Progress 01/01/16 to 09/30/20
Outputs
Target Audience:Undergraduate and graduate students in food science/food safety Academic and regulatory researchers working in the area of food processing and food safety Fresh produce industry professional Changes/Problems:As sonocatalytic coatings were found to be not durable following the proposed coating methods and therefore not further pursued. However, we found that high frequency ultrasound in combination with other GRAS compounds were able to reduce bacterial load on food contact surfaces such as stainless steel (manuscript in preparation). Using liposomes as a model for bacterial membrane, we demonstrated that synthetically created microbubbles in combination with high-frequency ultrasound can perturb lipid bilayers. However, despite numerous trials, we did not observe its antimicrobial effect when bacteria were used. We attributed this contradiction to: (a) inadequate number of microbubbles per bacterial cells to cause meaningful stress on the later, or (b) bacterial response to stresses generated by cavitating bubbles was able to repair the membrane damage. Nevertheless, this led to a possibility that if we presented other GRAS antimicrobials with microbubbles, we may be able to enhance its uptake by bacterial cells due to moderate stress exerted by microbubbles. We tested this hypothesis using carbon dioxide bubbles and carvacrol coupled with 1 MHz ultrasound treatment. This was found to be effective (Food Control 121 (2021) 107580). This led us to recognize the significance of gas bubbles in enhancing microbial detachment and inactivation during washing fresh produce. In our second no-cost extension, we hoped to complete the remaining experiments and publish more manuscripts. While we partially accomplished this, COVID-19 significantly affected our lab's ability to perform experiments and delayed preparation of manuscripts. We are optimistic that we will submit two more manuscripts based on the research in this project. NIFA support will be acknowledged. What opportunities for training and professional development has the project provided?1 undergraduate student, 5 graduate students and 4 postdoctoral students were involved in the project. Each of these participants is a co-author on publications that resulted from this project. How have the results been disseminated to communities of interest?Theses and Dissertations, Peer reviewed publications and conference presentations What do you plan to do during the next reporting period to accomplish the goals?As mentioned in the final annual report, we are still working on two more manuscripts (experiments completed) that will be submitted for review in the coming months, after the submission of this final report. NIFA support will be acknowledged.
Impacts
What was accomplished under these goals?
Aim 1 We demonstrated that Zinc oxide nanoparticles, Erythrosine B, and propyl gallate can enhance bacterial inactivation upon interaction with low-frequency ultrasound. We attributed this enhanced microbial inactivation to formation of reactive oxygen species and/or bacterial membrane damage. However, these materials did not coat the stainless steel surface as well as planned and therefore, their evaluation was performed in solution alone. In another appraoch, we employed high-frequency ultrasound (1MHz) and propyl gallate to inactive biofilm formed by L.innocua on stainless steel surface. The combined high-frequency ultrasound (HFUS) and propyl gallate showed a synergistic biofilm reduction where 5 log bacterial inactivation was observed after 30 minutes of treatment. The detached bacterial cells were also inactivated by the combined treatment. Crystal violet assay indicated a significant biomass removal rate, at 70 to 80%. SEM results confirmed the removal effect of the combined treatment along with the membrane damage features in the L. innocua cells after treatment. Treated biofilm showed a suppressed metabolic activity. In another investigation, we evaluated role of low frequency ultrasound with and without surfactants and sanitizers to improve decontamination of fresh produce and their impact on the quality of fresh produce. Gram positive Listeria innocua, and gram-negative Escherichia coli O157:H7 and a soilage microbe Pseudomonas fluorescens were tested in this study. The reduction in bacterial concentration from inoculated leaf samples increased with an increase in ultrasound treatment time until 10 min for the three strains. Compared to ultrasound alone, combination of ultrasound with surfactants did not significantly increase the removal of E. coli O157:H7 and L. innocua from the lettuce surfaces, however, the removal efficacy of P. fluorescens increased significantly. Simultaneous washing of multiple leaves using ultrasound showed a net bacterial reduction from lettuce leaves by approximately 0.5 log CFU/cm2 for all the selected strains compared to treatment of a single leaf. Similarly, combination of ultrasound with surfactants for simultaneous treatment of multiple leaves did not improve removal of E. coli and L. innocua from the surface of lettuce leaves, but improved removal of P. fluorescens by approximately 1 log unit compared to ultrasound alone. Ultrasound could significantly reduce the risk of potential cross-contamination by reducing bacterial attachment to the uninoculated lettuce surface. Combination of ultrasound with adequate active free chlorine could prevent the transfer of E. coli and L. innocua cells from the inoculated leaf sample to