Progress 06/01/17 to 11/30/20
Outputs
Target Audience:The target audience will the the food industry who manufacture and hold juices and beverages, testing laboratories that conduct challenge and validation studies and state and federal regulatory agencies enforcing Juice HACCP. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The findings of the project was originally scheduled to be trial in a High Pressure Processing and validation workshop at the Institute for Food Safety and Health, IIT in April 2020. This workshop was to give training to industry users and laboratory scientists who conduct studies using HPP. The workshop will also provide an overview of how challenge studies should be conducted for HPP treated juices. However, the workshop was cancelled due to COVID-19 and rescheduled for late 2021/early 2022. A session in partnership with the Cold Pressure Council was scheduled in September 2020 but due to COVID-19, the session was rescheduled to September 2021. The findings of the project will be presented to industry members so that they understand how to interpret and conduct appropriate challenge studies for their juice products. The project findings were also presented at the virtual Food Safety Summit 2020 in the session Validation of Non-thermal Technologies and the information was well received. How have the results been disseminated to communities of interest?The results have been disseminated to the scientific communities via the International Association for Food Protection annual scientific meetings and the IAFP European Symposium. PhD candidate, Catherine Rolfe, was awarded 3rd Place in the 2019 IAFP Developing Scientist Award for her work. Similarly, the information was also shared with FDA and industry at the IFSH Juice and Beverage Safety Task Force annual meeting. We have three manuscript in preparation or under review and will make those manuscript available to interested parties once they are published. The industry engagement from juice manufacturers, HPP equipment manufacturers and regulators have been fantastic with active engagement during the course of the project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The objectives for #3 was combined in part with objective #2 and #4 as the protocol for recovery were address in Objectives #2 and #3. The impact of recovery procedures post-HPP treatment were also addressed in Objective #2. The purpose of the project was to develop validation guidelines for HPP inactivation and post-HPP recovery of pressure resistant and matrix-adapted E. coli O157:H7, Salmonella spp., and Listeria monocytogenes in HPP-treated juices. Main objectives included: screen bacterial isolates for barotolerance and select isolates for validation studies, assess the effects of juice matrices and define HPP parameters, determine impact of recovery methods on enrichment procedures, define protocol for shelf-life analysis, and provide framework for guidance document related to HPP-treated juices. Ten strains of each microorganism were prepared in three growth conditions (neutral, cold-adapted, or acid-adapted) and assessed for barotolerance or sensitivity. Cold-adapted cells were prepared at 17°C. Acid-adapted cells were prepared in intermediate pH 5.0 broth. Approximately 6 log CFU/mL of bacterial strains were inoculated into buffer (pH 3.5). Pressure treatment applied at sublethal levels for the initial bacterial screening. Analyses were conducted 0h, 24h and 48h (4°C storage) post-HPP. Pressure resistant and sensitive strains from each (6 strains with neutral, cold-adapted, or acid-adapted growth conditions) were used to evaluate HPP inactivation with increasing pressure levels (200 - 600 MPa) in juice matrices (apple and orange). A 75-day shelf-life analysis was conducted on HPP-treated juices inoculated with acid-adapted resistant strains for each pathogen and examined for inactivation and recovery. Individual strains of E. coli O157:H7, Salmonella spp., and L. monocytogenes demonstrated significant (p <0.05) differences in reduction levels in response to pressure treatment in high acid environments. It was found that E. coli O157:H7 was the most barotolerant of the three microorganism in multiple matrices. Bacterial screening resulted in identification of pressure resistant strains E. coli O157:H7 TW14359, Salmonella Cubana, and L. monocytogenes MAD328, and pressure sensitive strains E. coli O15:H7 SEA13B88, S. Anatum, and L. monocytogenes CDC. HPP inactivation in juice matrices (apple and orange) confirmed acid adaptation as the most conservative preparation compared to neutrally grown and cold-adapted. Shelf-life studies conducted for 75 days in cold storage with mild temperature abuse reached a 5-log reduction in HPP-treated juices immediately following pressure treatment for L. monocytogenes MAD328, after 24 h in cold storage for S. Cubana, and after 4 days of cold storage for E. coli O157:H7 TW14359. Recovery of injured cells was observed for L. monocytogenes in orange juice but still maintained a 5-log reduction. These results suggest the preferred inoculum preparation for HPP validation studies is the use of acid-adapted, pressure resistant strains. At 586 - 600 MPa, critical inactivation (5-log reduction) was achieved during post-HPP cold storage, suggesting sufficient HPP lethality is reached at elevated pressure levels with a subsequent cold holding duration. The shelf-life studies concluded with unexpected results. The microorganism reoccurring during shelf-life studies for orange juice