HPP Studies: Microbial Challenge, Process Optimization, Process Validation and Shelf-Life

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04/02/2025 Added

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HPP Studies: Microbial Challenge, Process Optimization, Process Validation and Shelf-Life. Studies by members of the HPP and Engineering Services Laboratory of The Food Processing Center - Prashant Dahal, Yhuliana Niño, Aryany Peña and Grace Danao. 2025 High Pressure Processing and Dehydration Workshop

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[00:00:00.000]Hi everyone, thank you so much for hanging on to the end of the workshop. So this afternoon,
[00:00:11.240]it is my pleasure to introduce to you my team. So I have a very small team here at the University
[00:00:17.840]of Nebraska-Lincoln, but I think it's a mighty team. We do a lot of services for the food
[00:00:22.360]industry, so this afternoon I've asked them to share with you how we go about doing our
[00:00:28.200]microbial challenge, process optimization, process validation, and shelf life studies.
[00:00:34.700]So they're also going to talk a little bit about some of the microbial limits that are
[00:00:39.120]required when conducting these studies, and some of the nuances and observations that
[00:00:44.120]we've seen in the lab. So a little bit of things that we've learned over the years doing
[00:00:50.520]these studies. So parts of the team are, first you'll hear from Arjani Pena, then you'll
[00:00:56.720]hear from Prashanta Hall.
[00:00:58.160]Then Juliana Niño-Fuerte, who is my PhD student. And then finally, I'm going to come back right
[00:01:04.440]in as a bookend and talk a little bit about our shelf life studies.
[00:01:08.720]So just a little bit more about our laboratory. Here's the folks that you'll hear from today.
[00:01:14.340]So Juliana, Prashant, Arjani. We do have a student intern every once in a while. So we
[00:01:19.520]have Fiorella Guevara, who most of you have met during the workshop. So she's going to
[00:01:24.400]be around learning more about HPP technology and also free.
[00:01:28.120]So definitely I do provide quite a bit of consultation and workshops like this is kind
[00:01:38.880]of a great way for us to train the new leaders in the food industry when it comes to both
[00:01:43.400]HPP and freeze drying. Certainly we cover process optimization and as I mentioned, microbial
[00:01:49.920]challenge and validation and shelf life studies.
[00:01:52.880]So without further ado, I give the floor to Arjani Peña to start talking about microbial
[00:01:58.080]challenge studies.
[00:01:59.080]Thank you, Dr. Danao, for the introduction. So my name is Arjani Peña and I'm going to
[00:02:04.440]start talking about the different microbial challenge studies that we conduct here in
[00:02:08.840]the HPP lab. And I would like to start by defining what a microbial challenge study
[00:02:14.080]is. So a microbial challenge study, it's a laboratory experiment in which a food product
[00:02:20.320]is usually intentionally inoculated with microorganisms of interest to see how they behave under
[00:02:26.560]certain conditions.
[00:02:28.040]In our case, to evaluate how they behave under high pressure processing, HPP. Just as the
[00:02:35.300]other speakers have mentioned throughout the workshop, HPP is a technology that can be
[00:02:39.900]implemented to a wide variety of products while keeping their fresh attributes from
[00:02:46.520]pet food to dairy products to ready to eat meals for humans.
[00:02:51.740]So but we are talking about microbial challenges. So we definitely need to determine what are
[00:02:58.000]the organisms that we want to control in these products. What are those pathogens of concern
[00:03:04.320]and where do we get this information from? Maybe from historical testing of the products,
[00:03:11.960]data from outbreaks or recalls and also information from the scientific literature. For example,
[00:03:20.060]we observe in this slide some of the previous products such as fresh guacamole, pet food,
[00:03:27.960]deli meat and oysters and these products have been involved in recalls here in the
[00:03:33.180]United States. For example, fresh guacamole has been linked with potential Listeria monocytogenes
[00:03:40.560]contamination. These bacteria along with Salmonella have been associated with pet food. Salmonella
[00:03:48.460]as well is being found in orange juice. Deli meats have been associated with Listeria outbreaks
[00:03:55.200]and oysters with Vibrio.
[00:03:57.920]For sure we need and want to control these pathogens in these products, but how does
[00:04:03.640]actually HPP control or destroy these microorganisms? In the screen we see the different protein
[00:04:13.520]structures: primary, secondary, tertiary and quaternary structures. So what pressure does
[00:04:20.920]is to unfold and refold the secondary structure of proteins. And usually when the pressure
[00:04:27.880]is released, those damages or changes are irreversible. That means that the proteins
[00:04:34.300]are not able to conduct their functions, and that's translated into cell membrane damage,
[00:04:41.400]organelle and pH disruption, and in general it compromised several cellular functions,
[00:04:48.860]which means the bacteria are not healthy anymore. It's also important to mention that thicker
[00:04:55.480]cell walls are more resistant.
[00:04:57.840]to pressure, which means that gram-positive bacteria will be more resistant to high-pressure
[00:05:03.900]processing. Also, being bacteria living microorganisms, they have the ability to repair themselves,
[00:05:12.180]which means that sometimes we're going to observe recovery of them during storage period.
[00:05:18.820]On the other hand, being viruses not living microorganisms, they are relatively easy to
[00:05:25.480]inactivate using pressure.
[00:05:27.800]Also, they are mostly made of proteins.
[00:05:30.900]In fact, it's been found that high-pressure processing is an effective technology to inactivate
[00:05:37.400]viruses in high-risk products, such as fresh fruits and vegetables and seafood.
[00:05:44.440]In this table, we observed inactivation rates for human norovirus and hepatitis A. For example,
[00:05:52.920]we see a reduction of three log PFU of human norovirus in oysters.
[00:05:57.760]When implementing a pressure of 400 MPa for one minute.
[00:06:03.920]As well, a reduction of 4.3 and 4.7 of human hepatitis A in mashed strawberry and green
[00:06:13.280]onions when implementing 300 MPa for five minutes.
[00:06:18.920]The same virus, hepatitis A, was reduced for more than 4.6 logs in oysters.
[00:06:27.720]First we see difference because we are implementing different conditions and we are also talking
[00:06:33.480]about different products.
[00:06:36.120]And in fact, there are some intrinsic matrix factors that may have an influence in these
[00:06:42.000]reductions.
[00:06:43.000]For example, water activity and salt content.
[00:06:46.420]The higher the water activity, the higher the virus survival.
[00:06:50.680]Also the presence of salt provides a protective effect for viruses.
[00:06:56.560]Now that we are talking about salt.
[00:06:57.680]About these different factors, let's move on and see what are other elements that may
[00:07:02.740]have an influence on the reductions we observe when implementing pressure.
[00:07:07.940]And that's the case of the antimicrobials, which in fact have a synergistic effect when
[00:07:14.720]combined with pressure.
[00:07:16.900]In this study conducted in 2023, the effect of combining HPP and lactic acid was evaluated
[00:07:25.820]against salmonella in Roppe.
[00:07:27.640]And in this table, we observe the reductions obtained.
[00:07:34.080]For example, when implementing just the lactic acid without the addition of the HPP treatment,
[00:07:41.260]the reduction was around 1.1 log CFE per gram.
[00:07:45.560]On the other hand, when implementing the HPP treatment without adding lactic acid, the
[00:07:51.760]reduction was around 4.44.
[00:07:55.100]So in theory, if we combine
[00:07:57.600]the HPP treatment and the lactic acid, we will expect a reduction of around 5.5 log CFE.
[00:08:04.900]However, the reduction on all the replicates of this experiment was higher than the expected
[00:08:11.600]value.