uninoculated leaves. Exposure to ultrasound treatment for less than 10 min had no significant effect on the quality of lettuce. Aim 2 Using liposomal bilayers as a model for bacterial cell membranes, we investigated the extents to which high-frequency (1.0 and 3.3 MHz) ultrasound-induced microbubble cavitation phenomena are effective for bacterial inactivation. Förster Resonance Energy Transfer (FRET), acoustic scattering and a mathematical model were used to quantify the proximity of microbubbles to liposomes, establish if liposomes affect microbubble cavitation and propose bilayer alterations' mechanisms. Addition of a positive charge on microbubbles increased their proximity to liposomes through electrostatic attraction, however, dampened microbubble's oscillation compared to freely-floating microbubbles. Alleviation of energy transfer due to phospholipid mixing between microbubbles and liposomes established that close-proximity is necessary for bilayer alterations. Approximately 19% mixing of phospholipids was observed at 3.3 MHz due to microstreaming from stable cavitation, while inertial cavitation at 1.0 MHz increased this mixing to 50%. This method can damage bacterial membrane; but, whether bacterial defense mechanisms can attenuate this effect remains to be understood. Our experiments suggested that high-frequency ultrasound induced cavitation of microbubbles alone may not be effective in inactivating bacteria, but it may sensitize bacteria to further treatments by antimicrobials concurrently presented. To test this hypothesis, we compared the antimicrobial effects of ultrasound at 20 kHz (US 20 kHz) or 1 MHz (US 1 MHz) in combination with carvacrol, citral, cinnamic acid, geraniol, gallic acid, lactic acid, or limonene against E. coli K12 and Listeria innocua at a constant power density in water. Compared to the cumulative effect of the individual treatments, the combined treatment of US 1 MHz and 10 mM citral generated >1.5 log CFU/mL additional inactivation of E. coli K12. Similarly, combined treatments of US 1 MHz and 2 mM carvacrol (30 min), US 20 kHz and 2 mM carvacrol, 10 mM citral, or 5 mM geraniol (15 min) generated >0.5-2.0 log CFU/mL additional inactivation in L. innocua. The synergistic effect of citral, and US 20 kHz treatment was determined to be a result of enhanced dispersion of insoluble citral droplets in combination with physical impact on bacterial membrane structures, whereas the inactivation by US 1 MHz was likely due to generation of oxidative stress within bacteria. Combined ultrasound and citral treatments improved the bacterial inactivation in simulated wash water in presence of organic matter or during washing of inoculated blueberries but only additive antimicrobial effects were observed. In another investigation, we evaluated combination of high-frequency ultrasound (HFU) and a novel emulsifier, erythorbyl laurate (EL), against Listeria innocua and Escherichia coli O157:H7. Combining EL+HFU significantly (> 5 log CFU/mL, P<0.05) enhanced inactivation of both the bacteria compared to individual treatments. Aim 3 Based on the results of Aim 2 and to further evaluate the role of microbubbles, we explored the efficacy of low (20 kHz) or high (1 MHz) frequency ultrasound in combination with carvacrol and carbon doxide microbubbles as a treatment to decontaminate fresh produce using blueberries as a model system. We also evaluated its impact on the quality of blueberries during refrigerated storage. Blueberries inoculated with Listeria innocua, a model pathogen, were washed in presence of individual treatments by either 20 kHz ultrasound at (20 kHz), 1 MHz ultrasound at (1MHz), 2 mM carvacrol (CR), or carbonated water (CW) as well as their combinations at 20 °C for 10 min. Compared to individual treatments both 20kHz + CR and 1MHz + CR + CW led to significantly lower (P < 0.05) bacterial counts on blueberries' surface (~ 1 log additional inactivation). Quality of blueberries washed with these treatments was compared to those washed with sodium hypochlorite (SH) containing 100 ppm free chlorine, or water during storage. Unwashed blueberries were used as control. Blueberries washed by selected treatments had equal or lower (P<0.05) total aerobic and total mold and yeast counts compared to those treated by water, SH and the control. There was no influence of ultrasound washing processes on the total phenolic content and total anthocyanin content of blueberries, however, a significant but marginal impact on color and texture was observed. We identified several, relatively low-cost GRAS materials that can be used with low or high frequency ultrasound for enhanced bacterial detachment and inactivation in produce surface. While these compounds are more expensive than chlorine based sanitizers that are widely used, these treatments present potential benefits such as: (a) no formation of organochlorines, (b) potential re-use, and (c) potentially better removal and inactivation of bacteria. Both low and high frequency ultrasound are well-established and scalable technologies, but further investments are needed to scale these processes for produce washing.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Huu C.-N.; Yang X.; Rai R.; Tikekar R.; Nitin N. Synergistic inactivation of bacteria based on a combination of low frequency, low intensity ultrasound and a food grade antioxidant. Ultrasonics Sonochemistry.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Walsh M; Tikekar R.; Nitin N.; Wrenn S. (2020) Phospholipid Bilayer Responses to Ultrasound-Induced Microbubble Cavitation Phenomena. Journal of Food Engineering. https://doi.org/10.1016/j.jfoodeng.2020.110410
