The Effect Of Multifrequency Ultrasound On Alicyclobacillus Acidoterrestris Spores,vegetative Cells,bioactive Compounds And Enzymes In Orange Juice(Citrus Sinensis(L.)Osbeck)

Orange juice can be regarded as an ecosystem comprising multiple microorganisms such as pathogenic,spoilage and starter fermentation cultures.The spoilage by Alicyclobacillus is generally identified as flat-sour spoilage with distinct offensive odors of medicinal or antiseptic description.The antiseptic odors in spoiled orange juice are often mostly caused by the development of chemical taint compounds,in particular,2-methoxyphenol(guaiacol)and phenolic halogenated substances,namely,2,6-dichlorophenol and 2,6-dibromophenol.Pasteurized orange juices and concentrates recorded spoilage incidents world widely.It has been found that Alicyclobacillus acidoterrestris,the responsible microorganism,is able to withstand the processes of thermal pasteurization,hot filling that prevent the growth of other microorganisms.A.acidoterrestris induces off-odors and off-flavors in beverages that are often ignored by manufacturers prior to customer complaints.Off-odors and off-flavors,even at low levels,greatly impact the perceived purity of the juice,restrict the food industry’s future usage and reduce its consumer demand.Besides,all variables that regulate the behavior of A.acidoterrestris are the existence of phenolic substances,ethanol,salt content and preservatives,could changes the fresh-like characteristic of orange juice.As a result,manufacturing progress may not be sufficient to inhibit this microbe in all products while reducing organoleptic properties or the energy expense of the juices.Different new approaches as an alternative to conventional juice production have been studied in recent years.Low-frequency and high-power ultrasound waves are generally capable of inactivating bacterial cells and enhancing bioactive compounds of orange juice.Thermosonication(TS)is a promising alternative to thermal pasteurization for the processing of fruit juices.Recently,multifrequency sonication has been identified as an efficient strategy for optimizing cavitation behaviors and inactivating bacteria,and has a strong synergistic impact,in comparison to monofrequency ultrasound which has been employed in the majority of reported studies.This work has illustrated many new findings:the optimum conditions for mono-and dual frequency thermosonication inactivation of spores and vegetative cells of A.acidoterrestris,the bioactive compound enhancement and enzyme deactivation,the explanation of thermosonication mechanism of A.acidoterrestris inactivation,predictive kinetic modeling during the storage of the ultrasound treated orange juice and the illustration of germination process of A.acidoterrestris spores.Generally,this thesis was decomposed into six experimental chapters,and the main contents and results of the study are as described:1.TS was applied to optimize the quality characteristics of orange juice and to inactivate A.acidoterrestris vegetative cells using the Box-B ehnken design by varying the temperature(4575℃),time(20-40 min),and frequency(20-60 kHz).Results obtained showed that the optimal conditions for TS were 47℃,30 min,and 20 kHz.Based on higher R2 and lower standard deviation values,the quadratic model was chosen to predict the quality characteristics of the orange juice.The models generated of each response were highly significant(p<.001)with the coefficient of determination between the range of 0.986-0.996.The optimal conditions demonstrated high total phenolic contents(TPC)(490.85 mg GAE/100 ml),pectin methylesterase(PME)enzyme inactivation(94.77%)and low A.acidoterrestris vegetative cells’ load(1.00 log reduction).In addition,total flavonoids contents(38.50 mg/100 ml ER),ascorbic acid(441.70 mg/ml),color change △E(1.85),polyphenol oxidase enzyme(56%),and antioxidant capacity(FRAP,107.33 g/100 ml AEAC,DPPH,85.90%)of treated orange juice showed a significant(p<.05)difference compared to the control.Thus,TS significantly enhanced the quality of the natural orange juice.2.The effects of moderate thermosonication(MTS)on the quality quartet namely physicochemical,microbial,nutritional and sensory qualities of orange juice(OJ)inoculated with A.acidoterrestris(AAT)were studied during 24 days of storage at ambient and refrigerated temperatures.The bioactive compounds and antioxidant activity of OJ decreased with storage,while the pectin methyl esterase(PME)increased.Nonetheless,noticeable changes were observed from the 12th day of storage.There was no obvious(p>0.05)variation in pH and total soluble solids.To determine the nutritional and microbial quality characteristics of OJ during storage,non-linear kinetic curves were successfully fitted with least square fitting polynomial and four-parameter log-logistic distribution models.3.The E-nose sensors was efficient in discriminating between the aroma of non-treated and treated OJ based on linear discriminant analysis(LDA).AAT spoilage issues were characterized by taint production in which guaiacol is a predominant