[00:08:12.740]We observed reduction values higher than 6 and even higher than 7 log CFE per gram.
[00:08:19.880]So we can conclude that definitely there is a synergistic effect between HPP and lactic
[00:08:26.600]acid.
[00:08:27.560]Now, let's see what happened with other antimicrobials.
[00:08:33.120]This was an experiment conducted in our lab in which the effect of other organic acids
[00:08:38.920]in combination with HPP was evaluated to reduce salmonella in pork trims.
[00:08:46.460]The acids evaluated in this case were acetic, citric, lactic, and peracetic acid.
[00:08:54.880]We see their reductions just by the addition of the acids.
[00:08:57.520]Which were around 1 log CFU, the lowest 0.56, the highest 1.21.
[00:09:06.720]Now, on the other hand, when implementing just the HPP treatment, 300 MPa for 3 minutes,
[00:09:15.180]the reduction was 1.75.
[00:09:17.980]So if we combine the HPP with each of these acids, we will expect the sum of 1.75
[00:09:27.480]and the reduction obtained with the acids, which will give us values higher than 2 but
[00:09:35.720]lower than 3 log CFU per gram.
[00:09:38.820]But again, in most of the treatment combinations, we observed a higher reduction, with the exception
[00:09:47.060]of the peracetic acid, with a value of 2.08 log CFU per gram.
[00:09:54.480]So yeah, there is definitely a synergistic effect of
[00:09:57.440]combining HPP and antimicrobials, but it depends on the specific antimicrobial or acid that
[00:10:04.720]we are evaluating.
[00:10:07.560]Now let's continue and talk about the process optimization and validation studies.
[00:10:14.480]And a validation process is basically obtaining the evidence that a control measure or the
[00:10:21.180]combination of a control measure is effective to control a specific hazard.
[00:10:27.400]Where do we get this information from?
[00:10:30.460]We can use scientific literature, for example, previous validation studies, or we can conduct
[00:10:39.900]our own validation study itself, which we can actually conduct for you in our laboratory.
[00:10:47.200]We can also collect data during operating conditions or use mathematical modeling.
[00:10:57.360]If HPP is that killing step in a process, it must be validated to confirm its effectiveness.
[00:11:04.320]So we need to determine what are those parameters required to control a specific hazard.
[00:11:11.200]In our case, we need to determine what is that specific pressure and holding time required
[00:11:17.280]to reduce a pathogen by 5 log CFU per gram or by 99.999 percent.
[00:11:27.320]I'd like to point out that in the case of pressure, it's commonly used in industrial
[00:11:34.320]operation the maximum, which is 600 megapascals.
[00:11:38.980]And even though temperature might be another factor that influence those reductions, it
[00:11:45.200]is usually controlled by the total.
[00:11:47.920]So it is usually between 36 to 40 degrees Fahrenheit.
[00:11:54.040]So pressure is usually the maximum.
[00:11:57.280]Temperature is controlled.
[00:11:58.800]In our validation study, we're basically looking for that specific holding time required to
[00:12:04.320]control a specific pathogen of concern.
[00:12:08.960]So now I would like to call my lab mate, Prashant, who is going to continue talking about the
[00:12:14.020]step-by-step for this validation process and all the different considerations.
[00:12:20.760]All right.
[00:12:22.680]Thank you, Ariane, for defining what process validation study is.
[00:12:26.280]Now I would like to move forward.
[00:12:27.240]And talk about how do we conduct process validation studies in our lab.
[00:12:33.320]As you can see in the slide, it starts with the production.
[00:12:37.400]When the food is produced and packaged in a manufacturing unit,
[00:12:41.440]there is a chance of contamination right there.
[00:12:43.280]And after that, we put the food in the transportation truck.
[00:12:48.660]It goes to the coal store before it gets HPP pasteurized.
[00:12:52.300]So this time, between production to pasteurization,
[00:12:57.200]depends if you, as a manufacturer,
[00:13:00.020]if you have your HPP unit on your own,
[00:13:03.120]then this time will be short.
[00:13:05.160]Otherwise, if you are using the HPP tollers,
[00:13:08.120]this could be long, and this is a very critical factor.
[00:13:11.040]So now when your food retakes the HPP pasteurizing unit,
[00:13:14.860]it gets pasteurized.
[00:13:16.520]After that, you, as a manufacturer,
[00:13:18.740]you retrieve your HPP-treated samples.
[00:13:21.140]It goes back to the lab.
[00:13:24.200]There is some consideration in time as well.
[00:13:27.160]After HPP pasteurization happens,
[00:13:29.400]you load the food in the truck.
[00:13:31.440]It goes through distribution channel,
[00:13:34.220]and whether it goes to retail shop
[00:13:36.000]or whether consumer consumes it.
[00:13:38.040]That is what really happens in the industry.
[00:13:40.620]So now, in the validation report we create,
[00:13:44.220]we try to mimic this process.
[00:13:46.140]We try to mimic product, process,
[00:13:48.780]and especially the timing, as I already told.
[00:13:52.140]When we get the product,
[00:13:53.840]we inoculate with respective bacteria,
[00:13:56.220]similar to what really
[00:13:57.120]happens in the contamination phase.
[00:14:00.400]After that, we acclimate it
[00:14:02.520]at refrigeration condition for 24 to 72 hours.
[00:14:08.560]This time varies.
[00:14:10.060]It is dependent on what is the talk with the clients.
[00:14:15.040]It is a part of our consultation.
[00:14:17.200]How long is the time between production to pasteurization?
[00:14:22.240]How long it takes for you as a manufacturer is based on that.
[00:14:27.080]How long we acclimate the sample.
[00:14:30.620]Now we have samples which is acclimated.
[00:14:34.120]We HPP pasteurize it.
[00:14:36.380]And after that, here you can notice right here,
[00:14:40.620]we provide the pathogen inactivation data for day one.
[00:14:44.500]It is the next day after HPP.
[00:14:47.860]When you see different journal articles
[00:14:49.800]and different research papers, it talks
[00:14:52.600]about the reduction you get after HPP is you plate
[00:14:57.040]on the same day, which is day zero.
[00:14:59.280]However, in our validation report, we do day one,
[00:15:02.840]the next day after HPP.
[00:15:04.780]Here, we are trying to mimic the time
[00:15:08.360]that you retrieve your samples from HPP treated sample.
[00:15:12.980]It goes to lab.
[00:15:14.220]So we are trying to mimic this, and we are trying
[00:15:17.060]to make it more conservative by plating on day one.
[00:15:21.060]By doing so, we are also letting the bacteria to recover.
[00:15:25.260]Those bacteria which has been injured
[00:15:27.000]or subliterally injured after HPP, we are letting it
[00:15:30.400]to recover such that the data we get will be more conservative
[00:15:34.200]and perfect for validation studies.
[00:15:37.340]Now, after that, whether we leave for the pathogen die-off
[00:15:41.920]or whether we keep on monitoring,
[00:15:44.560]the reduction will be based on our consultation with the client.
[00:15:48.760]So all in all, our process validation studies we create is
[00:15:54.500]trying to mimic what really happens
[00:15:56.960]in the real-world scenario.
[00:15:59.620]Now, moving forward, let's talk about different bacterial
[00:16:02.700]strains and the conservative approach we take
[00:16:06.540]for stress adaptations.
[00:16:09.640]Strains. This is a very critical step.
[00:16:13.880]We select bacterial strains based on the historical outbreaks
[00:16:18.480]and also its relevance to the food product.
[00:16:21.780]In our lab, we have a plethora of bacterial strains.
[00:16:25.280]We make a cocktail of them.