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang H.; Wang S.; Goon K.; Gilbert A.; Huu C.; Walsh M; Nitin N.; Wrenn S.; Tikekar R. (2020) Inactivation of foodborne pathogens based on synergistic effects of ultrasound and natural compounds during fresh produce washing. Ultrasonics Sonochemistry. https://doi.org/10.1016/j.ultsonch.2020.104983
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang H.; Tsai S.; Tikekar R. (2020). Inactivation of Listeria innocua on blueberries by novel ultrasound washing processes and their impact on quality during storage. Food Control. https://doi.org/10.1016/j.foodcont.2020.107580
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang H.; Tikekar R.; Ding Q.; Gilbert A.; Wimsatt S.(2020). Inactivation of foodborne pathogens by the synergistic combinations of food processing technologies and food-grade compounds. Comprehensive Reviews in Food Science and Food Safety. 19(4), 2110-2138. https://doi.org/10.1111/1541-4337.12582
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Huang K.; Wrenn S.; Tikekar R.; Nitin N. (2018). Efficacy of decontamination and a reduced risk of cross-contamination during ultrasound-assisted washing of fresh produce. Journal of Food Engineering. 224, 95-104. https://doi.org/10.1016/j.jfoodeng.2017.11.043
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Dolan H.; Bastarrachea L.; Tikekar R. (2018). Inactivation of Listeria innocua by a combined treatment of low-frequency ultrasound and zinc oxide. LWT-Food Science and Technology. 88, 146-151. https://doi.org/10.1016/j.lwt.2017.10.008
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Bastarrachea L.; Walsh M.; Wrenn S.; Tikekar R. (2017). Enhanced antimicrobial effect of ultrasound by the food colorant Erythrosin B. Food Research International. 100, 344-351. https://doi.org/10.1016/j.foodres.2017.07.012
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Zhang H.; Tikekar R. Synergistic Effects of Ultrasound and Natural Antimicrobials against Listeria innocua and Escherichia coli K12. IAFP Annual Conference, Louisville, KY. T7-02 and 13th International Conference on Engineering and Food. Melbourne, Australia.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
M. Walsh, R. Tikekar, N. Nitin and S. Wrenn; Bilayer Alteration through Ultrasound Induced Cavitation of Microbubbles; Colliod and Surface Science Symposium of the American Chemical Society, 2019 June 16-19; Georgia Institute of Technology, Atlanta, GA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
M. Walsh, S. Alborzi, R. Tikekar, N. Nitin and S. Wrenn, Ultrasound-Induced Interactions between Bilayers and Bubbles, Colliod and Surface Science Symposium of the American Chemical Society, 2018 June 10-13; State College,Pennsylvania.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
M. Walsh, S. Alborzi, R. Tikekar, N. Nitin and S. Wrenn, Ultrasound-Induced Interactions between Bilayers and Bubbles, Biointerfaces International; 2018 August 14-16; Zurich, Switzerland.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Nitin N. Synergistic Non-thermal Food Processing Solutions for Enhanced Microbial Inactivation, International Nonthermal Food Processing Symposium, Zhejiang.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Nitin N. Novel Approaches for Improving Fresh Produce Food Safety, International Conference on Food Science and Technology,
Davis, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Bastarrachea L.; Tikekar R. Enhanced antimicrobial effect of ultrasound by the food colorant Erythrosin B. 13th Conference of Food Engineering, Columbus, OH.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Dolan H.; Bastarrachea L.; Tikekar R.; Synergistic interaction between low-frequency ultrasound and sonocatalytic compounds ZnO and Erythrosine-B to inactivate microorganisms. IUFOST-18th world congress of food science and technology, Dublin, Ireland.
|
Progress 01/01/20 to 09/30/20
Outputs
Target Audience:Academic researchers and graduate students Changes/Problems:University resarch labs were shut down in the middle of March 202 due to COVID-19 pandemic. It had significant impact on the experiments ongoing at the time. All the labs resumed research activities between June and Junly 2020, albeit with severe restrictions. This delayed completion of some of the experiments. Therefore, there are still three manuscripts which we had hoped to submit before the end of the project that are is still in preparation. NIFA support will be acknowledged in these manuscripts. What opportunities for training and professional development has the project provided?One PhD level graduate student (Martin Walsh) was trained in this project (Drexel). Dr. Walsh successfully defended his PhD dissertation in 2020. One PhD level student (Cuoung Huu- UC Davis) has his PhD dissertation based on ultrasound based treatments. One post-doctoral level research (Hongchao Zhang UMD) was also trained on this project. Three other graduate students had an opportunity contribute to a review paper led by Hongchao Zhang during this performance period. How have the results been disseminated to communities of interest?Peer reviewed publications What do you plan to do during the next reporting period to accomplish the goals?We will work on writing the manuscripts based on some of the recently completed studies.
Impacts
What was accomplished under these goals?
