Studies On Screening Of Fungus Producing Chymosin And Its Enzyme Properties

Studies on Screening of fungus producing chymosin and its enzyme properties is Supported by National High Technology Research and Development Program of China(No. 2006AA10Z306) and Department of Agriculture Modern Agriculture Industry Technology System construction special funds (No. nycytx - 0502).Aspergillus niger and Mucor pusillus could produce many kinds of hydrolytic enzyme outside the cells. Screening was based on the curd activity from three strains of Aspergillus niger and Mucor pusillus with lab collected and preserved. Many kinds of mutation methods was carried out on its parent strain Aspergillus niger J1. Aspergillus niger W2, W5, UV1, U5, C1, P-W1and P-W3 was obtained by mutagenesis and rational screening. Compare Aspergillus niger W2, W5, UV1,U5,C1,P-W1, P-W3 with Mucor pusillus G3, on the conclusions that Mucor pusillus G3 have higher production curd enzyme ability .So Mucor pusillus G3 was selected for studying solid-state fermentation technology. Curd enzyme of Mucor pusillus were also discussed in this dissertation by using leaching and purification. At the mean time, the curd enzyme from Mucor pusillus was studied in cheese production. This paper mainly dealt with five aspects:1. Breeding of the curd enzyme Producing Strains:(1) Compare strains growth curve with production curd enzyme curve, it is obviously that Aspergillus niger have markedly single colony and curd circle. Owing to Mucor pusillus mycelia growth too much quickly, it have only single colony in a short time but cannot observe curd circle. Aspergillus niger is more easily to be screened in Mutation. (2) Compare strains with production curd enzyme curve between Aspergillus niger and Mucor pusillus ,it is discovered that Aspergillus niger J1 and Mucor pusillus G3 have higher ability to produce curd enzyme in homologous series strains.So Aspergillus niger J1 and Mucor pusillus G3 were selected as experiment strains undertake next experiment.(3) Contrasting the curd activity with different strains rough extracts curd enzyme after solid state fermentation, discovered that curd enzyme of Mucor pusillus have higher activity than Aspergillus niger.2. Improvement of strain with high curd enzyme activity by mutation, rational screening. Preliminary screening was based on size of milk-clotting zones and the secondary screening was done on solid fermentation medium based on curd enzyme activity .By single factor and compound mutagenesis, screening the high yield strain of curd enzyme.(1) The Results of Single Factor Mutagenesis: Mutation was carried out on its parent strain Aspergillus niger J1 by UV, microwave, ultrasonic, HPEF and hyperpressure treatment and rational screening. Preliminary screening was based on size of milk-clotting zones and the secondary screening was done on solid fermentation medium. Some mutant strains W2,W5,UV1,U5,C1 with good genetic stability and high milk-clotting activity were screened.(2) The Results of Compound Mutagenesis: Mutation was carried out on its parent strain Aspergillus niger J1 by microwave-UV, ultrasonic- UV, HPEF-microwave and hyperpressure- microwave treatment and rational screening. None was screened by ultrasonic-UV compound mutagenesis. Aspergillus niger W-UV2, W-UV5, P-W1, P-W3 and C-W1 were selected by preliminary screening and the secondary screening was done on solid fermentation medium. Some mutant strains Aspergillus niger P-W1,P-W3 with good genetic stability and high milk-clotting activity were screened. (3) By single factor and compound mutagenesis, screening the high yield strain of curd enzyme are W2, W5, UV1, U5, C1, P-W1 and P-W3.There were selected as experiment strains undertake next experiment .3. A study on the solid state fermentation process for producing curd enzyme of mould.(1)Contrasting the curd activity with different strains rough extracts curd enzyme after solid state fermentation, discovered that curd enzyme of Mucor pusillus have higher activity than Aspergillus niger by mutagenesis. So Mucor pusillus G3 was selected as experiment strains undertake next solid state fermentation process experiment.(2)Base on the activity of rough extracts curd enzyme, systematic studies were carried out on the optimization of fermentation conditions for producing the enzyme by the strain. This optimal fermentation condition was established by single factor design and orthogonal design. That fermentation temperature is the most important factor in solid state fermentation process, next is fermentation time. The sequence is fermentation temperature>fermentation time>Solid-liquid ratio >inoculums size. The optimal experiment combination is A2B3C2D3. In other words ,the optimum condition were fermentation temperature 30℃,fermentation time 5d,Solid-liquid ratio 3:4,application of sample 3.