[00:16:26.920]Before inoculation, making cocktail of them,
[00:16:30.480]it provides a conservative outlook
[00:16:33.520]on what the most resistant strain would look like
[00:16:36.460]in the HPP treatment condition.
[00:16:39.340]Next up, we have acid adaptation of E. coli O157H7.
[00:16:45.900]When we are validating the fruit juices,
[00:16:48.580]which has pH less than 4.6,
[00:16:51.780]we generally acid adapt E. coli O157H7.
[00:16:56.020]Previous outbreak,
[00:16:56.880]related to the fruit juices,
[00:16:59.320]there is a presence of--
[00:17:02.220]or the outbreak itself is because of the acid-tolerant
[00:17:05.320]E. coli O157H7.
[00:17:07.320]So for that, we acid adapt E. coli O157H7
[00:17:12.700]and inoculate in the fruit juices.
[00:17:16.700]Next, we have cold adaptation of Listeria monocytogenes.
[00:17:21.500]Listeria, which grows favorably at 32 degrees Celsius,
[00:17:26.840]when it's inoculated to food matrix
[00:17:30.000]and refrigerated in the acclimation condition,
[00:17:32.640]acclimated for 72 hours.
[00:17:36.180]This difference in temperature, it may kill Listeria,
[00:17:40.120]or there's a term called cold shock.
[00:17:43.120]Now, after that food matrix is HPP treated,
[00:17:47.900]the reduction we get would not be a proper demonstration
[00:17:51.440]of what lethality are we actually being just with HPP.
[00:17:55.840]So to avoid cold shock,
[00:17:56.800]we cold adapt Listeria monocytogenes before inoculation.
[00:18:01.800]Next, let's talk about inoculation level.
[00:18:07.820]This is a very critical step as well.
[00:18:10.520]If we are aiming for five log reduction,
[00:18:13.180]then if we're aiming for five log reduction,
[00:18:18.220]then we inoculate at around seven log or even eight log,
[00:18:22.660]such that even with five log reduction,
[00:18:25.060]we have some bacteria to count.
[00:18:26.760]Keeping in mind that we always have one log of detection limit.
[00:18:32.600]If we are aiming for one to three log reduction,
[00:18:36.200]then we inoculate with three or four log of bacteria,
[00:18:41.100]such that having one log detection limit
[00:18:43.680]and with two log of reduction,
[00:18:45.680]we are still able to count some bacteria.
[00:18:49.780]Next up, we have surrogates here.
[00:18:53.360]Can we use surrogates in the validation studies?
[00:18:56.720]Well, there are processing facilities
[00:18:59.280]who want to use surrogates in the validation study
[00:19:02.920]that they do in the processing facilities
[00:19:05.560]because they don't want to use pathogens right there.
[00:19:08.360]But if you are using surrogates in that case,
[00:19:11.100]there are a few things you need to consider.
[00:19:14.480]For that, let's understand what surrogates are.
[00:19:18.380]FDA defines surrogates are non-pathogenic species
[00:19:22.420]and strain responding to a particular treatment in a manner
[00:19:26.680]equivalent to pathogenic species and strain.
[00:19:30.680]So, surrogates are the one that is equivalent to pathogen.
[00:19:34.720]However, if you are using surrogates in the validation
[00:19:38.560]study, there must be a margin of safety.
[00:19:41.720]As Jay et al. in their book explained that this margin of
[00:19:46.100]safety, the surrogate must be a tad more resistant
[00:19:51.140]than the pathogen.
[00:19:53.140]As you can see in this graph alongside,
[00:19:56.640]the number of surrogate at any time of the treatment must be
[00:20:00.840]higher than the number of pathogen,
[00:20:03.220]ensuring that if surrogate dies in a certain treatment,
[00:20:07.420]then pathogen would definitely die.
[00:20:12.060]However, again going back to the definition,
[00:20:15.120]we need to consider whether surrogate is determined based on
[00:20:19.320]a particular treatment.
[00:20:21.560]It is a treatment dependent, meaning a surrogate that
[00:20:26.600]is identified in the thermal treatment,
[00:20:30.300]it may not work in the cold pressure condition.
[00:20:33.740]Well, to our experience, there are some bacterial surrogates.
[00:20:38.340]They work well in HPP.
[00:20:40.420]But I would like to highlight enterococcus fascium,
[00:20:43.880]which is a recognized surrogate of salmonella
[00:20:46.460]in thermal condition.
[00:20:48.220]It doesn't behave well in HPP.
[00:20:50.960]In an experiment, which was conducted at 600 megapascals for
[00:20:56.560]a minute, it was able to reduce salmonella by five log.
[00:21:03.140]However, the same treatment, it only reduced effacium
[00:21:07.440]by around two logs or so.
[00:21:09.840]So they were very resistant, and this effacium,
[00:21:13.880]it cannot be used as a surrogate in HPP condition.
[00:21:18.320]However, in the future, if surrogates and pathogens
[00:21:22.380]are tested in the same HPP at the same
[00:21:26.520]time for the same amount of run,
[00:21:29.280]then we will identify the surrogate that
[00:21:32.280]will work best with the pathogen in the validation
[00:21:35.200]study that could be conducted.
[00:21:38.080]Now, moving forward, let's talk about what
[00:21:40.760]are the considerations of the conservative approach
[00:21:43.920]we use in the laboratory.
[00:21:47.980]So for accurate pathogen enumeration,
[00:21:50.960]or to avoid false negative, we have some consideration
[00:21:54.200]right here.
[00:21:55.740]As Ariani already said--
[00:21:56.480]as Ariani mentioned--
[00:21:57.980]HPP inactivates the secondary structure of the protein.
[00:22:03.040]It breaks the cell wall.
[00:22:05.660]So these cell walls, the bacteria
[00:22:07.940]cells which have broken cell walls,
[00:22:09.860]are sublithally injured, which means
[00:22:12.800]they have potential to grow back if they
[00:22:16.400]find the favorable conditions, like in the food.
[00:22:19.700]However, these are viable, but not culturable.
[00:22:23.300]So when they are plated in selective media,
[00:22:26.440]they may not grow because selective media
[00:22:29.180]is known to grow for only healthy cells.
[00:22:33.080]So in order to prevent such kind of underestimation
[00:22:36.660]that may happen in the lab, we use a thin agar layer
[00:22:40.720]or an additional layer of nutrient media
[00:22:44.160]to gently recover those sublithally injured shell
[00:22:47.820]and allow them to form colonies to ensure
[00:22:51.160]a more realistic assessment of microbial load.
[00:22:54.960]To put this in picture,
[00:22:56.400]we have a case study right here.
[00:22:59.500]Another slide.
[00:23:00.780]Here we study the effects of cultured media.
[00:23:04.600]This experiment was conducted in our lab
[00:23:07.840]where salmonella was inoculated in chicken
[00:23:11.940]and the recovery of sublithally injured shells
[00:23:14.960]after HPP was studied in four different kinds of plates.
[00:23:19.520]On the left-hand side, there's XLD,
[00:23:22.200]which is a selective media for salmonella.
[00:23:25.260]And on the right-hand side,
[00:23:26.360]there are three different forms of plate:
[00:23:29.060]overlay of XLD over triptychsoi agar,
[00:23:32.440]which is a nutrient media which allows the growth,
[00:23:36.100]thin agar layer of TSA over XLD,
[00:23:39.500]and TSA with 0.35 percent ferric ammonium citrate.
[00:23:45.120]So, how were the results?
[00:23:49.040]This table summarizes what we found.
[00:23:52.520]The highlighted part at 300 megapascals
[00:23:56.320]in the XLD media,
[00:23:58.460]Salmonella was enumerated to be around 4.29 log CFU per gram.