In this performance period, our primary goal was to complete any remaining experiments associated with ongoing studies and write and submit manuscripts to peer-reviewed journals. To that end we completed following studies. These studies were intiated in 2019 and completd in the first part of 2020 and therefore have similar abstracts as 2019 Progress Report. As we discussed previously, we did not observe increased microbial inactivation by inclusion of positvely or negatively chared microbubbles in high freqeuncy insonated water. We hypothesized that it may be due to poor interaction between microbubbles and microbubbles. To test this hypothesis, we explored how high-frequency (1.0 and 3.3 MHz) ultrasound-induced microbubble cavitation can damage a liposome bilayer, a model for bacterial cell membranes. Förster Resonance Energy Transfer (FRET), acoustic scattering and a mathematical model were used to quantify the proximity of microbubbles to liposomes, establish if liposomes affect microbubble cavitation and propose mechanisms of bilayer alterations. Positive charge on microbubbles increased their proximity to liposomes through electrostatic attraction. Electrostatic tethering dampened microbubble's oscillation compared to freely-floating microbubbles. Alleviation of energy transfer due to phospholipid mixing between microbubbles and liposomes established that close-proximity is necessary to impact bilayer architecture. Approximately 19% mixing of phospholipids was observed at 3.3 MHz due to microstreaming from stable cavitation, while inertial cavitation at 1.0 MHz increased this mixing to 50% . This method can damage bacterial membrane; but, bacterial defense mechanism can attenuate this effect potentially explaining why did not observe enhanced microbial inactivation. Nevertheless, these results indicate that interatcion of microbubbles wiht bacterial memrabes may weaken the later and increase the susceptibility of bacteria to other antimicrobials presented in the same environment. Our results from another study (abstract below), supports this possibility. We explored the efficacy of ultrasound in combination with carvacrol and carbonated water containing microbubbles of carbon dioxide as a treatment to decontaminate fresh produce using blueberries as a model system. We also evaluated its impact on the quality of blueberries during refrigerated storage. Blueberries inoculated with Listeria innocua, a model pathogen, were washed in presence of individual treatments by either 20 kHz ultrasound at (20 kHz), 1 MHz ultrasound at (1MHz), 2 mM carvacrol (CR), or carbonated water (CW) and their combinations at 20 °C for 10 min. Individual treatments resulted in around 2 log CFU/g L. innocua reductions on the blueberries' surface with 3-4 log CFU/mL viable bacteria remaining in the wash water. Comparatively, both 20kHz + CR and 1MHz + CR + CW led to significantly lower (P < 0.05) bacterial counts on blueberries' surface (~ 1 log additional reduction) with 2.6 and 2.9 log CFU/mL in the washed water. 20kHz + CR and 1MHz + CR + CW treatments were selected to further investigate their effects on blueberry quality compared to washing with sodium hypochlorite (SH) containing 100 ppm free chlorine, or water during subsequent 4 °C storage for up to 20 days. Blueberries washed by selected treatments had equal or lower (P<0.05) total aerobic and total mold and yeast counts compared to those treated by water, SH and the control. There was no influence of ultrasound washing processes on the total phenolic content and total anthocyanin content of blueberries, however, a significant impact on color and texture was observed. The findings showed that washing blueberries with novel combined ultrasound washing processes have the potential to enhance the safety and shelf-life of ready-to-eat blueberries. In another line of investigation focused on developing a synergistic antimicrobial processing approach using a combination of low frequency ultrasound and a food grade antioxidant, propyl gallate (PG). The experimental design included evaluation of the antimicrobial activity of this approach using a model gram positive and gram negative bacteria using the synergistic approach. Bacterial inactivation data was complemented by phenomenological characterizations of membrane damage, nucleic acid leakage and the role of oxidative stress in the synergistic antimicrobial activity. Studies using bacteria model systems was also compared with the results of perturbations in membrane structures using a lipid membrane model systems. The results of these studies illustrate that synergistic combination of US and PG significantly (more than 4 log) enhanced inactivation of both the model gram positive and the gram negative bacteria. The results also illustrate that the model gram positive had a higher resistance to inactivation compared to the model gram negative bacteria. The phenomenological studies illustrate significant role of membrane damage and the lack of oxidative stress in the synergistic inactivation of bacteria using the combination of low frequency US treatment. Evaluation using the model lipid membrane illustrate significant role of PG in rapidly inducing membrane damage but not significant synergistic increase in membrane damage with the US treatment and illustrate that the synergistic membrane damage observed in bacteria could be a result of both membrane damage and intracellular processes in bacteria. In summary, this study illustrate development of a novel synergistic antimicrobial approach using a combination of low frequency US and a food grade antioxidant. Success in developing low cost non-thermal processing approaches can significantly enable industrial adoption of these technologies for improving both food quality safety of diverse food products.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang H.; Tsai S.; Tikekar R. Inactivation of Listeria innocua on blueberries by novel ultrasound washing processes and their impact on quality during storage. Food Control. 121, 107580. Published online ahead of print. https://doi.org/10.1016/j.foodcont.2020.107580
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Walsh P.; Tikekar R.; Nitin N., Wrenn S. Phospholipid Bilayer Responses to Ultrasound-Induced Microbubble Cavitation Phenomena. Journal of Food Engineering, revision submitted.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang H.; Tikekar R.; Ding Q.; Gilbert A.; Wimsatt S. Inactivation of foodborne pathogens by the synergistic combinations of food processing technologies and food-grade compounds. Comprehensive Reviews in Food Science and Food Safety. 19(4), 2110-2138. https://doi.org/10.1111/1541-4337.12582. Work on manuscript revision completed in 2020.
|
Progress 01/01/19 to 12/31/19
Outputs
Target Audience:Acadmic researchers, graduate and undergraduate students, fresh produce industry, sanitation industry Changes/Problems:Our intitial approach of using phosopholid stabilized microbubbles with high frequency ultrasound to enhance microbial inactivation was not successful as no additional antimicrobial effect was observed. To understand the reason behind this, we performed additional experiments using phospholipid vesicles that mimic bacterial membranes. The results from these studies gave us some understanding of why we did not observe increased microbial inactivation. The dominant factors that would determine the potential antimicrobial action were (a) ability of microbubbles to seek and attach to bacteria, and (b) ability of bacteria to potentially repair changes in bacterial membrane as a result of bubble cavitation. A manuscript is being prepared to highlight this. Interestingly, we observed that cabonated water bubbles were able to enhance bacterial removal from fruit surfaces when combined with ultrasound treatment. This has created a potential to explore this approach further. Preliminary results are already being prepared into a manuscript. What opportunities for training and professional development has the project provided?1 undergraduate student, 4 graduate students and 1 postdoctoral student received training related to these projects. They are also co-authors on the publications submitted in this time period. How have the results been disseminated to communities of interest?Conference presentations and peer-reviewed publications What do you plan to do during the next reporting period to accomplish the goals?-Complete the remaining experiments around microbubbles and ultrasound treatments and finish submission of manuscripts -Perform preliminary cost-benefit analysis
Impacts
What was accomplished under these goals?
