Isolation,Identification,Fermentation And Application Of A Yak Milk Preferred Milk-clotting Enzyme

Chymosin (milk-clotting enzyme; EC 3.4.23.4) is a neonatal gastric proteinase and is of commercial importance in cheese-making industry. It belongs to the aspartic proteinase family or serine proteinase family. Milk-clotting enzyme (MCE) is widely distributed in many organisms and tissues with different physiological properties and functions. MCE can cause the coagulation of milk, and plays an important role during cheese maturation as well.As one of the most remarkable domesticated animals inhabiting altitudes ranging from 2500 to 5500 meters, yak has survived in the tough environments. Feeding on energetically saturated forage in the uncontaminated environment, yak provides organic food materials (milk, meat and fat). Tibetans have made yak milk into dairy products, in which yak cheese is the main and the most popular one. Yak milk is different from cow milk in its high content of total proteins, total caseins and the proportion of individual caseins, particularly high amount of β-casein. In addition, the processing of yak cheese requires neutral pH, from 6.0 to 8.0, and a high temperature, over 65℃, to reduce microbial contamination. As such, the traditional chymosin, which only shows high specificity for κ-casein, and is stable only up to 50℃ and pH 6.0, is of limited use in processing and production of yak cheese. In light of the high nutritional value of yak cheese and its unique position in Tibetan dietary, it is urgent to develop new sources of MCE with strong yak milk coagulation activity at 50℃ and neutral pH.For this reason, screening and researching of yak milk preferred milk-clotting enzyme were carried out. The main results of the dissertation are as follows:1. One wild-type B. subtilis named as QL-2 has been isolated and obtained from aged Qula in our previous work. This strain isolated from Tibet plateau was found to be a novel commercial source of MCE because of its high milk-clotting activity (MCA), short-period fermentation, and especially its substrate specificity. However, the production of MCE by this strain is still far from the need of industrial application. In order to improve its production performance, a mutant, B. subtilis YB-3 was screened following mutagenesis of the wild-type strain B. subtilis QL-2. After treatment with ultraviolet radiation, the MCE of B. subtilis YB-3 (200 SU/ml) was 2.5 times higher than that of the original strain (80 SU/ml).2. To further improve its MCE production, the culture medium was screened and optimized using the statistical design techniques. One-factor-at-a-time method was used to investigate the effects of carbon sources, nitrogen sources and initial pH on MCE production. The Plackett-Burman design was introduced to find out the most significant nutrients which contributed to the yield of MCE. Finally, response surface methodology (RSM) was applied to formulate a mathematical model to identify the optimum concentrations of the significant nutrients for MCE production. According to the optimized composition of 6.37 g/L beef extract, 11.07 g/L casein,1.14 g/L MgSO4, MCE of 1000 ± 3.52 SU/ml was obtained by three repeated experiments, which was very close to the predicted value of 1009.14 SU/ml. The pH-control batch fermentation using optimized culture medium in a 10-L bioreactor allowed a significant increase in biomass (50%) and MCE production (20%). The improvement in MCE production after optimization process can be considered adequate for large-scale applications, and the clotting activity of the MCE for their use in industrial cheese making.3. A milk-clotting enzyme named YS-1 was purified from a B. subtilis YB-3. The enzyme YS-1 was identified as a metalloproteinase. SDS-PAGE analysis of the purified enzyme gave a molecular weight of 42 kDa. A database search (the online Mascot MS-MS Ions search database from Matrix Science) identified 8 peptides that represent 44.67%coverage of the metalloproteinase sequence of Bacillus pumilus (gi56405351), which indicated that MCE YS-1 from B. subtilis YB-3 is a member of the metalloproteinase family. Analysis of the nucleotide sequence and its flanking DNA regions revealed the presence of an open reading frame (ORF) of 1563 bp, and the deduced amino acid sequence shows a pre-pro-protease of 521 amino acids. Sequence alignment with protein databank shows that the metalloproteinase from B. subtilis YB-3 has (516/521) 98.24%homology with metalloproteinases of Bacillus pumilus. When yak milk was a substrate, MCA of MCE YS-1 from B. subtilis YB-3 was 115%higher than MCA of metalloproteinase from B. subtilis. 4. The pH stability study showed that MCE YS-1 is highly stable in the wide pH range of 5.0 to 9.0. The enzyme retained about 90%activity in the pH range of 6.0 to 8.0. The thermal stability study showed that MCE YS-1 is highly stable in the wide temperature range of 20 to 80 ℃. The pH profile and the temperature profile showed that MCE YS-1 was most stable and active at wide pH range and tempreture range, indicating that MCE YS-1 would be quite suitable to be employed in yak cheese-making process.5. The substrate specificity of purified MCE YS-1 was further studied. MCE YS-1 and Rhizomucorpusillus enzyme digested κ-casein into a short peptide (MW 14,000), but in the case of MCE YS-1 an additional degradation product (MW 10,000) was generated. The results show that Rhizomucor pusillus enzyme appeared to be ineffective in digesting a-casein and β-casein. In contrast, MCE YS-1 completely degraded a-casein within 10 min, generating nonspecific hydrolysis products; similarly, MCE YS-1 completely degraded β-casein within 5 min, generating small peptides of the size of MW 13,000,16,000 and 20,000 Da. MCE YS-1 exhibited high substrate specificity to P-casein and yak milk casein, and it could led to a 75 percent more rapid coagulation of yak milk than that of cow milk, due to high β-casein content in yak milk.6. Miniature cheeses (yak milk cheese and cow milk cheese) were manufactured with MCE YS-1 from B. subtilis YB-3, and analyzed by a taste panel, who found acceptable both cheeses.In summary, MCE YS-1 from B. subtilis YB-3 might be appropriate for cheese making, especially for yak cheese making.