[00:24:05.600]In all other three forms of media,
[00:24:07.940]overlay, thin agar layer, and MTSA,
[00:24:10.840]the number were higher.
[00:24:13.340]The enumeration in OL was 4.93.
[00:24:16.740]In TAL, it was 4.84.
[00:24:19.540]In MTSA, it was 5.16 log CFU per gram.
[00:24:24.180]And why is this difference?
[00:24:26.280]It is because in XLD,
[00:24:29.020]it is only allowing the healthy salmonella shells to grow.
[00:24:33.520]However, all other three plates,
[00:24:35.860]they were allowing the recovery of sublithally injured shell.
[00:24:39.520]So this number accounted for healthy
[00:24:41.760]and sublithally injured shell.
[00:24:44.200]Same trait was observed in higher pressure
[00:24:46.400]at 450 megapascals.
[00:24:49.340]In XLD, we noted here it is less than 1
[00:24:53.900]because I already mentioned
[00:24:56.240]we have a detection limit of 1 log.
[00:24:58.380]Anything less than 1 will be reported as 1.
[00:25:01.840]But in overlay and MTSA in these cases,
[00:25:05.240]the enumeration was more than 1.
[00:25:08.680]So the conclusion is using an extra layer
[00:25:12.720]of nutrient media over selective media,
[00:25:15.220]it helps the growth of healthy and sublithally injured shell.
[00:25:20.800]In the second case study here,
[00:25:24.300]the same thing was done, but the pattern
[00:25:26.200]of pathogen recovery dynamics was studied for a longer time.
[00:25:31.840]On the right-hand side, there is a TSA plus XLD overlay,
[00:25:36.580]and on the left-hand side,
[00:25:37.720]there is a such selective media XLD.
[00:25:41.080]If you see the red line right here at day 21, in XLD,
[00:25:47.860]the enumeration of salmonella was found to be
[00:25:50.360]around 3 log CFU per gram, but in TSA plus XLD,
[00:25:56.160]it was around 3.5 with the standard deviation reaching
[00:26:00.700]up to 5 log CFU per gram.
[00:26:03.840]This means the layer of non-selective media,
[00:26:07.640]it allows the growth of bacteria to recover and repair,
[00:26:10.880]and it doesn't just happen day one.
[00:26:13.380]This is a pattern.
[00:26:14.580]There is a consistent recovery throughout 21 days.
[00:26:19.520]There is enhanced bacterial repair,
[00:26:21.580]and the recovery was seen in TSA plus XLD.
[00:26:25.160]All in all,
[00:26:26.120]in this case study,
[00:26:27.260]it means that if you are using just the selective media,
[00:26:31.360]it is allowing the growth of healthy shells.
[00:26:33.960]However, when you are using extra layer of nutrient media
[00:26:38.160]with the selective media,
[00:26:40.300]the healthy and subliterally injured shells are grown.
[00:26:44.100]So in our validation studies,
[00:26:46.360]we use the second kind of the place.
[00:26:50.000]We use selective media and non-selective media together
[00:26:54.280]in a thin agar layer form.
[00:26:56.080]Next up, I'll talk about the considerations we apply
[00:27:01.440]during the processing conditions.
[00:27:05.380]We always provide the lower end of operational parameters
[00:27:09.320]while performing the validation studies.
[00:27:13.140]Here, I'm going to talk about pressure drift.
[00:27:17.660]So what really happens in the real world?
[00:27:20.460]When we put the food samples in the HPP unit,
[00:27:24.320]if we are treating for 600 megabytes
[00:27:26.040]per minute, we're trying to build up to 600 megapascals
[00:27:29.100]for five minutes.
[00:27:30.720]Initially, it takes some time to build the pressure up
[00:27:33.720]to 600 megapascals.
[00:27:35.340]After that, it tries to hold at 600 for five minutes.
[00:27:40.920]However, there is a chance that when
[00:27:43.260]it's trying to hold at 600 megapascals,
[00:27:45.520]the pressure may drop a little bit before the intensifier
[00:27:48.560]clicks in and it raises to 600 again.
[00:27:52.200]This small drop in pressure is called pressure drift.
[00:27:56.000]And to capture this, we always go for lower end
[00:28:00.080]of operational parameters, and in this case, the pressure.
[00:28:03.640]Meaning, if we are performing a validation study
[00:28:06.480]for 600 megapascals, then we do the study at 590 megapascals,
[00:28:13.580]ensuring that if 590 megapascals can kill bacteria,
[00:28:18.760]then 600 megapascals will definitely do that.
[00:28:22.620]Next up, we have sensitivity to hold.
[00:28:25.960]What is the holding time?
[00:28:28.820]As Ariane already told, when you are using the tooler,
[00:28:33.600]they usually keep temperature around 4 degrees Celsius.
[00:28:38.140]And the presser, they always try to use 600.
[00:28:40.980]So they try to play with the holding time.
[00:28:43.840]And we all know there is a variability,
[00:28:46.780]biological variability in food metrics.
[00:28:49.420]The differences in composition of fat, protein.
[00:28:53.220]If you are dealing with pet food, the formulation,
[00:28:55.920]or even the presence of bones in the meat.
[00:28:59.560]All these things matter.
[00:29:01.600]So there is a sensitivity to holding time
[00:29:04.200]that we need to consider.
[00:29:06.260]To put this in picture, we have a case study here.
[00:29:11.600]In this paper, three different kinds of chicken was used.
[00:29:15.880]A chicken, S chicken, and R chicken with a varying degree
[00:29:19.480]of protein, fat, and moisture.
[00:29:22.020]They were inoculated with Listeria monocytogenes, Salmonella,
[00:29:25.880]and Ciga toxin-producing E. coli.
[00:29:28.720]And the treatment was performed at 586 megapascals for two,
[00:29:33.480]three, and four minutes at four degrees Celsius.
[00:29:36.760]So what did we get?
[00:29:39.060]In the table alongside, you can see the effect
[00:29:42.440]of treatment time on Listeria monocytogenes reduction
[00:29:45.460]was observed.
[00:29:46.600]In two-minute treatment, in a chicken,
[00:29:50.380]the Listeria monocytogenes was reduced by a log of 2.23.
[00:29:55.840]In three-minute treatment, the reduction was increased to 3.11.
[00:30:00.080]And in four-minute treatment, the reduction of L-mono
[00:30:03.000]was a log of 3.77.
[00:30:07.680]Here, the log difference with increasing the time,
[00:30:11.020]it may not seem very large, because there is just
[00:30:13.300]an increment from 2.23 to 3.11.
[00:30:16.300]It's around 0.9.
[00:30:17.940]But in reality, it's a 9-fold reduction.
[00:30:22.860]So this table establishes a fact that
[00:30:25.800]increasing the holding time increases lethality
[00:30:30.140]within the same formulation.
[00:30:33.140]However, besides holding time, the composition of
[00:30:37.980]protein, fat, and moisture, it plays a role in
[00:30:41.020]microbial resistance to pressure.
[00:30:43.520]Because when you compare one column with another
[00:30:46.960]column, you may find the difference.
[00:30:50.020]Let's see our chicken here.
[00:30:53.000]In the two-minute treatment, the reduction in
[00:30:55.760]hard chicken of the Listeria monocytogenes was 1.27.
[00:31:00.860]With increasing three-minute treatment, the reduction
[00:31:03.640]reached to 1.77, and increasing it to four-minute,
[00:31:08.240]reduction was 2.17 log CFU per gram.
[00:31:12.680]Here, again, it iterates the same thing.
[00:31:16.040]Increasing the holding time increases the reduction in
[00:31:19.220]bacteria.
