Inactivation Mechanism Of Acetobacter Sp.and Its Alcohol Dehydrogenase By Pulsed Electric Fields

In winemaking,various microorganisms adhered to the skins of the grapes will enter the fermentation process as the grapes are broken since raw materials(grapes)are not cleaned and sterilized.In particular,acetic acid bacteria(AAB) are a spoilage group of microorganisms in the wine industry,which are able to convert the ethanol produced by yeasts into acetic acid through its intracellular enzymes including alcohol dehydrogenase(ADH)and aldehyde dehydrogenase(ALDH),resulting in increasing volatile acidity in wine and thus inducing wine deterioration.Traditionally,sulfur dioxide(SO₂) is commonly used to prevent and limit microbial activity in winemaking,thanks to its antimicrobial and antioxidative properties.However,SO₂ has a limited effect on the viability of AAB and a certain concentration of the free SO₂ must be maintained to inhibit the growth of microorganisms.Moreover,overuse of SO₂ not only impair the quality of wine,but also poses a food safety risk.Additionally,growing consumers demand high-quality and natural foods without chemical preservatives.Therefore,it is very important for the development of wine industry to explore suitable SO₂ substitutes or alternative methods.In recent years,pulsed electric fields(PEF),as a novel non-thermal sterilization technology,has attracted increasing attention due to its good ability to inactivate microorganisms and enzymes in liquid foods without modifying the original quality of the food.However,it is difficult to implement?one size fits all?in the application of PEF to inactivate microorganisms in food since the sterilization efficiency of PEF is affected by many factors such as food product(medium)parameters and the characteristics of microorganisms.It is worth noting that the impact of these factors on the inactivation of AAB by PEF has not been reported in detail.In addition,the effect of PEF treatment on microbial intracellular enzymes is rarely reported until now.The underlying mechanism of the inactivation of microorganisms by PEF remains to be fully elucidated.Therefore,in the present work,the inactivation mechanism of Acetobacter sp.(one typical AAB associated with grapes and wine)and its alcohol dehydrogenase by PEF was investigated.Moreover,the effect of PEF pretreatment of grape juice(instead of SO₂) on the volatile acidity of wine after alcohol fermentation was explored.The specific research results are as follows:The inactivation effect and kinetics of PEF treatment on Acetobacter sp.were investigated.The results showed that the inactivation effect of PEF on Acetobacter sp.was enhanced with increasing electric field strength(10?25kV/cm) and treatment time(1.5?6.0ms),and the maximum inactivation was 3.15log.Compared with treatment time,the electric field strength was more important for the inactivation of Acetobacter sp.by PEF.Moreover,the inactivation of Acetobacter sp.increased with increasing initial treatment temperature(4?42?) and the maximum inactivation reached 4.97log.For grape juice and wine as processing media,it was found that the inactivation of Acetobacter sp.in low-conductivity media was higher than that in high-conductivity media.At the same time,the presence of ethanol in wine was found to have a synergistic effect on PEF sterilization.Furthermore,it was found that Acetobacter sp.in the exponential phase was more sensitive to PEF than those in the stationary phase.Besides,the kinetic analysis showed that the Weibull mathematical model was a good fit for the inactivation results.The influencing mechanism of ethanol adaption on the PEF resistance of Acetobacter sp.in the stationary phase was investigated.Results showed that ethanol(0%?9%) as a growth substrate could significantly inhibit the growth of Acetobacter sp.and the cells cultivated in the medium with relatively high ethanol concentrations were more vulnerable to PEF.Based on the results of gas chromatography-mass spectrometry,Raman spectroscopy and fluorescence polarization analysis,combined with the inactivation data of Acetobacter sp.by PEF,it was found that the membrane fluidity of ethanol-adapted cells was responsible for its PEF resistance.When Acetobacter sp.cells were grown in the medium with a high concentration of ethanol,the content of unsaturated fatty acids in the cell membrane increased,while the content of saturated fatty acids decreased.Simultaneously,the order degree of C–C and lateral packing order of membrane lipid chain became more disorderly.Due to this development,cell membrane became more fluid,which in turn made cell membrane of Acetobacter sp.more sensitive to PEF.Moreover,morphological analysis by scanning electron microscopy showed that the cells cultivated at relatively high concentration of ethanol were more vulnerable to PEF treatment,and thereby forming irreversible electroporation easily.The effects of PEF treatment on the cell membrane and intracellular enzymes of Acetobacter sp.were investigated.The results showed that the permeability of the cell membrane of Acetobacter sp.and the leakage level of intracellular substances such as nucleic acids,proteins and ions increased with increasing electric field strength(0?36kV/cm).Simultaneously,the order degree of C—C and lateral packing order of C—H in the phospholipid chain increased,leading to a decrease in cell membrane fluidity.Moreover,morphological analysis by scanning electron microscopy showed that the morphology of Acetobacter sp.cells was destroyed after PEF treatment.When the electric field strength was 36kV/cm,obvious holes appeared on the surface of Acetobacter sp.cells.Besides,the flow cytometry analysis showed that the cells with high activity of intracellular enzyme decreased with increasing electric field intensity.The effect of PEF treatment on the activity and structure of alcohol dehydrogenase(ADH)in Acetobacter sp.was investigated.The results showed that PEF treatment could significantly inactivate ADH in Acetobacter sp.and the inactivation level of ADH was a function of electric field strength(0?28kV/cm) and treatment time(0?4.5ms).Moreover,the analysis of Fourier transform infrared spectroscopy and circular dichroism spectra showed that the secondary structure of ADH changed after PEF treatment.With the increase of electric field intensity,the?-helical structure decreased,while random coil structure increased.Moreover,the analysis of UV absorption spectroscopy and fluorescence spectroscopy indicated that the unfolding of tertiary structure of ADH occurred after PEF treatment,which resulted in the change of the microenvironment of the aromatic amino acid residues,and some natural chromophores moved to the inner hydrophobic area of the molecule.Besides,the electrophoretogram produced by SDS-PAGE of ADH indicated that PEF treatment did not modify the polypeptide chain composition of ADH.Therefore,it can be inferred that changes in protein spatial conformation may be responsible for the inactivation of ADH by PEF.The effect of PEF pretreatment of grape juice(instead of SO₂) on the volatile acidity of wine after alcohol fermentation was investigated.The results showed that PEF treatment(18kV/cm,4.8ms)had no significant effect on the total sugar,Total acidity,soluble solid and p H of grape juice.However,compared to the treatment with SO₂ addition,PEF pretreatment promoted must fermentation,accelerated fermentation speed and significantly reduced the volatile acidity of the fermented wine(from 0.52g/L to 0.23g/L).Furthermore,a slight increase in alcohol content was observed in the PEF wine,while a significant decrease(43.32mg/L) in the content of total polyphenols was also observed.