Aim 1: Develop sonocatalytic coatings for a low-frequency sonochemical process for enhanced inactivation of spoilage microorganisms on food contact surfaces In the food processing environment, the presence of biofilms on food-contact and non food-contact surfaces can cause serious consequences to consumer health due to the risk of causing foodborne diseases and outbreaks. Biofilm depicts a challenge to the food industry due to its inherent resistant to disinfection methods. A novel synergistic bacterial inactivation using high-frequency ultrasound (1Mhz) and food additive propyl gallate was employed to inactive biofilm formed by L.innocua on stainless steel surface, a commonly used material in the food industry. The combined high-frequency ultrasound (HFUS) and propyl gallate showed a synergistic biofilm reduction where 5 logs CFU bacterial inactivation was observed after 30 minutes of treatment time. The detached bacterial cells were also inactivated by the combined treatment. Crystal violet assay indicated a significant biomass removal rate as a 70 to 80% of biomass from the biofilm network was removed. SEM results confirmed the removal effect of the combined treatment along with the membrane damage features in the Listeria cells after treatment. The treated biofilm showed a suppressed metabolic activity as represented by the resazurin assay where the peak of enzymatic activity was significantly delayed compared to the control sample. The combined HFUS and PG treatment allows the removal and inactivation of L.innocua biofilm with a complete bacterial inactivation both on the stainless steel surface and in the solution. Aim 2: Develop food-grade microbubble assisted high-frequency sonochemical process for enhanced inactivation of spoilage microorganisms in fresh and cut produce The ability of ultrasound-induced cavitation of microbubbles to impacts cells and bacteria is firmly established. However, in our experiments with high frequency ultrasound and microbubbles, we did not observed enhanced microbial inactivation. To understand the reason behind this, we examined the interactions of acoustically driven microbubbles (MBs) with neighbouring phospholipid vesicles (that mimic bacterial cell wall) (PVs) in two different architectures: MBs mixed with PVs and MBs tethered to the outer leaflet of PVs. These architectures allow us to study proximity effects for a variety of acoustic parameters, including driving frequency, peak negative pressure (PNP), and duty cycle. Using a combination of ultrasound attenuation and acoustic spectra, we identify threshold ultrasound intensities corresponding to stable and inertial cavitation and concomitant acoustic microstreaming and shockwave formation, respectively, for each architecture. Looking at the two different architectures revealed that the MBs mixed with PVs had no interference with MBs cavitation but when MBs were tethered to PVs the MB cavitation decreased at around 2 MPa. These results show that the proximity of the MBs and PVs have a significant effect on the MB cavitation. This is believed to be due to the proximity and the tethering of the MBs to the PVs. Additionally, using a combination of Förster resonance energy transfer (FRET) and ultrasound we investigated the phenomenon of pore formation, permanent damage to the PVs or phospholipid transferring between the MBs and PVs. The FRET pair was placed on the MBs and mixed with PVs in the two different architectures with variety of ultrasound parameters. These results showed only changes in the FRET when the MBs were tethered with PVs and had an increase in the change of FRET when the intensity of the ultrasound was increased. The increase in ultrasound intensity can be related to an increase in the cavitation of the MBs creating a greater chance in lipid transferring, pore formation, or permanent damage to the PVs. Using a combination of electrical sensing zone (Multisizer 4e Coulter Counter) and dynamic light scattering (DLS) with these results, we identify there that the MBs' lipids were transferred and mixed within the PVs' bilayers. The mechanism by which is not fully developed but we believe it to be from a combination of pore formation and lipid shedding from the MBs while they are oscillating. In another study, A synergistic combination between high-frequency therapeutic ultrasound (1Mhz) and food additive propyl gallate was used to inactivate bacteria in aqueous and clarified apple juice environments. Two strains of food-borne pathogen surrogates, a non-Shiga toxin-producing E.coli O157:H7 and L.innocua were investigated. The study showed that the combined high-frequency ultrasound (HFUS) and propyl gallate (PG) was highly efficient in inactivating the selected bacteria in both aqueous and acidic clarified apple juice, and a 5 to 6 logs CFU bacterial reduction within 10 to 15 minutes was observed. A strong synergistic interaction between HFUS and PG was verified by constructing the isobologram model. It appears that Gram-negative E.coli was more susceptive to the combined treatment than Gram-positive L.innocua represented by a shorter treatment time (lower inactivation rate constant) and a more potent cellular damage feature. SEM results