[00:31:20.120]It's kind of a rule of thumb.
[00:31:21.780]However, the same holding time didn't reduce
[00:31:25.720]Listeria monocytosis equally in all these forms of chicken.
[00:31:30.100]In four-minute treatment, in A chicken,
[00:31:34.220]L mono was reduced by 3.77.
[00:31:37.060]In S chicken, it was reduced by 2.08.
[00:31:40.360]And in R chicken, only the reduction was 2.17.
[00:31:44.380]So the factor of composition, protein, fat,
[00:31:47.680]and moisture plays a huge role here.
[00:31:50.380]Same trend was seen in Salmonella and E. coli.
[00:31:55.680]Let's say in Salmonella, the reduction you can see here.
[00:31:59.680]Increasing the time increases the reduction in bacteria.
[00:32:03.680]Even in E. coli, increasing the time increases the reductions
[00:32:07.680]within one formulation.
[00:32:09.680]However, there is a difference of reduction
[00:32:12.680]between every formulation right there.
[00:32:15.680]Here I would like to highlight one more thing.
[00:32:18.680]The reduction of Salmonella and E. coli for a certain treatment
[00:32:23.680]is higher than what we are seeing
[00:32:25.640]in the Listeria monocytogenes.
[00:32:27.640]For example, four-minute treatment,
[00:32:32.640]it reduced E. coli by 6.85 lox EFU per gram in a chicken.
[00:32:39.640]The same treatment in Salmonella,
[00:32:41.640]it reduced Salmonella by 6.84.
[00:32:45.640]However, the same treatment time of four minutes in a chicken
[00:32:49.640]reduced only 3.77 Listeria monocytogenes.
[00:32:53.640]Why is it so?
[00:32:55.600]It is because Listeria monocytogenes is a gram-positive bacteria.
[00:32:59.600]It has a thicker cell wall and it is more resistant to pressure.
[00:33:03.600]Compared to Salmonella and E. coli,
[00:33:06.600]we have lower pressure.
[00:33:09.600]So, we always encourage our clients
[00:33:14.600]to perform an optimization test before the validation study,
[00:33:18.600]so that we will find a proper time that is the lowest,
[00:33:22.600]such that we get a proper lethality
[00:33:25.560]for the formulation of the food we are doing the validation study for.
[00:33:29.520]After the optimization test,
[00:33:32.520]we encourage them to do validation study for a certain time.
[00:33:36.520]That was my time. Thank you.
[00:33:39.460]you now I would like to call Juliana to cover up about the better protective
[00:33:43.780]effect and she will also give a talk about self-life well thank you Prashant
[00:33:49.600]for the introduction so to continue our discussion about process validation I
[00:33:55.360]would like to introduce the concept of bar protection but a protection refers
[00:34:01.660]to the reduced effectiveness of HPP due to the inner and components of the food
[00:34:06.860]matrix in the case of ready-to-eat meat and poultry products this component is
[00:34:12.100]fat while the limited research affects this area we know from some authors that
[00:34:20.720]this effect may be due to the fact that fat rich foods may create low water
[00:34:25.800]activity zones which difficulties that pressure transmission in the product and
[00:34:31.100]other authors have stated that fat may create this physical shield which
[00:34:36.800]protects bacteria and even can absorb the pressure even though we know this
[00:34:44.480]information from the literature there is a contradictory information since some
[00:34:50.660]studies have reported butter protection when increasing the fat content of their
[00:34:56.040]matrices all their authors have stated the opposite effect or even not effect
[00:35:01.640]at all coming from fat so to talk about this
[00:35:06.740]I would like to refer to my lab mate Ariane she already discussed that we
[00:35:12.680]don't necessarily need to have our own study for validation purposes we can
[00:35:17.780]also rely on the literature and different set of studies or in a
[00:35:22.260]particular study for validation purposes so I would like to tell you or
[00:35:28.220]to share with you my experience when looking at the literature during my
[00:35:32.480]masters so I conducted a scoping review
[00:35:36.680]and my research question was how does fat level affect inactivation of
[00:35:41.600]listeria species in HPP treated RTE foods I made my investigation in three
[00:35:48.560]different product categories RTE meat and poultry products RTE seafood and
[00:35:53.600]cheese and we mainly focused our investigation on inactivation of
[00:35:58.160]listeria monocytogenes and listeria innocua so we searched on six different
[00:36:05.240]databases and
[00:36:06.620]after the duplication processes we found 603 different papers and at the
[00:36:13.940]end just 16 of them reported the fat content of their matrices so at the end
[00:36:21.320]we observed eight papers related to meat and poultry products five papers about
[00:36:27.380]seafood and just four papers related to cheese in here we established our first
[00:36:33.980]conclusion that was that few researchers
[00:36:36.560]report the fat content in microbial challenges studies involving HPV the
[00:36:44.720]following graph shows the probability distribution for inactivation of the
[00:36:48.920]serious species in ready to eat meat and poultry products we couldn't find enough
[00:36:53.380]data related to the remaining product categories and the main studied products
[00:36:58.880]in here were your ham sausages mortadella and turkey products fat content of these
[00:37:06.500]products vary from 1.73 to 28.8 percent and as you can see in the graph we
[00:37:15.440]divided these food matrices into groups above 15 percent depicted in pink and
[00:37:22.060]below 15 percent depicted in blue the pink one refers to the high fat content
[00:37:28.580]and the blue one to the low fat so as you can see we can also observe these
[00:37:33.440]shapes that are called violins
[00:37:36.440]and we can also observe that there's wide distributions in them this is due
[00:37:41.160]to the fact that we are plotting data points from different studies that don't
[00:37:46.160]necessarily share processing conditions or product characteristics so for
[00:37:52.620]example the holding time of these studies vary from 0.1 to 15 minutes and
[00:37:57.960]the fluid temperature from 10 to 17 Celsius degrees to read correctly this
[00:38:05.080]graph it is important
[00:38:06.380]to focus on the triangle in the middle that represents the most probable log
[00:38:11.100]reduction to be obtained for each condition we also have three different
[00:38:15.880]ranges of pressures so the first one is 300 to 450 megapascals second one goes
[00:38:23.300]from 500 to 600 and the last one from 727 to 800 in the first two cases we
[00:38:31.900]observed that the log reductions are greater in the low fat content
[00:38:36.320]matrices such as in this case and in this case but in the last case we
[00:38:44.380]observed the opposite effect however this difference is minimum and in here
[00:38:50.760]we can definitely observe that pressure diminish the effect of fat however these
[00:38:56.640]conditions are not usually used at the industry level we mostly use the second
[00:39:01.700]condition and that's why we
[00:39:06.260]obtained our second conclusion which is as fat content of an RTE meat and
[00:39:11.120]poultry product increases the inactivation of listeria species appears
[00:39:15.220]to decrease then we conducted a microbial challenge study since we
[00:39:22.880]wanted to confirm the main hypothesis from the scoping review so we started by
[00:39:28.800]developing our samples which were pork meat emulsions so we used pork loin we used
[00:39:36.200]pork belly and we mix these meats with salt water sodium phosphate sodium
[00:39:42.860]nitrite and sodium erythorbate as meat block bases and we mix it with different
[00:39:48.500]proportions or ratios of the meats to get meat emulsions of 5% 15% and 25%
[00:39:57.340]then we surface inoculated samples using a two strain cocktail of listeria inocua
[00:40:06.140]until reaching a concentration from 10 to the 6 to 10 to the 7 CFU per gram
[00:40:10.820]then we treated these samples using 300 megapascals 450 and 600 megapascals for
[00:40:22.400]three minutes using chilled water for enumeration purposes we use dsaye and after that we calculated
[00:40:29.300]our low reductions the following graph represents the microbial challenge
[00:40:36.080]study results so at the 300 megapascal side we found that pressure was too low to yield an
[00:40:45.200]appreciable log reduction in the case of the 600 megapascals we observed that the microbial counts
[00:40:52.700]were below the detection limit in all the cases and also we noticed that these results were
