennet is strongest, and any slight change in the acidity of the raw milk can significantly affect the activity of rennet. Most of the chymotrypsin activity comes from trypsin, while a small portion comes from bovine pepsin (although the active ingredient in porcine chymotrypsin is porcine pepsin). The optimal pH for trypsin is 5.4, while the optimal pH for pepsin is lower than that for trypsin.
(2) Temperature: The optimal temperature for rennet is 42 ℃. (At 55-60 ℃, the enzyme itself is destroyed) because the milk temperature significantly affects the coagulation rate. When the milk temperature is 30 ℃, the coagulation time of raw milk is 2-3 times that of 42 ℃. However, in actual cheese production, the milk temperature is usually maintained at 30-33 ℃. Firstly, considering the optimal temperature for lactic acid bacteria (for example, the optimal temperature for Streptococcus is around 30 ℃ and cannot exceed 40 ℃); The second reason is that the solidification rate of the clot is too fast at higher milk temperatures, making subsequent cutting more difficult.
(3) Ca2+concentration: Casein converted by rennet can only coagulate when free calcium ions are present in the raw milk. Therefore, the concentration of calcium ions will affect the coagulation time, coagulation hardness, and whey excretion.
8. Coagulation process
There are three types of casein in raw milk: α S-casein β- Casein and К- Casein, the first two are easily affected by Ca+2 to form precipitates, while the latter not only stabilizes but also has the effect of inhibiting the precipitation of the former. The coagulation of raw milk by rennet is divided into two stages: first К- Casein breaks down into by-products К- Casein; Secondly, the deputy К- Casein and α S-Casein and β- Casein precipitates under the action of Ca+2.
9. Additive amount
The usual dosage is 100kg of raw milk with 20-40 milliliters of rennet solution added. The coagulation time is inversely proportional to the amount of rennet used. If the amount is doubled, the coagulation time is halved.
The excretion of whey is not affected by the amount of rennet, and experiments have shown that the amount of rennet is not related to the moisture content and pH of cheese after 24 hours. However, the amount used affects the ripening of cheese. If the amount is large, the breakdown of protein will accelerate during the ripening period; At the same time, due to the breakdown of protein by rennet to produce bitter peptides, a large amount will also increase the bitterness of cheese.
The determination of curd strength is carried out using the Arima method. 5ml, 100g/L of skimmed milk is taken and kept at 35 ℃ for 5 minutes. Then, 0.5ml, lg/L of papaya curd enzyme solution is added and quickly mixed evenly. The time T (s) from adding the enzyme solution to coagulation is accurately measured. The enzyme amount of 40min coagulated 1ml, 100g/L of skimmed milk is defined as one Soxhlet unit. Coagulation strength=(2400/T) x (5/0.5) x D (dilution ratio).