was L. monocytogenes which was previously thought to be the most sensitive to pressure application compared to E. coli O157:H7 and Salmonella spp. Further, modifications to the E. coli O157:H7 recovery and enrichment procedures in both apple and orange juice did not prove substantial for sublethally injured cells. Salmonella results were as predicted with continued loss in viability following pressure treatment and lack of recovery following refrigerated storage. Limited research has been conducted on the effects of pressure application on membrane permeability and injured cell recovery. The disruption of the cell membrane during HPP treatment may lead to altered response mechanisms against selective agents included for enrichment procedures. Pressure induced cell damage may allow for better recovery of injured cells that have undergone stress adaptations. A thorough understanding of the effects of high pressure on E. coli O157:H7 membrane structure and function is necessary to develop proper recovery mediums and procedure. In addition, exploration of how L. monocytogenes stress adaptation influences recovery during cold storage conditions would provide beneficial guidance for shelf-life applications. Further genetic studies would be advantageous to conceptualize the gene expression following acid adaptation, pressure treatment, and cold storage, especially in a complex juice matrix. In relation to guidance development, the information generated from the project is being synthesized into manuscript and guidance document are also being developed with FDA collaborators. Consumer interest in functional beverages and minimal processed juices has led to increasing research of juice products treated with minimal processing. HPP has been shown to provide effective inactivation of pertinent microorganisms in high acid juice products to comply with the FDA Juice HACCP rule in various studies. The current study demonstrated the variability associated with individual strains of E. coli O157:H7, Salmonella spp., and L. monocytogenes when treated in high acid food environments. The results of this research propose barotolerant and acid-adapted strains of juice-associated pathogens should be used in cocktail inoculations for validation studies of HPP-treated juice. A pressure level of 586 MPa achieved a 5-log reduction of E. coli O157:H7 after 4 days in cold storage for apple and orange juice, however, data suggests increasing pressure and increasing hold times may provide greater inactivation. Further research on target pressure and hold time combinations in relation to different juice types would be beneficial. Additional assessment of acid-adapted E. coli O157:H7 enrichment methodology from high acid juices would provide reassurance of complete inactivation.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Rolfe C, Lee A, Anderson N and Black G. (2020). Influence of acid adaptation, cold adaptation on barotolerance on survival of E. coli O157:H7, L. monocytogenes, and Salmonella spp. during HPP treatment of apple juice. J. Food Protect. 83:1-288.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Lee A. Novel processing technologies Validation, Application, Regulation. Food Safety Summit Conference and Expo. Rosemont, IL, May 4-7, 2020.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2020
Citation:
Catherine Rolfe PhD thesis - submitted and degree awarded Dec 2020
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Progress 06/01/18 to 05/31/19
Outputs
Target Audience:The target audience include juice and beverage companies both domestic and international who exports to USA or manufacture juices and beverages in USA, HPP toll processers, HPP equipment manufacturers. The project results have been presented at the Cold Pressure Council where members are from the juice and beverage industry. The results have been presented at scientific conferences and meetings such as IAFP Europe and IAFP Annual Meeting in USA. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project allowed for the professional development of PhD student, Catherine Rolfe and exposed her to several opportunities to present her work. She is a finalist at the 2019 IAFP Annual Meeting Developing Scientist competition. How have the results been disseminated to communities of interest?Yes. The results have been disseminated to industry partners and members of the Cold Pressure Council. The project was also pick up for a writeup by Food Quality and Safety magazine. What do you plan to do during the next reporting period to accomplish the goals?In the September 2019 meeting with industry partners to discuss the initial draft of the guidance document. IT will also help formulate subsequent research activities and streamline the work involved in objectives #3 and #4. Some work may be needed to use another juice matrix such as orange juice to confirm results seen in apple juice as orange juice has a different organic acid. The data will then lead to bacterial recovery procedures and if the recovery procedures are impacted by the juices. Similarly, establishing additional data using another juice with a different organic acid will allow better evaluation of survival or inactivation during a shelf-life study since the pertinent microorganism may change with a different organic acid. For example, in the apple juice study, E. coli O157:H7 seem to be the pertinent microorganism that survive post-HPP storage but not L. monocytogenes. However, in orange juice, the pertinent microorganism may change.