demonstrated a notable morphology change and deformation in treated E.coli cells compared to the intact L.innocua cells. Intracellular thiol reduction data suggest that the mechanism of bacterial inactivation was mainly due to acoustic cavitation generated free radicals that were toxic to bacterial cells. This observation was further fortified by the protective effect on the tested bacteria by the addition of antioxidants. The combined HFUS and PG facilitates the use of ultrasound as a killing step and enables more production of value-added "green label" and organic product that benefits both the food producer the consumers. Aim 3: Evaluate the shelf-life of produce and risk of cross-contamination from the proposed technologies and perform cost-benefit analysis We explored the efficacy of ultrasound in combination with carvacrol as an alternative treatment to decontaminate fresh blueberries and extend their shelf life. Blueberries inoculated with Listeria innocua (~ 5.0 log CFU/g) were washed by the combinations of 2 mM carvacrol (CR), ultrasound at 20 kHz (US20), ultrasound at 1 MHz (US1), and carbonated water (CW) compared to individual treatments and the control (water wash) at 20 ± 3 °C for 10 min. Effective combined treatments were selected to further investigate their effects on blueberry quality compared to no treatment and 100 ppm Chlorine (CH) during subsequent 4 °C storage for up to 20 days. Individual treatments resulted in around 2 log CFU/g L. innocua reductions on the blueberries' surface with 3-4 log CFU/mL viable bacteria remaining in the wash water. Comparatively, both US20 + CR and US1 + CR + CW led to significantly lower (p<0.05) bacterial counts on blueberries' surface (~ 1 log additional reduction) and in the washed water (>1.7 log additional reductions). Blueberries washed by US20 + CR and US1 + CW + CR also had equal or lower (p<0.05) aerobic (1-2 logCFU/g) and mold and yeast (1-3 log CFU/g) counts compared to those washed by CH. The lightness of blueberries washed by US20 + CR and US1 + CW + CR was lower (p<0.05) than other groups after 7 days. Further, significantly lower (p<0.05) firmness was observed for blueberries washed by US20 + CR compared to other treatments. However, total phenolic and anthocyanin content of blueberries remained comparable (p>0.05) as the unwashed blueberries. The findings showed that washing blueberries with ultrasound combining with carvacrol or carbonated water have potential to increase safety and shelf-life of ready-to-eat blueberries.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Zhang H.; Wang S.; Goon J.; Gilbert A.; Huu C; Walsh M; Nitin N.; Wrenn S.; Tikear R. Inactivation of foodborne pathogens based on synergistic effects of ultrasound and natural compounds during fresh produce washing. Ultrasonics Sonochemistry. Work completed in 2019, manuscript submitted in 2019, accepted in Jan 2020, in press. https://doi.org/10.1016/j.ultsonch.2020.104983
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Zhang H.; Tikear R. Synergistic Effects of Ultrasound and Natural Antimicrobials against Listeria innocua and Escherichia coli K12. IAFP Annual Conference, Louisville, KY. T7-02 and 13th International Conference on Engineering and Food. Melbourne, Australia. .
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Zhang H.; Tikekar R.; Ding Q.; Gilbert A.; Wimsatt S. Food pathogen inactivation by the synergistic combinations of food processing technologies and food grade compounds. Comprehensive Reviews in Food Science and Food Safety, in review.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
M. Walsh, R. Tikekar, N. Nitin and S. Wrenn; Bilayer Alteration through
Ultrasound?Induced Cavitation of Microbubbles; Colliod and Surface Science Symposium of the American Chemical Society, 2019 June 16-19; Georgia Institute of Technology, Atlanta, GA.
|
Progress 01/01/18 to 12/31/18
Outputs
Target Audience:Food industry, fresh produce industry, scientists, regulators and academicians Changes/Problems:-A significant challenge we encounteed this year was to identify operational envelope where high frequency ultarsound can achieve inactivation of microbes. -Microbubbles, while ruprture lipid bilayers upon high frequency ultrsound induced cavitation, do not inactivate microorganisms. We are investigating what may contribute to this difference in effect. What opportunities for training and professional development has the project provided?- 3 graduate students and 2 postdoctoral students worked on this project in 2018 collaboratively. Exoeriments were performed jointly between UMD, Drexel and UC Davis. -Students also had an opportunity to present their findings at a scientific meeting -One manuscript is presently in preparation. How have the results been disseminated to communities of interest?Conference presentations What do you plan to do during the next reporting period to accomplish the goals?- Complete the ongoing experiments on ultrasound enhanced inactivation of microbes - Evaluate the impact of successful processes on produce quality and shelf-life and perform cost-benefit analysis - Write manuscripts and present findings at scientific reports
Impacts
What was accomplished under these goals?