compound.Measuring of guaiacol content by HPLC and GC-MS has been recognized as a primarily effective strategy in diagnosing AAT induced spoilage in OJ.Furthermore,terpenes,alcohol and partially aromatic compounds were the main spoilage indicators of OJ during storage based on E-nose analysis and confirmed by HS-SPME-GC/MS analysis.Thus,MTS significantly extended the shelf life of the quality quartet of natural OJ at 4℃.E-nose-GC/MS fusion offered odor fingerprints to AAT microorganisms that can be used as spoilage index without using traditional food analysis techniques.The proposed approach can be used as an alternative tool for rapid detection of spoilage microorganisms in OJ.4.Ultrasound(US)and thermosonication(TS)treatments were used to investigate the inactivation of A.acidoterrestris,nutrients and enzymes of orange juice.The optimized results of TS(using Box Behnken design-response surface methodology)and further comparison with different modes of US(mono-and multi-frequency)revealed that 20/40 kHz,24 min and 64℃ were the best optimum conditions.The AAT spores and vegetative cells were inactivated by 2 and 4 logs,respectively,without deteriorating orange juice contents.In addition,AAT inactivation indicated an inversely proportional relationship with ROS production.FTIR and UV-Vis spectroscopy characterization confirmed the existence of ROS in treated orange juice and LF-NMR analysis confirmed the inactivation of AAT spores.The findings illustrated the successfully use of dual-frequency ultrasound technology for fruit beverages,including inactivation of microorganisms and enzymes,and promoting beneficial changes in physical properties without any significant effect on the quality of ascorbic acid.5.A.acidoterrestris was proposed as an index of pasteurization design for high-acid fruit products due to spore resistance and spoilage outbreaks in fruit juices.This study aimed to determine the effectiveness of pulsed multifrequency ultrasound to minimize AAT spores and vegetative cells in aqueous suspension.For this research,an investigation of the reactive oxygen species and antioxidant activity was performed to examine the effect of temperature and frequency on AAT spore inoculation.Total decreases in AAT bacteria were 5.99 and 5.74 Log CFU/mL for vegetative cells for both dual-frequency ultrasound(DFUS)and dualfrequency thermosonication(DFTS),respectively;and 5.90 and 5.38 Log CFU/mL in spores for both DFUS and DFTS,respectively.The loss of the percentage of cells in ultrasound(US)and thermosonication(TS)treatments was inversely associated with the rate of O2·-and H2O2 development.The fluorescence microscopy revealed a higher bactericidal efficacy of DFTS compared to the DFUS and control.SEM and TEM demonstrated ultra-structural modifications such as the interruption of cell walls by cavitation and pores in the membrane structure of the AAT bacteria induced by sonoporation.Several TS frequencies of 20/40/60,20/40,and 20 kHz treated spores had higher electrical conductivities than untreated ones,with an improvement of 7.94,5.68,and 3.72%,respectively.FTIR spectroscopy revealed major changes in the spectral region of membrane fatty acids and proteins of AAT.Simultaneously,AAT inactivation specific energy rate was significantly reduced using dual-frequency ultrasound,which provided the strongest performance than using mono-frequency ultrasound.The significant results of this work proposed pulsed DFUS as an alternative application to mono-frequency US in beverage industries.6.The use of dual-frequency thermosonication(DFTS)is considered an effective technique to inactivate A.acidoterrestris spores and to minimize nutritional loss and sensory quality of orange juice(OJ).DFTS could induce the germination bacterial spores making it more susceptible to inactivation.Nutrients such as L-alanine,L-valine and asparagine,glucose,fructose and potassium ion mixture(AGFK)may also trigger germination.The objective of this work was to decide whether active agents namely DFTS,nutrients(L-alanine and the AGFK mixture)and non-nutrient germinants(calcium dipicolinate,Ca-DPA)could influence the amount of the spore’s germination.The behavior of A.acidoterrestris spores was studied by AFM,electrical conductivity and protein content.AAT spores inactivated by 4 logs of A.acidoterrestris in OJ and complemented by L-ala showed equal germination percentages,as with the addition of no germinant.However,AAT spores in both treated and untreated citrate buffer solution and OJ samples germinated with L-ala significantly in DFTS-treated OJ.The germination potential can therefore be established as:L-alanine>no germinants>Ca-DPA>AGFK>L-valine.The findings showed that the degree of germination of A.acidoterrestris spores depended on the strain and medium,and that heat of DFTS often increases the spore germination rate.In summary,this research identified a practical and beneficial application of multifrequency thermosonication by inactivating A.acidoterrestris and pectin methylesterase and increasing the antioxidative activity of bioactive compounds in orange juice.