Impacts
What was accomplished under these goals?
The objectives accomplished were objectives #1 and #2. During the second year of the project, data on individual strain behavior in combination with high pressure treatment in buffer and apple juice matrices were collected. Using the bacterial strain selection conducted in Year 1 of the project, growth of individual strains ofE. coliO157:H7,L. monocytogenes, andSalmonellaspp. in various trypticase soy broth with 0.6% yeast extract (TSBYE) conditions were collected (Table 1). The pH levels were recorded at the end of incubation in the presence of pH neutral TSBYE at 37°C (24 h incibation), acid-adapted growth conditions with pH 5.0 TSBYE at 37°C (24 h incibation forE. coliandSalmonella, 48 h incubation forL. monocytogenes), and cold-adapted growth conditions with neutral TSBYE at 17°C (48 h incubation). The most HPP resistant and most HPP sensitive strains tested to date from the culture collection for E, coli O157:H7, Salmonella spp. and Listeria monocytogenes were selected for further study in apple juice using bacterial cells derived from three different growth conditions - neutral pH growth medium, cold adapted cells and acid adapted cells, and pressure treated at 200 - 600 MPa. The samples were stored 24 and 48 h post HPP treatment and microbiological evaluated at 24 and 48h. E. coliO157:H7 exhibited barotolerance compared toSalmonellaspp. andL. monocytogenes. In BPW neutral growth conditions,E. coliO157:H7 strain TW14359 demonstrated resistance (<3 log reduction) andE. coliO157:H7 strain SEA13B88 was most sensitive. Acid-adaptedL. monocytogenesstrain MAD328 had <1 log reduction whileL. monocytogenesstrains CDC and ScottA were most sensitive in BPW.Salmonellaisolates grown in neutral and acid-adapted conditions had comparable results (<1.5 log reduction), while cold-adaptedS.Cubana andS. Montevideo showed resistance compared to other strains. Bacterial inactivation in apple juice showed similar strain inactivation to BPW but incremental barotolerance was observed. Progressive loss of viability occurred from all post-HPP storage samples.The currentresults suggest HPP is an effective inactivation method forE. coliO157:H7,L. monocytogenesandSalmonella, however, matrix composition, bacterial strain selection and preparation methods and process parameters can influence overall inactivation levels.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
International Association for Food Protection (IAFP) European Symposium, April 25, 2019, Nantes, France Bacterial strain selection for the validation of high-pressure-treated juices [Technical Presentation]
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
Cold Pressed Juice is a Hot Trend, May 23, 2019, Food Quality and Safety (https://www.foodqualityandsafety.com/article/cold-pressed-juice-trend/?singlepage=1)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
2019 Cold Pressure Council Annual Conference, March 26-27, 2019 - Update on the Juice Task Force Progress for All [Invited Presentation] (pmmi.docsend.com/view/qdzz4wp)
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Progress 06/01/17 to 05/31/18
Outputs