Synergistic Effects of Ultrasound and Natural Antimicrobials Against Listeria innocua and Escherichia coli K12 Fresh produce washed with chlorine-based sanitizers are often ineffectively disinfected causing serious foodborne disease outbreaks. The objective of this study is to investigate the potential of using ultrasound and food grade natural antimicrobials to improve fresh produce sanitation processes. Inactivation capacities of a 20kHz ultrasound probe device (US20) or a 1MHz ultrasound therapy device (US1) combined with 9 different natural compounds(Carvacrol, Citral, Cinnamic acid, Geraniol, Lactic acid, Linoleic acid, Limonene, Gallic acid and Octanoic acid) was studied in water solutions (containing 2-5% ethanol) against Listeria innocua or E. coli K12 (stationary phase, 106-107 CFU/mL) at the power density of approximate 200 W/L at 22 ± 2 °C. Results showed that after 5 min both ultrasound treatments alone did not reduce bacterial population (p>0.05), expect for US20 which reduced E.coli K12 by 1.5 ± 0.1 log. For synergistic treatments, US20 together with Carvacrol (2mM), Citral (10mM) or Geranoil (5mM) induced additional 2.5-3.0 log reductions of Listeria innocua compared to the additive inactivation from US20 and each antimicrobial alone in the end of 15 min. US1 with Carvacrol (2mM) induced additional 1.0 log reduction at 15 min and 3.0 log reductions at 30min of Listeria innocua. US1 with Citral (10mM) or Geranoil (5mM) also showed potential synergistic antimicrobial effects for E.coli K12. Antimicrobial effect mechanism was evidenced by increased intracellular oxidative stress of each synergistic treatment (e.g. relative fluorescent value increased from 0.85 ± 0.08 to 2.02 ± 0.47 for Cavacrol, p<0.05). While no changes were observed for single ultrasound or antimicrobial treatments as well as the negative controls (gallic acid). Additionally, morphology of treated bacteria was also measured by TEM to understand the physical damage to bacterial cellular structures.Preliminary evaluation of treated produce showed no visible damage to the quality of produce. Further investigation is ongoing. Microbubble assisted, high-frequency induced inactivation of bacteria: understanding the mechanism using lipid bilayers The mechanism by which ultrasound-induced cavitation of microbubbles affect the phospholipid bilayers of bacteria were observed using a combination of Förster resonance energy transfer (FRET), ultrasound harmonic imaging, and electrical sensing zone (Multisizer 4e Coulter Counter) with microbubbles (MB) and negatively charged phospholipid vesicles (PVs) to mimic bacteria. Observing the ultrasound harmonics of the MBs help understand if the MBs are oscillating linearly or non-linearly at various pressures. Ultrasound harmonic imaging was used to look at the cavitation behavior of neutral MBs, positively charged MBs, and positively MBs mixed with PVs. These results revealed that the PVs had no interference with MBs cavitation. Next, FRET efficiency was examined in two different methods: FRET pair on the PVs or the acceptor on the PVs and donor on the MBs. Both these methods showed no change in the FRET efficiency in the presence of high frequency ultrasound with either a focused transducer or therapeutic ultrasound machine (Intelect Legend Transport Ultrasound). We concluded that the interactions of the FRET pair is to close with each other where any alteration to the phospholipid bilayer from the microbubble cavitation will not display any change in the FRET efficiency.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
M. Walsh, S. Alborzi, R. Tikekar, N. Nitin and S. Wrenn, "Ultrasound-Induced Interactions between Bilayers and Bubbles," Colliod and Surface Science Symposium of the American Chemical Society, 2018 June 10-13; State College, Pennsylvania.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
M. Walsh, S. Alborzi, R. Tikekar, N. Nitin and S. Wrenn, "Ultrasound-Induced Interactions between Bilayers and Bubbles," Biointerfaces International; 2018 August 14-16; Zurich, Switzerland.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Synergistic Non-thermal Food Processing Solutions for Enhanced Microbial Inactivation, International Nonthermal Food Processing Symposium, Zhejiang, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Novel Approaches for Improving Fresh Produce Food Safety, International Conference on Food Science and Technology, Davis, CA.
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Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Scientists working in the area of food science, processing and safety Food industry professionals Graduate students and post-doctoral associates Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Provided training opportunities for three postdoctoral scholars and two graduate student How have the results been disseminated to communities of interest?Peer reviewed publications What do you plan to do during the next reporting period to accomplish the goals? Analyze the interactions between multilamellar liposomes and microbubbles with FRET to expand the characterize of the interaction of bacteria and microbubbles Investigate non-clinical ultrasound's effect on the leakage of small unilamellar liposome vesicle and multilamellar liposomes in the presence of neutrally and positively charged microbubbles Measure the destruction of E. Coli when under non-clinical ultrasound with microbubbles Characterize the antimicrobial activity of sono-activated food grade compounds and understand the mechanistic aspects of synergy between compounds and ultrasound. Determine the influence of both high frequency and low frequency systems on sono-catalytic activity of food grade compounds Develop spectroscopic assays to assess biological damage induced by the synergistic combination of US and food grade compounds
Impacts
What was accomplished under these goals?
Observed the effects of Erythrosin B and Zinc Oxide had on the cavitation of microbubbles in the presence of ultrasound Detected the surface tension of Erythrosin B solutions Measured the leakage of a fluorescent dye within a liposome from neutrally charged microbubbles and positively charge microbubbles' cavitation with clinical ultrasound to relate to bacteria destruction Examined the interaction of neutrally and positively charged microbubbles with negatively charged liposomes using fluorescence resonance energy transfer (FRET) Assessed the mole percent of Dimethyldioctadecylammonium's effect on the zeta potential of the microbubble Measured the influence of ultrasound for enhancing removal of both human and plant pathogens from fresh produce surface Assessed the combination of ultrasound and chlorine for improving sanitation of fresh produce Estimated the level of shear force required to achieve more than 1 log removal of inoculated microbes from fresh produce Discovered novel sono-activated food grade compounds for inactivation of E.coli and listeria in aqueous environment Developed spectroscopic assays to characterize interactions of ultrasound and sono-activated compounds using model lipid carriers
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Dolan H.; Bastarrachea L.; Tikekar R.(2018). Inactivation of Listeria innocua by a combined treatment of low-frequency ultrasound and zinc oxide. LWT-Food Science and Technology. 88, 146-151.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Bastarrachea L.; Walsh M.; Wrenn S.; Tikekar R. (2017). Enhanced antimicrobial effect of ultrasound by the food colorant Erythrosin B. Food Research International. 100, 344-351.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Huang K.; Wrenn S.; Tikekar R.; Nitin N. (2018). Efficacy of decontamination and a reduced risk of cross-contamination during ultrasound-assisted washing of fresh produce. Journal of Food Engineering. 224, 95-104.