(3) The nutrient medium component of Mucor pusillus G3 were optimized .The wheat bran, rice bran and soya-bean cake etc,10 kinds of substrate were optimized by Plackett-Burman design .The result of solid state fermentation showed that wheat bran, (NH₄)₂SO₄ and KH₂PO₄ are major factors affect producing curd enzyme in solid state fermentation. Then the centers were definited by method of path steepest ascent design. This optimal nutrient medium component was established by Box-Behnken design. Using the medium containing 15.50g bran, 0.15g (NH₄)₂SO₄, 0.08g KH₂PO₄, the enzyme yield was 1200 U/ml under the optimum condition, which was 44.9% higher than that under non-optimized fermentation conditions.4. Taking the curd activity as parameter, optimized preparation and purification of rough extracts curd enzyme.(1)By single factor design and analysis, the optimization leaching parameters are sodium chloride concentration 4%, leaching time 2h, leaching temperature 30℃,rate of rotation 150rpm.(2)By single factor design and analysis, the optimization ammonium sulfate salting-out parameters are ammonium sulfate concentration 70%, salting-out time 8h.(3)By single factor design and analysis, the optimization ethanol precipitation parameters are ethanol concentration 65%, ethanol precipitation time 4h.(4) Contrast ammonium sulfate salting-out with ethanol precipitation taking the curd activity as parameter; draw conclusions that ethanol precipitation is more effective in purification. On the other hand, enzyme liquid with salting-out has higher ammonium sulfate concentration. We should operate dialyzing and ultrafiltration in order to desalting. But enzyme liquid with ethanol precipitation contains hardly mineral salt. Experiment is concise so ethanol precipitation was selected as preliminary purification.(5)The curd enzyme coefficient of recovery is higher by ethanol precipitation and freeze dehydration; the curd enzyme purity is higher by gel filtration and anion exchange chromatograph but its coefficient of recovery is lower.5. Enzyme properties and applied research of Mucor pusillus curd enzyme : taking the curd activity as parameter, inspect some factors influencing curd activity of Mucor pusillus; contrast curd enzyme of Mucor pusillus with commodity curd enzyme, inspect viscosity and texture differences in simulating cheese production. (1)It was studied that curd activity of Mucor pusillus at different temperature: the optimum milk-clotting temperature was 65℃; it was studied that curd activity of Mucor pusillus at different pH: the optimum milk-clotting pH was 5.0.That explained curd enzyme of Mucor pusillus thermostability is well below 60℃and its suitable for acid environment.(2)It was discovered that EDTA could improve clotting milk in a narrow range. When the concentration of EDTA was exorbitant, skimmed milk appeared flocculation phenomenon .While ethanol had the opposite effect, with the increase of ethanol concentration; the activity could be weakened steadily. SDS could improve clotting milk in low concentration, but it could weaken clotting milk in high concentration. Any concentration of mercaptoethanol had remarkable effect on promoting clotting; Ca²⁺,Mg²⁺,Fe³⁺ and Mn²⁺ had tremendous effect on promoting clotting in a certain concentration range ,while K⁺, Na⁺ and Li⁺ had faint effect on promoting clotting in a certain concentration range; Co²⁺, Zn²⁺,Cu²⁺ and Ni²⁺ could inhibit clotting milk, while Pb²⁺ had the opposite effect in low concentration. Under the similar experimental conditions, SO₄^2- had more significant influence to curd enzyme of Mucor pusillus than Cl-.(3)It was discovered that PBS, acetic acid buffer and citric acid buffer had remarkable effect on promoting clotting under the similar pH and concentration experimental conditions; with the increase of milk concentration, the activity could be improved steadily. On the condition of concentration above 12.5%, the activity maximum later tended to be stable; pH of law milk had a great effect on curd activity, with the increase of pH the curd activity become lower gradually; Curd activity become extremely low when pH of raw milk was 7.8.(4)By simulating cheese production, volume of addition with curd enzyme had an effect on viscosity when milk is clotting. There had different gel system because curd enzyme of Mucor pusillus and commodity curd enzyme had different curd characteristics. Cheese texture was determined by TPA model. The higher hardness value of cheese by using curd enzyme of Mucor pusillus as milk coagulant; two cheeses had lower glutinosity and similar cohesiveness. So it is showed that had little difference of texture in cheese production as milk coagulant.