[00:40:58.820]sensitive to the level of inoculation for instance we inoculated higher the samples of the 25 fat
[00:41:06.020]which resulted in higher log reductions in the second scenario related to 450 megapascals we
[00:41:15.920]obtained this parabolic trend that you can see here so as expected the lowest log reduction
[00:41:22.840]was obtained at the 25 percent fat the highest was obtained at the 15 percent fat and in the
[00:41:31.580]middle we found the results for the five percent fat content
[00:41:35.960]so then we obtained our third conclusion which was that there is a threshold between 15 percent
[00:41:44.420]and 25 percent fat above which fat provided this matter protective effect so now I would
[00:41:52.820]like to discuss chef life studies so as we learn throughout the workshop and our
[00:41:58.880]different sessions the inactivation can be a factor that high pressure can manage
[00:42:05.900]not only for pathogens also for spoilage microorganisms and enzymes that can
[00:42:11.280]affect the quality of the product over time so the determination of chef life
[00:42:18.780]of the product it's crucial to have in mind three different key aspects which
[00:42:24.500]are safety nutritional value and sensory in the case of safety it is important to
[00:42:30.500]assess that our product is free of pathogenic bacteria or any other microbial
[00:42:35.840]hazard that can compromise people's health or the product itself in the case
[00:42:44.540]of the nutritional value it is important to preserve the micronutrients
[00:42:48.320]and other components of the foods such as the antioxidants which are very
[00:42:52.640]important for some products and in terms of sensory we will need to preserve
[00:42:57.300]color flavor and smell to enhance people's appearance appealing sorry for
[00:43:05.780]product and consumers acceptability now I would like to share with you the key
[00:43:13.440]steps in determining shelf life so in first place we have to define the
[00:43:19.020]objective so we need to know if are we dealing with a new product or maybe a
[00:43:24.220]reformulated product or maybe new packaging or a new process then we
[00:43:32.000]identify the mode of deterioration which can be
[00:43:35.720]logical or due to oxidation or any other chemical change this is helpful to
[00:43:43.760]identify the key attributes to monitor over time this can be microbial
[00:43:48.840]enumeration or chemical analysis such as the formation of byproducts to finally
[00:43:57.800]select the test methods that can be aerobic play counts or coliforms
[00:44:03.160]testing or even lipid oxidation
[00:44:05.660]so for example if we have a fat rich food we can check for fat stability over
[00:44:11.280]time then we establish our storage conditions so in the vast majority of
[00:44:18.600]HPP products it's going to be refrigeration but in some cases it can
[00:44:23.580]also be room temperature after that we set the target endpoint and the testing
[00:44:32.520]frequency so ideally this testing
[00:44:35.600]frequency is more than five points plus an additional point beyond the expected
[00:44:41.900]shelf life for the product after that we determine the appropriate test and
[00:44:48.080]control samples and we perform the shelf life study we collect the data and we
[00:44:54.260]analyze the results to finally monitor and confirm the shelf life of our
[00:44:59.120]product as Ariane already mentioned in her section
[00:45:05.540]there are some factors affecting HPP effectiveness but in case in this case
[00:45:10.760]we also have some factors affecting shelf life so she already discussed that
[00:45:15.860]there are factors that affect pathogenic inactivation but guess what these factors
[00:45:21.560]also contribute to the inactivation of spoilage microorganisms and enzymes so
[00:45:27.380]I'm just going to summarize them so we have water activity fat content pH antimicrobial
[00:45:35.480]agents and the type of microorganism enzymes some typical applications of HPP to extend
[00:45:44.060]the chef life are RT meats pet food juices baby food dips fruit purée and seafood
[00:45:52.400]and we could observe some of these examples during the hyperbaric pilot plan demo so
[00:45:59.720]they share with us some products that are typically HPP'd in the industry and
[00:46:05.420]they also share insights of some conditions that are used for these purposes and expect
[00:46:11.420]the chef life for each of them so in the following slide you can see four different products so we
[00:46:19.520]have raw marinated meat cured salami cooked pet food and freeze-dried pet food most of these
[00:46:28.280]products were treated at 87 000 psi for three minutes with the exception of the cure salami that
[00:46:35.360]was pressurized for four minutes and the expected chef life at refrigeration conditions was up to
[00:46:43.040]55 days for the raw marinated meat 90 days for the cure salami and 60 days for the cook pet food
[00:46:51.440]in the case of the instinct pet food which was freeze-dried we observed a chef life from one
[00:46:59.360]to two years but this product is shelf stable
[00:47:05.300]we have another set of four examples so we have fruit puree from once upon a farm
[00:47:10.660]rte meal from fuchsia rte overnight oats from mush and guacamole from good foods these four products
[00:47:19.620]were hpped at 87 000 psi for three minutes and the expected shelf life on the refrigeration was
[00:47:27.620]60 days for the fruit puree 45 days for the rte meal and 40 days for the oats and
[00:47:35.240]for the guacamole they also provide examples about daily meat products humus and cooked crab meat
[00:47:44.360]so same conditions of hpp as the previous examples were applied in here
[00:47:48.920]but the expected chef life was 90 days for daily meat 60 days for the humus and 28 days
[00:47:57.640]for the cooked crab meat all of these under refrigeration conditions finally the
[00:48:05.180]they also provide insights about additional seafood products so we have lobster tail
[00:48:10.280]marinated salmon and banded oysters the hpp conditions implemented here were 29 000 psi for
[00:48:18.360]two minutes and in the case of the lobster and the marinated salmon the expected chef life
[00:48:23.680]was 40 14 days excuse me on the refrigeration they also share that for the banded oysters
[00:48:30.960]these conditions are helpful for shocking and extraction
[00:48:35.120]purposes but they also mentioned that these conditions um keep under control vibrio and
[00:48:41.960]norovirus so the expected shelf life for them is 21 days on ice and refrigeration so now i would
[00:48:50.260]like to call dr canal who is going to talk about case studies related to shelf life thank you
[00:48:58.420]juliana i think those case studies or examples of shelf life that you shared is really a really
[00:49:05.060]good lead-in to some of the case studies that i'm going to share here and these are actual
[00:49:10.560]data that we collected for some of our clients in our own laboratory i'm going to mask a little
[00:49:17.160]bit about exactly which products they are but i'll provide some background if you will so this
[00:49:22.640]very first case study with an rte meat so it's a fully cooked smoked beef brisket hpp treated at
[00:49:30.360]590 megapascal for three minutes with the intent to store a
[00:49:35.000]four degrees c or just refrigerated temperatures for a period of about hopefully 70 days that's
[00:49:41.640]the intended or anticipated shelf life of the product but this is a product that's going to
[00:49:46.440]be used in food service so one of the first things that we do is when we do these shelf life studies
[00:49:53.000]we typically do two replications so two replications of the run hpp run each one
[00:50:00.360]containing probably two sub samples hopefully each run also
[00:50:04.940]has product coming from two different lots of production so this allows us to incorporate as
[00:50:11.420]many variation of the product as much as possible also when it comes to the shelf life so day zero
[00:50:18.380]here is really the day where we inoc we don't inoculate the product but we just hpp the product
[00:50:24.300]just as it comes to us and it'll be in the final packaging because packaging is going to be a huge
[00:50:29.580]part of the shelf life itself so as you can see on the day zero data
[00:50:34.880]the blue line tells us the control so that is for samples that are not
[00:50:38.960]hpp treated at all and then the green line represents what happens when it's hpp treated
