Identification Of A Novel Milk Clotting Enzyme-Producing Strain Quambalaria Cyanescens And Enzyme Characterization And Application To Cheese Production

Cheese industry consumes the largest amount of raw milk every year. The major biological function of rennet, the key enzyme in cheese production, is its restriction digestion ofκ-casein at Phe105-Met106, leading to solidification of milk. Rennet is an important and widely used enzyme in cheese production, which ranks the second in terms of yield and accounts for 15% of the total enzyme preparation used in cheese production all over the world. Currently, the most widely used rennet is extracted from the fourth compartment of the stomach of calves. With the increase of demand for rennet, more and more calves are being slaughtered for rennet each year, which resulted in the continued decrease of calves and the increasing price of rennet. It’s becoming imperative for humans to find a substitute for rennet with wide variety of resource and lower cost. Microorganism is characterized by short production cycle and easy to culture. It is believed that microbial milk clotting enzyme is the most promising substitute for rennet, the direction for future new milk clotting enzyme development with great potential. The aim of this study is to screen a highly milk clotting enzyme-producing strain. Serial study of this strain was carried out with the purpose to provide some new literature for milk clotting enzyme development. The main results are as follows:1. A strain QY229, which was able to produce an enzyme with high milk-clotting activity, was obtained from red kojic rice. This strain was identified as Q. cyanescens based on morphology and the sequence of ITS-rRNA gene.0.02 mM pepstatin A, a specific inhibitor for aspartic protease, could inhibit the milk-clotting activity of fermentation broth from Q. cyanescens, indicating that milk-clotting enzyme from Q. cyanescens was a aspartic protease.2. After purification by DEAE Cellulose-52 ion-exchange chromatography, Hiprep 16/10 DEAE FF ion-exchange chromatography and Superdex 75 10/300 GL gel filtration, a single chromatographic peak with high milk-clotting activity was obtained. Milk-clotting enzyme was purified with the purification fold of 24.1 and the recovery of 19.1%。Enzymological study showed that this enzyme was stable between 30-40℃, with optimal milk clotting temperature of 40℃. The milk-clotting activity was stable between pH4.5～6.5, with optimal pH 5.0. The activity of milk-clotting enzyme could be increased by Ca2+, Mg2+, Zn2, Mn2+and Fe2+, and the greatest effect was observed in Ca2+treated group; while K+, Na+, Cu2+could inhibit its clotting activity. Casein hydrolytic ability showed calf rennet and milk clotting enzyme from Q. cyanescens wass not significant differences.3. The preparation time for the simple spore suspension was determined as 6 days based on the observation of spore forming process. Fatality rate of Q. cyanescens by ultraviolet mutation and DES was investigated.The mutation time was determined as 240S for UV and 40min for DES, respectively. Mutation in Q. cyanescens QY229 was induced by UV or DES alone, or by UV+DES, or by DES+UV. A mutant strain DU10 with 105.16% higher yield of milk-clotting enzyme than that of original strain was finally obtained. Genetic analysis showed that this strain was genetically stable.4. The medium formula and fermentation conditions were optimized in order to improve the enzyme yieldfrom DU10. After single factor experiments, the methodology of Plackett-Burman designing method was used to screen the key factors rapidly from the related 12 factors, including compositions of the medium, initial pH, rotation speed, culture temperature and culture time etc. The results showed that glucose and rotation speed had a positive effect on the yield of milk-clotting enzyme (p< 0.05), initial pH had a negative effect (p<0.05), and no significant effects was observed for other factors (p>0.05) Then the key factors (glucose, rotation speed and initial pH) and possible interactions among factors were further investigated using Box-Behnken Response Surface Methodology (RSM). Fermentation conditions were optimized by a second-order polynomial equation and given as follows:the culture medium composed of 36g/L glucose, 4g/L peptone,5g/L yeast extract,0.5g/L CaCl2,2.5mg/L MnSO4, Tween-80 0.8g/L, the initial pH 4.6, amount of medium per shake flask 50mL/250mL,1%(V/V),1% inoculum amount(V/V), incubation temperature 30℃, shaking speed 188 r/min, and incubation time 96 h. Under optimal conditions, the yield of milk-clotting enzymes was 2.5 times higher than the original condition and the maximum activity reached 6.10IMCU/mL.5. The technological parameters to make cheese with milk-clotting enzyme from Q. cyanescens were optimized as follows:50IMCU/L milk-clotting enzyme,0.02% CaCl2 holding time 40min and coagulating temperature 35℃. The texture profile of cheese was analyzed by TA-XT.PLUS/30 food texture analyzer from Stable Micro Strfems. The results show that texture parameters of cheese produced using enzyme from Q. cyanescens were not significant different from those of cheese solidified by rennet. Dynamic rheological parameters of milk added milk-clotting enzyme were characterized by CVOR150 rheometer from Malvern. The results show that rheological parameters of milk added different MCEs were not significant different.