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Genetically engineered rennet ferment preparation and application of

Author: ZhengLi
Tutor: WangZuo；YangZhenNai
School: Jilin University
Course: Food Science and Engineering
Keywords: Genetic Engineering Recombinant Pichia Ferment Rennet Cheese
CLC: TS252.53
Type: Master's thesis
Year: 2011
Downloads: 175
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Abstract

Cheese is considered to be China's dairy industry to develop new growth points. Cheese rennet is the enzyme necessary for the production process, which not only make the milk clotting, and affect the maturation of cheese flavor and texture formation. With the rise in world production of cheese, rennet for cheese production increasingly short, a lot of researchers have begun to seek alternative rennet. Wild microbial fermentation using rennet Although the production cycle is short, low cost, however, microbial rennet proteolytic enzyme activity than bovine animal rennet curd higher thermal stability is higher, and thus lead to the production The cheese is easy to produce a bitter taste. In China, the shortage cheese with rennet, rely heavily on imports, resulting in higher production costs cheeses. Produced by genetic engineering techniques rennet has become a new hotspot, China has independently developed genetically engineered rennet has important significance. Pichia pastoris as rapid development in recent years, genetically engineered expression vectors, high security, a target protein yield, easy to scale production, etc., have been used to express a variety of proteins. In this study, the laboratory recombinant Pichia pastoris (including Mucor pusillus rennin gene) expression Mucor pusillus rennet. To study the optimization of enzyme production conditions, enzyme purification, enzymatic properties and applications in cheese production. The main results are as follows: (a) producing recombinant Pichia pastoris fermentation conditions rennet experimental results show that: Recombinant Pichia 24h after induction into the stable phase, induction 216h, the total number of bacteria and reached the maximum number of viable cells, Then enter the decline phase; recombinant Pichia pastoris at 192h when the production of chymosin activity reached the maximum that 300.0SU/mL, and milk clotting activity and proteolytic activity ratio (C / P value) reaches the maximum that is 27.91, while the proteolytic When activity is relatively low in the induction 192h, only 12.00u/mL. Electrophoretic analysis of recombinant Pichia produced chymosin molecular weight of about 47kDa. (2) determined by single factor experiments in Pichia pastoris medium BMGY optimal growth conditions: culture temperature 30 ℃, pH of the medium 5.2, rotation speed 280r/min, medium shake bottled liquid volume of 5% ( 12.5mL/250mL). (3) In the production of rennet optimization process of fermentation conditions by single factor experiments and Plackett-Burman experimental screened three pairs of recombinant Pichia pastoris producing rennet important influential factors: Shake bottle liquid medium Cover (A), the amount of methanol added (B) and the initial pH of the medium (C). Box-Behnken response surface using experimentally derived regression equation: Y (rennet activity) = 472.20-42.81A-8.03B 24.43C 55.34AB-16.99AC-47.57BC-52.59A2-60.01 B2-131.34C2. On this basis to determine the optimum value of the three: shake flask culture liquid volume 13.1% (32.65mL/250mL), the amount of methanol added every 24h, adding 1.5% 6.2 initial pH of the medium. In optimal conditions, the enzyme activity was maximum 491.7SU/mL, and verification of experimental results. (4) identified by genetic engineering method for purifying chymosin: the 80% saturation (0 ℃) ammonium sulfate precipitation, enzyme sample was dissolved in 0.1M phosphate buffer (pH5.8), by the DEAE-52 ion-exchange layer analysis dextran G-75 gel chromatography, enzyme purity of 89.3 times, recovery of 53.77%. Electrophoretic analysis showed that the purified enzyme a single protein band. (5) Characterization of the results showed that: genetically engineered rennet optimum temperature is 55 ℃, the optimum pH was 5.4. Enzyme activity at acidic pH conditions higher activity at alkaline pH conditions decreased when the enzyme is stable at pH6.0. (6) the curd results show that: with the increase of added amount of rennet curd shortened; with increasing concentrations of skim milk curd time gradually becomes longer, add skim milk curds promote 0.05M CaCl2 formation. The viscosity of the raw milk curds and texture analysis results show that the product of genetic engineering rennet and rennet curd has similar properties. (7) the use of commodity rennet and rennet were genetically engineered produce Cheddar cheese, after comparing the mature sensory, chemical and microbiological indicators. The results showed that both the indicators cheese close. Although the use of genetically engineered rennet cheese produced proteolysis during ripening enzyme levels slightly above the use of goods produced cheese, but the former does not produce significant bitterness. Obtained in this study can be used in genetic engineering rennet cheese production.

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