[00:50:46.320]so we assume that on day zero right that the day right before they're hpp treated that they're
[00:50:52.880]probably going to have the same level of background microflora in this particular case the
[00:50:58.560]client was really interested in seeing apc psychotrophs lactic acid bacteria and yeast and
[00:51:04.820]so as you can see without hpp treatment on the aerobic plate counts or apc you see this
[00:51:13.420]gradual progression towards higher numbers of hp of the apc over time as a rule of thumb every time
[00:51:22.480]you have counts that are above 10 to the 6 or 6 logs that's really a sign that the product has
[00:51:28.400]already spoiled so here it looks like probably even right around day 56 post hpp
[00:51:34.760]you can kind of consider this product as being at the end of its shelf life but for this particular
[00:51:41.580]case the client was really interested to see what would happen if we were close to twice as long as
[00:51:47.840]the intended shelf life so which is why you're seeing data go all the way up to about 126 days
[00:51:54.060]when it's hpp treated you definitely see aerobic plate counts stay well below 10 to the 6 cfu per
[00:52:04.700]and in fact it's just kind of staying or hovering around that 10 to the 4. and again that's pretty
[00:52:10.100]typical for a lot of food products out there essentially also if something has been refrigerated
[00:52:15.320]for quite a bit of time psychotropes are usually enumerated very similarly to aerobic plate counts
[00:52:22.340]we would normally use petrofilms when we're conducting these studies and then for the psychotropes
[00:52:27.440]we would then just lower the temperature when we're incubating the plates so in this particular case
[00:52:34.640]again as you can see typically psychotropes are going to be a little bit lower than your
[00:52:38.660]aerobic plate counts and we're no means close to the 10 to the 6 CFU per gram
[00:52:46.040]yeast and molds compared to aerobic plate counts and psychotropes are really easy to
[00:52:51.320]inactivate with HPP especially in a ready-to-eat product like this so notice that even untreated
[00:52:59.180]with pressure those numbers for yeast and molds are definitely lower in the
[00:53:04.580]aerobic plate counts and then it's even further lower and actually probably below
[00:53:09.200]detection limits but we're reporting a one log CFU per gram for yeast and molds from
[00:53:15.020]zero all the way up to 56 days now remember HPP is a pasteurization technique at some
[00:53:22.640]point unless we wipe out any of these bacteria they're bound to show up at some point during
[00:53:29.420]shelf life so for yeast and molds we see it come back up above detection limits
[00:53:34.520]for yeast and molds by the time we get to 84 days post HPP treatment and then I want to highlight
[00:53:42.020]over here the last quadrant for lactic acid bacteria the data over there you can see that
[00:53:49.220]lactic acid bacteria definitely is also suppressed by HPP those numbers remain lower than if we had
[00:53:57.360]not treated it with pressure at all but lactic acid bacteria is also one of those spoilage micro
[00:54:04.460]organisms that are really hard to kill with HPP and it just so happens that in this particular
[00:54:10.580]food product there's a lot of components that are in the marinade that you know within the
[00:54:17.720]formulation of the smoked beef brisket so it also kind of remains pretty low so I think I don't
[00:54:23.300]remember exactly what the manufacturer or the client decided at the end but I think they did
[00:54:27.800]dial down the shelf life of this product rather than the anticipated or expected 70-day shelf life
[00:54:34.400]to about 50 days just because that's when they can guarantee that the HPP product had it been
[00:54:40.520]refrigerated the entire time after HPP it would have that level of or that length of a shelf life
[00:54:49.760]obviously if they were distributing the product frozen that would seize all microbial activity
[00:54:59.540]so then that could actually buy them a little bit of time if they really were trying to get
[00:55:04.340]up to 70 days perhaps adding 10 days of being frozen plus 50 days or so of being refrigerated
[00:55:13.700]then that can allow them that's one strategy they can use in order to get the anticipated or
[00:55:19.640]expected 70-day shelf life on this product moving on this was a really fun project somebody wanted
[00:55:27.920]to make baba ganoush so instead of us roasting the the eggplants which is the main component
[00:55:34.280]of the baba ganoush dip we actually ended up using a canned roasted eggplant itself so as
[00:55:40.940]you can imagine making this product no heat treatment at all we were just basically mixing
[00:55:46.580]and whipping up the all the ingredients together and at the end of the day we always start off
[00:55:51.800]with something that is very common in the HPP industry you saw it in the slides that
[00:55:58.220]Juliana presented to you this 590 megapascal for three minutes is usually a really good
[00:56:04.220]Gateway for anybody who wants to start using HPP to extend the shelf life of their product so the
[00:56:11.900]intent here is this particular plant-based dip is still going to be refrigerated stored at 4 degrees
[00:56:18.440]C after HPP treatment so looking at this is no longer an animal protein-based product it's now
[00:56:26.600]plant-based looking at the data once again the green curves show you what happens when you treat
[00:56:34.160]pressure the blue lines are right above it show you what happens to the control samples when you
[00:56:39.140]don't apply pressure aerobic plate counts for the most part tracks similarly whether or not it's HPP
[00:56:45.500]or not but the green line at least is always below the control samples yeast and molds in
[00:56:51.680]a product like this totally wipe them out probably we are reporting a one log CFU per
[00:56:59.000]gram throughout the shelf life of the product because that is the detection limit of our method
[00:57:04.100]when we played it for yeast and molds the good news is for these kinds of products a lot of your
[00:57:09.380]plant dips point of spoilage would be they start smelling fermenty right and that's usually that's
[00:57:15.740]because if there's some lactic acid bacteria in the sample and what we're noticing at least in
[00:57:21.440]this particular case is we're not seeing an outgrowth on the lactic acid bacteria one of the
[00:57:29.420]things that the client for this particular project was really looking for I think they would have been
[00:57:34.040]a five-day shelf life or even if pushing it a 60-day shelf life and as you can remember from
[00:57:40.640]Juliana's slides that that's very typical for HPP treated plant-based dips this particular
[00:57:47.120]client wanted to understand is there going to be any noticeable color change to the product
[00:57:53.480]so our lab can do these types of testing too we would measure L star A star B star values
[00:57:59.720]and then we would calculate what is that color change which is you know in
[00:58:03.980]food science we would call that the Delta E and as you can see over the next couple of weeks from
[00:58:10.640]zero days to 74 days of refrigerated shelf life there's hardly any change those two curves the
[00:58:18.380]blue line and the green line sit on top of each other so this particular product even
[00:58:23.720]though it may be past the shelf life if they were looking for 45 to 60 days
[00:58:27.680]still actually looks as if the way it looked just right on day one which I think is kind
[00:58:33.920]of a really powerful and interesting way of also extending the shelf life so we're not just doing
[00:58:39.620]it from a microbial quality standpoint but we're also retaining or able to retain the look of the
[00:58:46.940]product smoothies or beverages another very popular category with HPP so this one happens to
[00:58:54.740]be a strawberry banana smoothie treated at 590 megapascals for three minutes and stored refrigerated
[00:59:01.760]after HPP treatment
[00:59:03.860]the combination of acids that are naturally occurring in strawberry and bananas is actually able to work with HPP so that different from the previous examples aerobic plate counts are really suppressed so they just stay below right until they you know just kind of grow exponentially but that is now looking at for this particular case after 91 days of refrigeration yeast and molds one again very easy
[00:59:33.800]to suppress especially within the first 90 days of the shelf life of the product
