Mechanistic And Methodological Studies On Pasteurization Of In-shell Walnuts Using Radio Frequency Energy

Raw walnuts are increasing in popularity among consumers due to their high nutritional values.However,many outbreaks have been associated with contaminated tree nuts or nut components added to foods.Contaminants on in-shell walnuts can survive for prolonged periods and multiply rapidly under suitable conditions.The safety of walnuts is related to human health.Thus,an efficient method for pasteurizing in-shell walnuts is needed.As a relatively novel thermal treatment,radio frequency(RF)energy has been widely used for the pasteurization of agricultural products.RF heating can maintain product quality while meeting the pasteurization requirements.However,it lacks in-depth systematic research on the distribution of pathogens in in-shell walnuts,mechanistic and methodological studies on pasteurization of in-shell walnuts using RF energy.In this study,we chose in-shell walnuts as the representative food to conduct mechanism research on RF pasteurization.The objectives of this study include:(1)the high-throughput sequencing technology was used to analyze the bacterial diversity on the surface of in-shell walnuts in different regions and storage times and investigate the possible bacterial pathogens on the in-shell walnuts surface;(2)the influence of temperatures,water activity and heating rate on the thermal death kinetics of target pathogens was evaluated using a heating block system(HBS);(3)the morphological change and the response triggered by water activity and heating rate of the target strain were studied based on molecular biology and transcriptome analysis;(4)the protocol of RF pasteurization for in-shell walnuts was developed and validated,and the influence of RF treatment on in-shell walnut quality and microorganism survival was investigated;and(5)the pasteurization mechanism of RF heating on pathogens at cell and gene expression levels according to molecular biology and transcriptomics was determined.The main results are as follows:(1)The bacterial diversity on in-shell walnuts surface was investigated by high-throughput sequencing(HTS)based on the bacterial 16S rRNA hypervariable region V4.The results showed that the composition of the bacterial community on the in-shell walnuts surface was similar,while the abundance varied among these walnuts in different regions and storage times.Proteobacteria,Cyanobacteria,Firmicutes and Actinobacteria were the most dominant phylum.Enterobacteriaceae was the dominant one at the family level.Some genera belonging to the opportunistic pathogens were detected,and Staphylococcus also existed in many samples.The distribution of bacteria in walnuts was similar to that in soil,water,and some field crops,indicating that walnuts could be contaminated by the external environment during harvesting and processing.(2)A heating block system(HBS)was used to compare the heat resistance of three representative strains in walnut shell powders.The most heat-resistant strain was chosen to conduct the thermal death kinetics at different temperatures,water activity and heating rate.The results showed that the D values of S.aureus ATCC 25923 increased with decreasing water activity at the same temperature.The D values of S.aureus ATCC 25923 were not significantly different among 1,5 and 10oC/min,while the D values increased with decreasing heating rates(<1oC/min).At the heating rate of 0.1°C/min,the population reductions of S.aureus ATCC 25923 could reach to 2.91(±0.1)log CFU/g during the come-up time from26°C to 56°C.The population reductions of S.aureus ATCC 25923 during the come-up time from 44°C to 50°C had a slight increase,which may be caused by the heat-resistant protein produced by S.aureus ATCC 25923.The inactivation under non-isothermal conditions was better fitted by Weibull distribution(R~2=0.97 to 0.99)than first-order kinetic model(R~2=0.88to 0.98).(3)The mechanism of the heat resistance of S.aureus ATCC 25923 affected by water activity and heating rate was explored by scanning electron microscopy,sublethal damage detection,and transcriptome sequencing.The results showed that the cell integrity of S.aureus ATCC 25923 inoculated in samples was damaged after heat treatment,especially in samples with high water activity values.Based on the results of RNA sequencing(RNA-seq)analysis,differentially expressed genes(DEGs)were mainly related to transcriptional regulator,energy metabolism,membrane transporters,membrane and cell wall synthesis.The cells in samples with low water activity prevented external damage by stimulating the synthesis of cell wall components and inhibiting membrane function related metabolism.The suppression of energy metabolism in samples at lower heating rate indicated that cells under this condition needed to reduce metabolic activity to survive.(4)A protocol for pasteurizing in-shell walnuts by using radio frequency heating was established,and the influence of RF treatment on in-shell walnut quality and microorganism survival was evaluated during storage.The results showed that the RF heating uniformity of in-shell walnuts was worse at higher moisture content.While the uniformity of RF heating was improved by combining with hot air,product mixing and holding for 40 min during treatment.The optimized RF treatment protocol for in-shell walnuts involved pre-heating between an electrode gap of 16.0 cm,followed by drying for 40 min between an electrode gap of 19.0 cm with the RF system continuously turned on for 30 s and then turned off for 1 min to hold the target temperature,and the process finished with 14 min forced air cooling of the walnuts in a single layer.This RF treatment protocol produced more than 4 log reductions of S.aureus ATCC 25923.The mean FA and PV values of samples after RF treatment were slightly higher than those of controls but within the acceptable range(PV<1.0 meq/kg and FA<0.6%)required by nut industry.Peroxide values of RF treated samples increased to more than 1 meq/kg during storage at 35°C for 20 days but showed no significant differences from controls while fatty acid values of both the control and RF treated samples were below 0.6%during storage.There were no significant differences in L*values of ground kernel color after RF heating,drying and storage.A rapid initial decline for all control samples was observed at two storage conditions of 30oC,30%relative humidity and 30oC,65%relative humidity.While the populations of S.aureus ATCC 25923 in control samples gradually tended to stabilize with the increase of storage time.The population of S.aureus ATCC 25923 in RF treated samples was gradually reduced to below the detection limit(1 log/nut)and there was no growth of S.aureus ATCC 25923 during the storage period.(5)The pasteurization mechanism of radio frequency heating on S.aureus ATCC 25923was explored by scanning electron microscopy,sublethal damage detection,extracellular ATP detection,and transcriptome sequencing.The results showed that the cell integrity of S.aureus ATCC 25923 was severely damaged after RF treatment.The degree of damage was more severe with increasing RF heating time.According to the results of transcriptome analysis,differentially expressed genes(DEGs)were mainly related to nucleic-acid metabolism,translation,energy metabolism,cell membrane transport,cell membrane and cell wall biosynthesis.After RF treatment,the cell wall synthesis was depressed and cell membrane homeostasis was disrupted.Meanwhile,the translational activity and energy metabolism were also affected,which eventually led to the death of S.aureus ATCC 25923.