Non-Thermal Processing of a Protein Functional Beverage Using Pulsed Electric Fields: Escherichia coli Inactivation and Effect on Proteins
Round 1
Reviewer 1 Report
Manuscript ID: beverages-1797480
In the article entitled: “Non-thermal processing of a protein functional beverage using pulsed electric fields: Escherichia coli inactivation and effect on proteins” was study the influence of pulsed electric fields for Escherichia coli inactivation suspended in protein shake beverage. Square bipolar pulses field of various intensities were applied to investigate the effect of different PEF conditions on the microbial population and the proteins relevant to this functional beverage.
This article has been written in a compact manner. From the methodological point of view, the employed measurement techniques are appropriate to the adopted objective of the research work. The results obtained may have practical application.
Title
The title and the aim of the study are clearly constructed.
Abstract
The abstract includes the aim of the study, methods used in the experiment and contain the principal results and conclusions.
Introduction
The introduction describes the matter of the experiment and states the problem being investigated. However, the effect of PEF on the properties of liquid food products is poorly noted.
Methods
The data is well collected. The methods are described in detail, in the way which permits the research to be replicated. The sampling is appropriate and adequately described. Statistical analysis of measurement results has been used. However, this does not follow directly from the text of the work (except for the captions under the drawings). Maybe the publication should be supplemented with this element?
Results and Discussion
They correctly interpreted and described the significance of the results for the research.
The effectiveness of the PEF method in the inactivation of Escherichia coli has been demonstrated. Also, it was found that the use of a pulsed electric field causes less protein denaturation than heat treatment. At the same time, both methods cause different conformational changes of proteins. Therefore, there is a lack of at least a sentence of the assumption as the PEF method affects bioavailability of the tested preparations. Although the authors announce further research, this is missing.
References
Generaly the references are accurate but there are no references to research from the last few years.
Language
The article is correctly written.
Author Response
Thank you for your comments. Please see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
General remarks
The paper describes the results of some experiments to use PEF for inactivation of E.coli in a diluted commercial beverage and the influence of PEF on denaturation of proteins in model dispersions. It is no clear why the authors use this beverage. Although, they explained why they have to dilute the beverage for PEF, such a step is not relevant for any practical applications. Furthermore, authors do no explain why they consider only the E. coli bacteria. What is the reason behind it? The additional studies on the highly concentrated protein dispersions (WPC) are not related to the beverage, or at least, this is not clear and it is not explained. Furthermore, different temperatures were used for thermal treatment for diluted beverage and model protein suspensions. Why?
The methods are not well described. Especially, a systematic overview of the treatment conditions, e.g. relationship between flow rate and frequency on one hand and resulting number of pulses and specific energy input is missed.
Most if not all of the results are either well known (for both PEF and thermal treatment) or expectable, e.g. influence of temperature.
Special remarks
L 11: What is the meaning of ‘70%v/v protein shake’ here? This is not clear.
L 16,17: This sentence can be removed, because this is known and expectable. Abstract should be focused on new findings.
L 19: As already mentioned above, the parameter ‘900 Hz’ or ‘400 Hz’ is useless here. Either specific energy or number of pulses should be given here.
L 46: Why E.coli?
L 46,47: The conformational protein changes in the beverage were not investigated (see above). Therefore, this cannot be given as a study objective.
L 68: This quite important statement for the study should be backed by an independent reference.
L 73,76: The description of the treatment cell is not clear. The electrode distance is set to 15 mm or 1.5 cm, but the treatment cell is only 5 mm to 8 mm. What is between the electrodes and the treatment cell? Is it also electrode material? Furthermore, the generator is able to deliver up to 30 kV. Using the electrode distance of 1.5 cm, it would result in a maximum field strength of less than 20 kV/cm.
L 92: Giving only the nutritional composition is not sufficient. At least, the ingredients list has to be added. What was the pH value of the beverage? See remarks to L 119.
L 97: How was the electrical conductivity measured?
L 106: What is the meaning of ‘One loop’ here?
L 106-114: Reference is needed here. What are the producers of the materials used?
L 116: What is the meaning of ‘BacTac application’?
L 119: How was the pH-value set? Why it is important to be given here in the thermal inactivation experiments? Was it different to PEF treatment with respect to pH value?
L 155: Where are the outlet temperatures for each experiment? All outlet temperatures should be given.
L 162-168: Why were these proteins and concentrations chosen? Please explain! Why such a high concentration for whey protein, although the total protein content in the beverage was only 17.5 g/100g before dilution? It is well known that protein concentration influences denaturation behavior, too.
L 175: Why here BSA?
L 176: Why were only the treated samples centrifuged, but no the control?
L 192-193: What is the meaning of ‘(Write the volume)’ here?
L 199: What is the meaning of the Greek sum symbol here?