[00:59:39.140]and then same thing with lactic acid bacteria so this is a very classic
[00:59:43.880]example most of your beverages whether or not it's a fruit juice or a mixture
[00:59:50.780]of fruit and vegetable juices or even in this case having some kind of a smoothie
[00:59:55.880]this is something that you should expect you should expect at least 60 days
[00:59:59.720]depending on the ingredients you're using you might be able to push it to 90
[01:00:03.740]and depending on the pH of the product you might even be able to stretch it out
[01:00:07.980]to a hundred and twenty days one of the things that we always try to do when it
[01:00:13.800]comes to juices is to also look for a pH change because pH is usually a critical
[01:00:19.040]control point in juice production so same way as you would read it before the
[01:00:24.620]blue line represents control untreated samples and then when you what do you
[01:00:31.580]call this when you treat it with pressure you will
[01:00:33.680]see the green line as you can see here it tracks pretty well now one of the
[01:00:38.920]things that I always advise people or always try to remember is that pH is
[01:00:44.920]also just like your microbial counts are reported in a log scale so even though
[01:00:51.920]you might say oh a pH change of 3.9 to 3.7 that's only a difference of 0.2 well
[01:00:59.180]guess what a difference in pH of 0.3 is actually
[01:01:03.620]a factor of 2 so remember the equation for pH pH is equal to the negative log of
[01:01:09.620]the concentration of your H+ ions so it's a little bit flipped so a change in
[01:01:14.960]pH of plus 0.3 means that you've actually halved or cut in half the
[01:01:21.600]number of H+ ions this one we're seeing a drop so it's a pH change
[01:01:26.220]of negative 0.2 or negative 0.3 so then actually we've kind of doubled
[01:01:33.560]if you will or at least lost half of the what do you call this lost half of the
[01:01:40.340]H+ ion so it's becoming more and more actually it's an increase doubling of
[01:01:45.740]the H+ ion so it's becoming more and more acidic over time and I think that
[01:01:51.020]is also indicative of the fact that there's some lactic acid bacteria
[01:01:55.220]already growing in the background but we weren't able to see it yet right up
[01:01:59.420]until after day 91 so that's something to always remember
[01:02:03.500]pH change or pH count numbers are on a log scale as well so the differences are
[01:02:10.500]a lot bigger than you think it is finally my fine my example here is
[01:02:15.440]actually from a research paper that Prashant found for us which I think is
[01:02:18.860]great this is all about working with raw
[01:02:22.580]yellowfin tuna fillet so this one was a research study so it's not necessarily a
[01:02:29.000]real application of somebody putting out a yellowfin tuna
[01:02:33.440]fillet out there that has been HPP'd for an extended shelf life but when you're
[01:02:38.060]dealing with seafood products there's a couple of things to keep in mind so
[01:02:42.020]first if you remember from Juliana's examples most of the time in real life
[01:02:48.560]real application when we're using HPP to extend the shelf life of seafood
[01:02:53.060]products we're looking at pressures that are well below 600 megapascal if you're
[01:02:59.300]applying too high of a pressure you start seeing that this
[01:03:03.380]coloration or cooked appearance of a raw seafood product so in psi we're talking
[01:03:10.760]about things that are our pressures that are about 29,000 psi but then if we're
[01:03:17.420]talking about in megapascals range we're really looking at somewhere around 300
[01:03:22.560]megapascal so if you look at this particular slide the first one as you
[01:03:28.140]can see the circles on top that's the control no HPP immediately below that
[01:03:33.320]the open circles 200 megapascal so we're looking at the upside down triangles so
[01:03:38.940]it's a tracking about one log below the control untreated one so there is some
[01:03:45.500]reduction on aerobic play counts but not so much we don't see those reductions
[01:03:50.760]being coming more significant until we reach let's say the 400 megapascal
[01:03:57.580]treatment but that's fine we're not looking for inactivation of pathogens
[01:04:02.000]here we're
[01:04:03.260]looking at reducing the microbial load of the background microflora so in this
[01:04:08.920]particular case even though we're only demonstrating or seeing a one log
[01:04:12.220]reduction that's really a ten times factor in the reduction of aerobic play
[01:04:18.480]counts so that's a measure for us that we can say if we treat it in commercial
[01:04:23.300]setting where should we should expect a ten times reduction in aerobic play
[01:04:28.360]counts when we're talking about microbial quality but seafood is just a
[01:04:33.200]measure for us that we can say if we treat it in commercial setting where
[01:04:37.160]should we should expect a ten times reduction in aerobic play counts when we're
[01:04:40.040]talking about microbial quality but seafood is just a measure for us that we
[01:04:42.020]can say if we treat it in commercial setting where should we should expect a
[01:04:43.980]ten times reduction in aerobic play counts when we're talking about
[01:04:45.040]microbial quality but seafood is just a measure for us that we can say if we
[01:04:46.040]treat it in commercial setting where should we should expect a ten times
[01:04:47.040]reduction in aerobic play counts when we're talking about microbial quality
[01:04:48.040]we can say if we treat it in commercial setting where should we should expect a
[01:04:49.040]ten times reduction in aerobic play counts when we're talking about microbial
[01:04:50.040]quality we can say if we treat it in commercial setting where should we should
[01:04:51.040]expect a ten times reduction in aerobic play counts when we're talking about
[01:04:52.040]microbial quality we can say if we treat it in commercial setting where should
[01:04:53.040]to kind of render or call a fresh seafood product as still being quote-unquote fresh so as you can
[01:05:00.560]see when it's untreated the control darkened circles and then also treated with 200 megapascal
[01:05:08.480]open circles those are the top two curves that you're seeing and within six days of refrigeration
[01:05:15.420]the product is no longer fresh that makes sense probably a lot of you have experienced this at
[01:05:20.180]home you buy a salmon fillet you leave it in your refrigerator with the intent of cooking it
[01:05:25.020]later tonight but you forget you forget the next night again and a week later if you smell it
[01:05:30.460]you're like ew that's no longer fresh however if we were to hpp it at least at 300 megapascal or
[01:05:38.060]29 000 psi as juliana showed earlier we see the suppression of this total volatile basic nitrogen
[01:05:46.180]so we can actually extend the shelf life of that refrigerated
[01:05:49.880]product anywhere from nine to even 12 days so that's great i mean two weeks in the refrigeration
[01:05:56.800]in the refrigerator and who would have thought it right that you can achieve that level of
[01:06:01.640]freshness for something refrigerated and finally another measure and this one really is for chemical
[01:06:07.120]safety one of the things that we have to keep track in a fresh seafood product
[01:06:11.900]is how much histamine has been produced so the good news here is hpp is really
[01:06:19.580]really good at inactivating histamine and keeping the level slow so in any one
[01:06:25.040]of these pressure treatments even all the way up to about nine days or even
[01:06:30.320]12 days of refrigeration post HPP we've suppressed that histamine it's well
[01:06:37.140]below the five milligram per 100 gram which is the limit standard by the US
[01:06:42.220]FDA so that's another benefit of HP being seafood items so when it comes to
[01:06:49.280]seafood always think that your microbial quality chemical quality and physical
[01:06:54.600]qualities need to be assessed in a shelf life study so that's my last slide and I
[01:07:01.020]just want to tell everybody this is a great team to work with it's always a
[01:07:04.840]pleasure to work with these young folks we do believe that working under
[01:07:09.520]pressure is our business and we hope you have enjoyed this presentation
[01:07:15.280]Thank you.

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HPP Studies: Microbial Challenge, Process Optimization, Process Validation and Shelf-Life

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