Target Audience:In Sept 2017, the project team interacted with the External Advisory Board, comprising industry members,Juice Product Association and FDA, to discuss and refine project plans and protocols and to ensure outcomes of the work stayed relevant to the industry. The project was also part of the larger IFSH Juice and Beverage Safety Task Force and as a whole, agreed to screen bacterial strains currently used in challenge and validation studies for high pressure processed juices. The discussion resulted in Dr. Lee presenting the project to members of the Juice Product Association Annual Meeting in November 2017 at Atlanta. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The current project provided training and professional development for a PhD candidate, Ms. Catherine Rolfe. How have the results been disseminated to communities of interest?The results have been discussed with FDA. The project team will present the data in the coming IFSH Juice and Beverage Safety Task Force meeting on 25 Sept 2017 at the Marriott South West Chicago, Burr Ridge, IL as part of the IFSH Annual Meeting. A depper dive on the results obtained to date will be presented to the High Acid Workgroup within the Task Force and next steps of the project will also be discussed. What do you plan to do during the next reporting period to accomplish the goals?The project team intends to continue working through the objectives of the projects. Work on bacterial strain screening occured using a buffered system and model juice using a low pH high acidsingle strength example of apple juice and another using high pH (
Impacts
What was accomplished under these goals?
Screen bacterial isolates for HPP resistance, select final sets of bacterial isolates for validation studies and quantify the effect of juice matrices on bacterial inactivation In the first year of the project, Objective 1 on screening bacterial isolates for HPP resistance or barotolerance was conducted with 15 bacterial isolates comprising ofE. coliO157:H7 (F4546, M-11-0450i-1, Sakai, SEA13B88, and TW14359),Salmonellaspp. (Anatum, Cubana, Montevideo, Newport J1894, Saintpaul, and Typhimurium) andL. monocytogenes(1838, CDC, F2365, MAD328, and ScottA) strains.Pressure resistance was determined through inoculation into buffered peptone water (BPW) adjusted to pH 3.5 ± 0.1 using hydrochloric acid (HCl) at an inoculum level of approximately 6 log cfu/mL followed by sublethal pressure treatment, 500 MPa forE. coliO157:H7 strains and 200 MPa forSalmonellaspp.andL. monocytogenesstrains, for 180 seconds with an initial temperature of 4°C. Analysis was performed immediately following pressure treatment, 24h and 48h after pressure treatment (stored in 4°C) to allow for recovery of sublethally injured cells. Evaluation of matrix-adapted strains was also performed by growing the bacterial inoculum in three different conditions: trypticase soy broth with 0.6% yeast extract (TSBYE) at pH 7 incubated in 37°C overnight, TSBYE at pH 7 incubated in a 17°C waterbath for 48h, and TSBYE at pH 5 incubated in 37°C overnight (48h forL. monocytogenesstrains). TheE. coliO157:H7 strains demonstrated the most barotolerant of the three microorganisms (Figure 1). Following pressure treatment at 500 MPa with growth conditions in pH neutral broth and 37°C incubation overnight, theE. coliO157:H7 TW14359 strain demonstrated the greatest resistance with a 2.94 log cfu/mL reduction after day-of analyses.Under equal conditions,E. coliO157:H7 SEA13B88 showed to be the most sensitive with 5.75 log cfu/mL reduction.In growth conditions for acid adaptation, similar results were observed withE. coliO157:H7 TW14359 demonstrating the most resistance (3.62 log cfu/mL reduction) andE. coliO157:H7 SEA13B88 demonstrating the highest sensitivity (5.82 log cfu/mL reduction). For the cold-adapted cells,E. coliO157:H7 Sakai showed the greatest resistance towards high pressure with 4.12 log cfu/mL reduction after day-of analyses.E. coliO157:H7 SEA13B88 continued to show the highest sensitivity. Figure 1.Effect of HPP on inactivation ofE. coliO157:H7 grown in various growth conditions of (a) 37°C in pH neutral TSB, (b) 17°C in pH neutral TSB, and (c) 37°C in pH 5 TSB. HPP treatments were conducted