L 229: Why here 71°C? Why only the WPC dispersion? What means ‘come up time of 40 s’ here? How was it determined?
L 228-233: Reference?
L 240: How was the final dispersion temperature (2-5°C) measured?
L 244-252: This is mostly well known and expectable. Reference would be sufficient.
L 249-250: Sentence need revision.
L 260: Either the diagram or the D value of 0.43 min for treatment 50 °C is wrong. According to the diagram, it took 6 min (from 4 to about 10 min) to reach a 1 log reduction. Therefore, the D value should be in a comparable range and not very close to the ones for 53 and 55 °C where the lines are much steeper. Compare it with literature values.
L 273: This diagram makes no sense. Frequency should be replaced by specific energy input as can be found in most of the PEF publications.
L 287: An electrical field strength of 25 kV/cm is already mentioned in line 265 and in the diagram. Why here ‘also’?
L 286-295: Data (for the second field strength?) have to be given here in a table or diagram.
L 297-300: This is well-known basic knowledge and can be removed.
L 301: Effect on what?
L 326-327: This statement about reaching 1 log reduction in 0.43 min at 50 °C is not supported by figure 5. See the remarks to L 260.
L 332-340: If the right parameters for an appropriate characterization of the PEF conditions would be delivered (see remarks above), a comparison of the results would be very well possible and should be added here.
L 337: What is the meaning of ‘frequency of the PEF treatment’? Is it related to the number of pulses per treatment or the number of treatments?
Author Response
Thank you for your comments. Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Although the authors did a good job to improve their paper, some issues remain, which have to be clarified.
Firstly, the characterization of the treatment conditions is still an issue. As proposed by me and as it can be find in nearly all publications about treatment of food by pulsed electric fields (PEF) specific energy or energy density (as in the paper) is a very suitable parameter for it. Giving only the frequency as it is still the case in the modified abstract does not provide necessary information about the treatment. On the other hand, the authors themselves show the specific energy input in figure 6 for one flow rate. Therefore, I recommend to provide the values about this parameter in the paper and to use this parameter to compare the effects with the state of the art in PEF treatment.
The choice of E. coli as the relevant microorganism should be backed by a reference.
There still a lack of data given by the authors to evaluate the results. As mentioned in the first review, the output temperatures for all treatment conditions should be given, e.g. in a table and not only some of them within the text.
The response of the authors regarding the protein concentrations, which were much higher in the solutions than in the diluted beverage, is not comprehensible. Addition of protein to obtain a desired electrical conductivity in the solution does not seem a favorable approach. Small amounts of salt are much more effective in modifying conductivity. At least a reference should be given here for their approach.
Author Response
Third submission
Answers to reviewer 2
Thanks for taking the time to review this manuscript. Please find below the answers your comments.
Firstly, the characterization of the treatment conditions is still an issue. As proposed by me and as it can be find in nearly all publications about treatment of food by pulsed electric fields (PEF) specific energy or energy density (as in the paper) is a very suitable parameter for it. Giving only the frequency as it is still the case in the modified abstract does not provide necessary information about the treatment. On the other hand, the authors themselves show the specific energy input in figure 6 for one flow rate. Therefore, I recommend to provide the values about this parameter in the paper and to use this parameter to compare the effects with the state of the art in PEF treatment.
Thank you for your comment. We added a chart for the outlet temperatures at two different flowrates and different frequencies. The information available in the manuscript is indicating that the treatment at the conditions selected maintain the temperature below 40 degrees in all cases. We added the dissipated power for two different flowrate and at 25kV/cm line 420-422, and we added the reference for in depth optimisation of this specific unit for more information.
The choice of E. coli as the relevant microorganism should be backed by a reference.
Thanks for your comment. A reference is added as well as the paragraph:
E coli (namely the shiga toxin producing strain) was chosen by analogy with raw milk hazards, as this pathogen has been recommended by European Food Safety Authority (EFSA, 2022) and used as reference to design safe pasteurization processes, both thermal and non-thermal (e.g. high-pressure processing, PEF) pasteurization [23].
There still a lack of data given by the authors to evaluate the results. As mentioned in the first review, the output temperatures for all treatment conditions should be given, e.g. in a table and not only some of them within the text.
Thanks for your comment. Figure 7 is added for this purpose.
The response of the authors regarding the protein concentrations, which were much higher in the solutions than in the diluted beverage, is not comprehensible. Addition of protein to obtain a desired electrical conductivity in the solution does not seem a favorable approach. Small amounts of salt are much more effective in modifying conductivity. At least a reference should be given here for their approach.
Thank you for your comment. The concentrations of whey protein solution (0.4 g/mL) and SPI protein dispersion (0.14 g/mL) were chosen to be close to the diluted protein beverage protein concentration (0.12 g/mL). This explanation was added to the paper in Section 2.3.1. Preparation of whey and soy protein solutions. This is added to the manuscript line 203-206.
