Study On Screening, Conditions For Enzyme Production, Enzymatic Characteristics And Application Of Rennet Producing Bacteria From Gannan Pastoral Area

Milk-clotting enzyme is very important in the production of cheese. The enzymes occur inanimal tissues, higher plants and microorganisms. For a long time, calf rennet, the conventionalmilk clotting enzyme obtained from the fourth stomach of suckling calves, is the most widelyused coagulant in cheese-making all over the world to manufacture most of the cheese varieties.The worldwide reduced supply of calf rennet and the ever increase of cheese production andconsumption have stimulated the research for milk clotting enzyme (MCE) from alternativesources to be used as calf rennet substitutes. Microbial rennet appears to be more promisingbecause its production is cheaper, its biochemical diversity is greater, and its geneticmodification is easier. Attention has been focused on the production of milk-clotting enzymesfrom microbial sources for use as rennin substitutes.In this study, we isolated and screened the microorganism with high milk-clotting activityfrom the soil sample collecting form Gannan Pastoral Area in Tibetan Plateau in China. Then thestrains obtained in the secondary screening were identified. On basis of mutation by UV andDES, the culture conditions, fermentation medium and enzyme properties were studied. And theproperties and application in rennet caisen of this milk-clotting enzyme were studied. The mainresults obtained are as follows:1. Screening and isolation of bacteria producing milk-clotting enzymeAn efficient chymosin producing strain GN4.1,was screened out from6bacterial strainsisolated from the soil of yak grazing district in Gannan County of Gansu Province, using caseinmedium,whose chymosin and proteolysis activities were1011.6SU/ml and14.6U/ml after48hshaking fermentation(140r/min)at37℃in wheat bran medium,respectively. This strain wasidentified as Bacillus amyloliquefaciens by the means of bacterial morphological featureobservation, physiological and biochemical characteristics measurement and16S rDNAsequence analysis.Furthermore, the growth and enzyme production curves of this strain wereplotted.The optimum levels of temperature and pH range of were37℃,7.0~8.0respectively for thegrowth of the strain. The strain was aerobic and the production of milk-clotting enzyme fromBacillus amyloliquefaciens is very little at the beginning of logarithmic growth phase, butincrease sharply after18h. The production of protease is an isochronous progress together withthe milk-clotting enzyme.2. Mutation of Bacillus amyloliquefaciens GN4.1by Ultraviolet and Diethyl sulfateBacillus amyloliquefaciens GN4.1producing chymosin was mutated by Ultraviolet andDiethyl sulfate to improve the ability of producing chymosin. The best mutation time for UV treatment were60seconds, and the best mutation time for Diethyl sulfate treatment were30minutes.After mutated by Ultraviolet，a mutant UV-1with high activity of chymosin and lowerproteolysis was obtained. Compared with the original strain, the activity of chymosin wasincreased by23.2%，the proteolytic activity was reduced by23.3%. With UV-1as the startingstrain，the mutagenesis was adopted with DES．After screening repeatedly，a mutant DES-6withhigh activity of chymosin and lower proteolysis was obtained. Compared with the original strain,the activity of chymosin was increased by40.2%，the proteolytic activity was reduced by22.39%. Genetic stability test showed that its milk-clotting activity was1417.2-1428.7SU/mL in8generations, indicating that this strain had stable genetic characteristics．3. Optimization of culture conditions and nutritional parameters for MCE productionThe optimum physical conditions and nutritional parameters for milk-clotting enzymeproduction were studied by single factor testing in the present study. The results showed that theoptimum levels of temperature, inoculum size, agitation speed and initial pH of the medium were37℃,3%, and230rpm respectively for the production of milk-clotting enzyme. The maximummilk-clotting activity was observed using wheat bran juice concentration18g/100mL. Amongvarious carbon source and organic nitrogens, skim milk powder and sucrose were found to be thebest in stimulating milk-clotting enzyme production.Two statistical methods were applied to optimize medium components to improve theproduction of the milk-clotting enzyme by Bacillus amyloliquefaciens DES-6. The regressionanalysis of the Plackett–Burman design shows that low levels of X3(glucose), X4(FeSO47H2O),X5(MgSO47H2O), X6(MnSO47H2O), and X7(ZnSO47H2O) enhanced milk-clotting production,whereas high levels of X1(wheat bran), X2(skim milk powder), and X8(Na2HPO4) resulted inhigh milk-clotting activity.Three significant independent variables [X1(wheat bran), X2(skim milk powder), andX8(Na2HPO4)] were selected and further optimized using the Box–Behnken design to determinetheir optimal concentrations. From equations derived by differentiation, we obtained themaximum point of the model. The maximum predicted value of milk-clotting activity was3313.7SU/mL. The optimized medium were as follows：17.25%wheat bran juice,3.31%skimmilk powder,5%glucose,0.1%FeSO47H2O,0.1%MgSO47H2O,0.1%MnSO47H2O,0.1%ZnSO47H2O and1.52%Na2HPO4.The milk-clotting enzyme activity of3326.7SU/mL in theoptimized medium was achieved after48h of cultivation, which was very close to the predictedvalue of3313.7SU/mL. The good correlation between these two results validates the model andthe existence of an optimal value.4. The properties of MCE from Bacillus amyloliquefaciens The molecular mass of the enzyme was56.1kDa as determined by SDS-PAGE. Theoptimum temperature and pH for the enzyme was60°C,5.5. It was fully inactivated after20minat65°C. The low level of thermostability of the B. amyloliquefaciens GN4.1enzyme made iteasy to inactivate, which allows the whey to be reused. The enzyme was stable in a relativelywide range of pH5.5–9.0, with maximum stability at pH6.5showing that the B.amyloliquefaciens enzyme was stable in the acidic to neutral range. The low pH sensitivity isvery important for the coagulant because the use of highly pH-sensitive rennet can lead toreduced yields and defective cheese due to a soft coagulum at cutting. The milk-clotting activityreached a maximum when the skim milk concentration was increased to50g/L. Different metalions had different effects on milk-clotting activity. Na+and K+had only a slight inhibitory effect,whereas Cu2+, Zn2+and Sn2+inhibited the milk-clotting activity significantly. In contrast, Mn2+had a significant stimulatory effect on milk-clotting activity, whereas1mM Li+and1mM Mg2+promoted the activity slightly, but had no effect when present at concentrations of only5mM or10mM.5. Application of MCE in the production of rennet caseinRennet casein was prepared from the fresh milk using bacteria rennet as coagulant and thetechnology was optimized by single factor test and response surface methodology. The optimalcoagulation parameters were as follows: temperature36.5℃, pH6.2and0.37mL bacterial rennetper500g skim milk. Under the optimized conditions, the yield of rennet casein reached3.53±0.02%and the relative error was0.19%compared with the predicated value. Thephysicochemical index and functional properties of the products were not significantly different(P>0.05). The technique of FTIR was used to determine and compare the rennet casein producedwith bacterial rennet and calf rennet. The spectra of rennet casein produced with bacterial rennetand calf rennet were similar. The results of physical and chemical test, functional properties testand infrared spectrum scan showed that there was no obvious difference between the twoproducts, suggesting that bacteria rennet from Bacillus amyloliquefaciens could be used as calfrennet alternatives in the production of rennet casein. The research can provide a reference forfull utilization of bacteria rennet.