at 500 MPa for 180s at an initial temperature of 4°C with analyses performed following treatment. TheSalmonellaspp. isolates showed dissimilarities compared to theE. coliO157:H7 andL. monocytogenesstrains with notable differences between the neutral and acid-adapted cells compared to the cold-adapted. A sublethal pressure of 200 MPa was used in order to recoverSalmonellaspp. after HPP treatment.Salmonellaspp. grown in neutral broth with incubation overnight at 37°C showed minimal variance between the strains,S.Cubana demonstrated the highest resistance (0.82 log cfu/mL reduction) andS.Anatum was the most sensitive to pressure (1.32 log cfu/mL reduction) following day-of analyses. Using acid-adapted cells,S.Anatum andS.Cubana were most barotolerant with similar log reductions of 0.66 log cfu/mL and 0.68 log cfu/mL, respectively (Figure 2). The least resistant strains wereS.Newport J1894 with 1.09 log cfu/mL reduction andS.Typhimurium with 1.07 log cfu/mL reduction. An increase in loss of viability was observed when cold-adapted cells were used whereS.Montevideo demonstrated the highest resistance with 3.51 log cfu/mL reduction andS.Saintpaul was the most sensitive with 4.68 log cfu/mL reduction. Figure 2.Effect of HPP on inactivation ofSalmonellaspp. grown in various growth conditions of (a) 37°C in pH neutral TSB, (b) 17°C in pH neutral TSB, and (c) 37°C in pH 5 TSB. HPP treatments were conducted at 200 MPa for 180s at an initial temperature of 4°C with analyses performed following treatment. ForL. monocytogenesstrains, pressure was applied at 200 MPa to achieve a sublethal treatment.L. monocytogenesgrown in neutral broth and incubated overnight at 37°C showed the highest resistance to 200 MPa withL. monocytogenesMAD328 being the most resistant with 2.41 log cfu/mL reduction and the most sensitive strain beingL. monocytogenesCDC with 4.06 log cfu/mL reduction after day-of analyses (Figure 3). Similar behavior was observed from the acid-adapted cells.L. monocytogenesMAD328 demonstrated barotolerance andL. monocytogenesScottA demonstrated sensitivity with reductions of 0.97 log cfu/mL and 3.43 log cfu/mL, respectively. However, inoculum levels of 6 log were difficult to achieve in an acidic growth environment. Average control inoculum of acid-adaptedL. monocytogenesranged from 5.36 to 6.05 log cfu/mL. The cold-adapted cells presented an alternative pattern withL. monocytogenes1838 showing the highest resistance at 0.91 log cfu/mL reduction andL. monocytogenesScottA least resistant with 4.11 log cfu/mL reduction. Figure 3.Effect of HPP on inactivation ofListeria monocytogenesgrown in various growth conditions of (a) 37°C in pH neutral TSB, (b) 17°C in pH neutral TSB, and (c) 37°C in pH 5 TSB. HPP treatments were conducted at 200 MPa for 180s at an initial temperature of 4°C with analyses performed following treatment. For allE. coliO157:H7,Salmonellaspp. andL. monocytogenesstrains evaluated in this study, recovery analysis performed after 24h and 48h of cold storage (4°C) post-HPP treatment did not deliver higher yields of recovery. Progressive loss of viability was observed. Current work consists of additional trials using 100% apple juice from concentrate at a pH of 3.5 to assess the effect of a juice matrix compared to BPW. Pressure resistant and sensitive strains ofE. coliO157:H7 TW14359,E. coliO157:H7 SEA13B88,S.Cubana,S.Anatum,L. monocytogenesMAD328 andL. monocytogenesCDC will be used.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Enhancing the Safety of High Pressure Processed Juices and IFSH Juice and Beverage Safety Task Force, Juice Products Association Annual Meeting, Atlanta
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