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:The target audience for the inevstigation performed during current reporting period is scientific community working in the area of food processing, and food safety Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the reporting period, the project directly involved 2 graduate students and 3 post-doctoral associates across the three collaborating research labs. This project formed a MS thesis for one graduate student. How have the results been disseminated to communities of interest?The results obtained were presented at: Conference of Food Engineering, 2016 IUFoST conference, Dublin, Ireland, 2016 One manuscript is under review, 2 manuscripts are being prepared for submisison in january 2017. What do you plan to do during the next reporting period to accomplish the goals?1. Focus on coating the materials identified to work synergisticlaly with ultrasound on food-contact surafces and evaluate their efficacay. 2. Conduct studies to evaluate the microbubble assisted ultrasound technology for inactivating microorganisms.
Impacts
What was accomplished under these goals?
Aim 1: During this reporting period, the synergistic combination of the food colorant Erythrosin B (FD & C 3) (0, 25, and 50 mM) and low frequency ultrasound (20 kHz, 0.86 - 0.90 W mL-1) was evaluated against Listeria innocua. Although Erythrosine B was antibacterial by itself, the inactivation rate significantly increased in a concentration dependent manner upon exposure to ultrasound and followed a sigmoidal behavior. The inactivation rate in a sequential treatment, where L. innocua was sonicated for 4 minute followed by exposure to 25 mM Erythrosin B, was comparable to that obtained by the simultaneous treatment, indicating complementary mechanisms of inactivation. Fluorescence microscopy showed attachment of Erythosin B to the cells, which may explain its intrinsic antimicrobial property. Other mechanism may include decrease in the cavitation threshold of water by addition of Erythrosin B, resulting in more effective cavitation. The study offers a proof-of-concept of a novel approach to complement ultrasound treatment for enhanced microbial inactivation. During this reporting period, the efficacy of an alternative sanitation method combining low-frequency ultrasound with zinc oxide (ZnO) in inactivating Listeria innocua was determined. 6 log L. innocua was treated with 20 or 40 mM ZnO, and sonicated (20-100kHz, 43-45 W, 120 mm) at room temperature for 0-30 min. Combined treatment with 40 mM ZnO and 8 min. ultrasound resulted in >5 log reduction in L. innocua. The combined treatment's antimicrobial mechanism appears to be Reactive Oxygen Species (ROS)-mediated. Addition of histidine, a known quencher of hydroxyl radicals and singlet oxygen, significantly attenuated the antimicrobial effect of the combined treatment. ZnO nanoparticle's also play a physical role in the treatment's antimicrobial effect. ZnO nanoparticle size measurements showed a decrease in particle size from ~250 nm to ~100 nm after sonication, while SEM images of L. innocua suspended in 40 mM ZnO imply that ZnO associates with the bacteria. Aim 2: Several microbubbles have been developed, and preliminary investigation regarding their efficacy to inactivate microorganisms has been explored. However, due to technical errors, the study was not successful. We will primarily focus on this aim in the second year. Aim 3: During this reporting period, the possibility of application of ultrasound in decontamination of pathogenic and spoilage microorganisms from fresh lettuce surface was demonstrated. The exposure to ultrasound treatment with distilled water for 10 min resulted in an enhanced removal of bacteria from lettuce leaves. The maximum populations of bacterial removed from lettuce surface were 2.61 ± 0.13 log, 2.23 ± 0.12 log, and 1.10 ± 0.06 log CFU/cm2 for E. Coli, Listeria and P. fluorescens.Compared to ultrasound treatment alone, Ultrasound in combination with 0.1% of Tween-20 or 0.1% of SDS did not significantly increase the removal of E. coli and L. innocua from the lettuce surfaces, however, the efficacy of reduction against P. fluorescens increased significantly in the presence of surfactants.After the extended incubation period, the resistance of E. coli and L. innocua to ultrasound treatment increased significantly. But there was no significant difference in the P. fluorescens cell persistence on the lettuce surface between 1 h and 24 h incubation. Compared to single-leaf treatment, large-mass ultrasound caused a 0.5 log units of decrease in decontamination efficacy. Meanwhile, ultrasound decreased the percentage of bacterial attachment to the uninoculated lettuce surface, suggesting the potential risk of cross-contamination during large-mass treatment could be reduced by ultrasound. Combination of ultrasound and free chlorine (15 ppm) was demonstrated to be able to prevent the cross-contamination during the washing process.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Enhanced antimicrobial effect of ultrasound by the food colorant Erythrosin B. Journal of Food Engineering
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Enhanced antimicrobial effect of ultrasound by the food colorant Erythrosin B
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Synergistic interaction between low-frequency ultrasound and the sonochemical compounds zinc oxide and erythrosin B
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