Study Of Thermosonication Blanching Treatment On Carrot Slices And Its Potential Mechanisms

The conventional blanching method of fruit and vegetable is to immerse the fresh or cut materials in hot water at a temperature within 90-100?.This method is limited in many aspects,for instance,long processing duration,high blanching temperature,low heat transfer efficiency,high content of nutrient loss,huge damage of cell structure,etc.In order to mitigate the limitations,ultrasound was applied to hot water blanching as a novel blanching method,namely thermosonication(TS).Firstly,carrot was selected as the raw material and peroxidase(POD)activity was used as the index,screening and optimizing the conditions of TS treatment to investigate the inactivation effect on carrot POD.Meanwhile,the effect of TS on the cell structure was also studied to reveal its mode-of-action on the product quality.Furthermore,the inactivation effect of TS on the horseradish peroxidase(HRP)was investigated.The relationship between the variations in conformation of HRP and its activity was established by means of the correlation and quantitative mathematical analysis to deeply find the potential denaturation mechanisms.This novel blanching method is expected to drop the restrictions caused by the hot water blanching,and lay a solid foundation for industrial application of TS treatment and development of the related ultrasonic devices.Main investigations and conclusions are as follows:(1)The inactivation effect of TS treatment with different frequency modes on the carrot POD was investigated.The denaturation mechanisms were revealed from the changes in the expression level of related carrot POD gene and the radicals indued by ultrasound during TS blanching.The quality characteristics of carrot were also evaluated.The results showed that TS treatment enhanced remarkably the inactivation efficiency of carrot POD,with a dual-frequency mode of 22/40 k Hz showing the most significant effect,which was 65%lower than the residual POD activity of hot water(HW)blanched samples.The total carotenoid contents(11.5-13.0 mg/100 g fresh weight)of thermosonicated carrots all exhibited significant increase when compared to the fresh one.Color attributes a*,b*and C*values increased accordingly,indicating that a brighter color of carrot was obtained after TS blanching.Additionally,the POD involved regulation of carrot whiteness index(WI)value notably decreased after TS treatment,and the lowest WI value was obtained by the dual-frequency of 22/40 k Hz,which was consistent with the results of POD activity.The mechanical research results showed that the POD-related gene expression was down-regulated by TS blanching,which was contrary to the thermally treated samples.Electron paramagnetic resonance(EPR)spectra shown that the formed free radicals from the ultrasound-induced decomposition of water played important roles in the POD inactivation during TS treatment.(2)The potential inactivation mechanisms of TS on the POD activity were deeply investigated.For this purpose,HRP was selected as the model enzyme and the effectiveness of various ultrasonic frequencies on the residual activity and structure of HRP was studied by means of UV-Vis spectrometry,fluorescence spectrometry,circular dichroism(CD)spectrometry,atomic force microscopy(AFM)and etc.Pearson correlation and stepwise multiple linear regression(Stepwise-MLR)were used to acquire the structure-activity relationship of HRP during TS treatment.Results showed that the structure of porphyrin ring was destroyed during TS treatment and the prosthetic group was ejected from the active site of HRP.The apo-enzyme was unfolded,and the hydrophobic amino acids as well as sulfhydryl groups were exposed.With the TS treatment proceeding,part of the apo-enzyme was cleaved and other part was aggerated because of the hydrophobic attraction induced by the exposed hydrophobic groups.Besides,the corresponding roughness value(R_a)also decreased after TS treatment.These changes all significantly lowered the binding capacity of enzyme towards substrate,which further caused the decrease in the enzyme activity.Pearson correlation analysis showed that Rz value and?-helix content were positively corelated with HRP activity,while relative tryptophan fluorescence intensity,relative ANS fluorescence intensity and free sulfhydryl content were all negatively correlated with HRP activity.Mathematical equations of HRP inactivation under TS treatment with different frequency modes were established using Stepwise-MLR,the corresponding R² ranged in 0.852-0.998and p values were all lower than 0.05,indicating that these equations could better reflect the quantitative relationship between the specific structural changes in HRP and its activity during TS treatment.(3)Effect of the TS blanching on the quality of carrot was investigated.For this purpose,studies of TS treatment with different frequency modes on the carrot cell membrane,cell water content and distribution were carried out using electrical impedance spectroscopy(EIS)with equivalent circuit model,low-field nuclear magnetic resonance(LF-NMR),malondialdehyde(MDA)content determination and etc.Results showed that the impedance and phase angle of carrot tissue decreased after TS treatment.Tri-frequency mode displayed the most significant effect on the EIS parameters amongst all tested conditions.Meanwhile,the parameters calculated by the modified Hayden equivalent circuit model displayed the same trend.In detail,the intracellular resistance(R_i)increased after TS treatment,while the extracellular resistance(R_e)and the cell membrane capacitance(C_m)values decreased,which indicated that TS treatment destroyed the permeability and integrity of carrot cell membrane,and also affected the balance of the osmotic pressure inside and outside the cell.These alterations fastened the proton exchange between the bound water,semi-bound water and free water in carrot cells and the surrounding environment,which expressed as the related relaxation times(T₂₁,T₂₂,T₂₃)gradually decreased after TS treatment.Moreover,the content of bound water and semi-bound water decreased,while the content of free water increased,which was conducive to the water discharge in the subsequent treatment.Additionally,the MDA content of carrot cell decreased after TS treatment,implying that the oxidative damage of cell membrane was also reduced.(4)Effect of TS treatment with different frequency modes on the carrot cell structure and cell wall composition was investigated in terms of the alterations of texture,rheological properties,cell wall polysaccharides,neutral sugar content of pectin,degree of esterification(DE),cell calcium content and etc.Results showed that TS treatment significantly increased the hardness value(4.4%-10.3%)of carrot tissue at 60?,while the hardness value decreased at TS treatment performed at 70?.Under such condition,the storage modulus(G')of carrot tissue was higher than loss modulus(G''),indicating that the cell structure of carrot tissue still remained rigidity.Compared to the cellulose and hemicellulose,TS treatment showed a more significant effect on the content and structure of pectin.After treatment,the content of chelator-soluble pectin(CSP)fraction decreased,and the contents of water-soluble pectin(WSP)and sodium carbonate-soluble pectin(NSP)fractions increased.At the meantime,the branching degree of rhamnogalacturonan(RG)in WSP increased,and the linearity of homogalacturonan(HG)in CSP was also enhanced.The DE of carrot cell wall alcohol insoluble residue(AIR)showed the opposite tendency of hardness value,combined with the AIR calcium content increased,which was beneficial to the formation of crosslinking structure between the de-esterified pectin and calcium ions,and such a structure was better to retain the texture properties of carrot tissue.