Physiological Response Mechanism To Environmental Stress Of Acetobacter Pasteurianus And Improving The Survival During Freeze-Drying

Acetic acid bacteria was used as the main industrial strain for acetic acid fermentation, many kinds of environmental factors such as ethanol, acetic acid, freeze drying can exert different stress on it. Acetic acid bacteria can form a set of complete and unique regulating system on their own when they faced with various kinds of stresses, so they can adapt to the complex and changeable environment. This study took acetic acid bacteria as test subject, analyzing the regulatory mechanism of the level of membrane and metabolism to stress response via studying the physiological response to stresses. Moreover, the strategies on improving the activity of ready-to-use acetic acid stater culture were studied. The main results were as follows:(1) This strain CICC 7015 was treated as the research subsject to elucidate the physiological response mechanism to ethanol and acetic acid stress cultivation from the perspective of membrane and metabolism. Ethanol stress decreased bacterial polysaccharide content, increased plasma membrane fluidity and decreased plasma membrane permeability. The H+-ATPase activity was highest at the ethanol concentrates of 3% and 5%, then distinctly decreased at the concentrations of 7% to11%. And the intracellular ATP concentrations decreased along with the ethanol concentration increased and were much lower than the control level when ethanol concentrations from 7% to 11%. Acetic acid stress caused the decrease of bacterial polysaccharide, the increase of plasma membrane fluidity and permeability. There was a positive correlation between membrane-bound H+-ATPase activity and acetia acid concentration, the enzyme activity with was 2.28 times as the control at acetic acid concentrations of 2.0%. Meanwhile the intracellular ATP concentration showed a negative correlation with acetia acid concentration, it decreased from the 0.148(control group) to 0.008 mmol·mg-1(protein) at acetic acid concentrations of 2.0%.(2) The influences of carbon source, exogenous energy auxiliary substrate and exogenous fatty acid on plasma membrane physiological characteristics and intracellular ATP level were investigated to probe into the physiological response of strain CICC 7015 to medium. When different carbon sources were used to incubatethe strain, ethanol reduced bacterial polysaccharide content; and ethanol and glycerol increased the plasma membrane fluidity to some extent; the lowest membrane permeability when glucose acted as sole carbon source; compared with glucose as sole carbon source, plasma membrane H+-ATPase activity was significantly increased when the other carbon sources incubated the strain; the intracellular ATP content was the highest when glycerol as sole carbon source. Then added exogenous energy auxiliary substrate including citric acid, malate and succinic acid in the medium, it was revealed that there was no significante effect on bacterial polysaccharide formation; however they increased the plasma membrane fluidity and permeability; the H+-ATPase activity and ATP concentration increased at the same time. And exogenous fatty acid had no significant correlation with the synthese of bacterial polysaccharide; oleic acid, linoleic acid increased the fluidity and plamitic acid, stearic acid decreased the fluidity; they all made the permeability decreased; and compared with the control,plamitic acid and oleic acid improved H+-ATPase activity significantly, yet stearic acid reduced its activity; moreover, unsaturated fatty acids promoted the formation of intracellular ATP more than saturated fatty acids.(3) The influences of protective agents and medium components on freeze-drying viability were studied. The ten kinds of protective agents were firstly studied, and dried skim milk showed the best effect(72.92%) which had significant difference with the control at 0.05 level. Therefore dried skim milk was selected to use for the next freeze-dying tests. Freeze-drying viability after ethanol and acetic acid stress pre-incubation showed: the viability after ethanol stress distinctly reduced compared with control; acetic acid stress incubation distinctly increased freeze-drying viability compared with control. The freeze-drying viability(73.61%) was the highest when carbon source was the combination of glucose and glycerol. The medium added with energy auxiliary substrate citric acid, malate and succinic acid made the freeze-drying viability higher than control significantly. The four kinds of fatty acids significantly enhanced the viability compared with control at 0.05 level and unsaturated fatty acids increased the viability more than saturated fatty acids.