Review Reports - Effect of Modified Atmosphere Packaging (MAP) and UV-C Irradiation on Postharvest Quality of Red Raspberries

Round 1

Reviewer 1 Report

Title: Effect of modified atmosphere packaging (MAP) and UV-C irradiation on postharvest quality of red raspberries

This study investigated the ability of UV-C radiation, passive modified atmosphere packaging and the combination of both technologies to preserve raspberries at 6 °C. The results showed that combining MAP with UV-C radiation prolonged the shelf life of red raspberries and maintained a higher content of bioactive compounds. This research provided a new sight and method to preserve postharvest red raspberries. However, the experimental work and the analysis of the experimental results are not in-depth enough. Publication of manuscript is recommended with major revisions.

Abstract: MAP with approximately 6% O₂ and 16% CO₂only appears in Abstract and Conclusions, and not find in the experiment. What led you to this conclusion? (Line 20)
Introduction: Do the two modified atmosphere packaging conditions refer to two films with different transmission? (Line 89-90)
Materials and Methods: “After the application of the corresponding UV-C treatment polyethylene terephthalate (PET) trays were manually filled with 0.15 kg of red raspberries.” “Approximately 150 g of raspberries were packaged in 750 mL polypropylene trays using as a lid a heat-sealed P-Plus microperforated film.” What are the impacts of the two packaging materials on the experiment? (Line 107 and 124)
Materials and Methods: How did the clamshell containers for control samples affect microbiological quality? (Line 111 and 132)
Results and discussion: Please check the 3.3 and 3.5 section subheads. (Line 321 and 449)
Results and discussion: What does an acceptable limit of 4 log CFU cm^-2 refer to? Mesophilic aerobic bacteria counts? Is there a corresponding limit of aerobic mesophilic and yeast and mold? (Line 363-366)
Conclusions: Please revise the conclusion in paragraphs. Conclusions are not just about summarizing the key results of research, it should highlight the insights and the applicability of your results for further work.

Minor points and other suggestions:

The manuscript still needs to be proofread carefully for grammar and format errors such as line 19, line 35, line 137, line 222 and line 534.

Line 337-338 and 296-300: This sentence is difficult to understand and needed to be modified.

Author Response

Reviewers 1 suggest that minor to moderate language changes might be required along the manuscript. Thus, the version revised by the authors has been checked by a native English colleague to improve language and style.

The reviewer also suggest that every section of the manuscript can be improved, so the authors have taken into account the general and specific comments to detail the methods, when necessary, clarify the results of the research and strengthen the conclusions obtained from them. Finally, after a careful proofreading, all the changes made in the manuscript are marked up using the Microsoft word track changes tool, and the answers to the referee are given below each comment.

REVIEW REPORT 1

1) Abstract: MAP with approximately 6% O₂ and 16% CO₂ only appears in Abstract and Conclusions, and not find in the experiment. What led you to this conclusion? (Line 20)

These concentrations refer to the final composition of the packages head space (Figure 2) at the end of shelf life of the batch UV2MAP2, considered the one which better preserves raspberries quality. However, it is not the gas composition since the beginning of the study so these values have been changed in the manuscript for the transmission rates of MAP2 microperforated film to avoid misunderstandings. The resulting sentences are:

Line 20) “… packaging film with transmission rates of 902 mL O₂ d^-1 and 785 mL CO₂ d^‑1 …”

Line 541) “… that combination of a suitable MAP film, which transmission rates meet fruit respiration rate, with a pretreatment of 4 kJ m^-2 of UV-C radiation …”

2) Introduction: Do the two modified atmosphere packaging conditions refer to two films with different transmission? (Line 89-90)

Yes, it does. With conditions the authors mean two different head space O₂ and CO₂ composition which are the result of merging raspberry respiration rate with the transmission rates of the selected film.

The sentence has been clarified as follows:

“…, two lidding films with different transmission rates and …”

3) Materials and Methods: “After the application of the corresponding UV-C treatment polyethylene terephthalate (PET) trays were manually filled with 0.15 kg of red raspberries.” “Approximately 150 g of raspberries were packaged in 750 mL polypropylene trays using as a lid a heat-sealed P-Plus microperforated film.” What are the impacts of the two packaging materials on the experiment? (Line 107 and 124)

We apologize for the mistake in the sub-section 2.1. In both cases, the sentences (line 107 and 124) refer to the same trays and the packaging material was PP, as well as the material of the clamshell containers. Thus, there were no differences between the trays used for UV-C treated and UVMAP samples and the clamshell containers. It has been corrected in the manuscript.

4) Materials and Methods: How did the clamshell containers for control samples affect microbiological quality? (Line 111 and 132)

The clamshell containers were macroperforated, so the gas concentration inside the packages was that of the atmosphere. Aerobic mesophilic bacteria did not noticeably change during storage. Counts for controls increased less than 1 log unit during the study, and MAP1 was the only batch which showed significant differences with control samples at the end of their shelf life, with an increase of 0.6 log CFU cm^-2. Regarding mold incidence, total mold counts increased (1 log unit) as well as rot incidence. Similar values were obtained for UV1, UV2, MAP1 and MAP2 samples, but a reduction of total mold counts was observed in combined treatments, being 2 log units less for UV2MAP2, probably due to lower O₂ concentration. Finally, an increase in total yeast counts was observed in all the treatments, except for UV1 and UV2 samples, which showed 1 and 0.8 log unit less, respectively. MAP and combined treatments counts were slightly higher than those of the control.

Additionally, we have noticed that line 111 and 132 tell the same to the reader, so we have removed sentence in line 132 from the manuscript.

5) Results and discussion: Please check the 3.3 and 3.5 section subheads. (Line 321 and 449)

Thank you for noticing that typing mistake. Subsection 3.5. heading has been corrected as follows:

“3.5. Influence of UV-C radiation, MAP and combined treatments on bioactive compounds”

6) Results and discussion: What does an acceptable limit of 4 log CFU cm^-2 refer to? Mesophilic aerobic bacteria counts?

Yes, it does. Considering that currently there are not criteria available for fresh berries regarding food safety, the authors considered acceptable 4 log CFU cm^-2 of mesophilic aerobic bacteria. They are generally considered indicators of microbiological load of foodstuffs to evaluate, for example, the quality of raw materials, whether an abuse of temperature has occurred or to set products shelf life.

Is there a corresponding limit of aerobic mesophilic and yeast and mold? (Line 363-366)

There is also no limit for yeasts and moulds. Although they increased during storage, the batches which presented higher counts were not those which the authors consider more suitable for the preservation of raspberries. However, a limit for mould and yeast counts may be stablished based on the relation between microbial counts and the appearance of off-flavours.

7) Conclusions: Please revise the conclusion in paragraphs. Conclusions are not just about summarizing the key results of research, it should highlight the insights and the applicability of your results for further work.

This section has been modified, expecting that the changes have improved the conclusions of the study.

Minor points and other suggestions:

- The manuscript still needs to be proofread carefully for grammar and format errors such as line 19, line 35, line 137, line 222 and line 534.

Thank you for the suggestion. These mistakes given as an example, as well as others found during revision, have been corrected to improve the language, style and format of the manuscript.

- Line 337-338 and 296-300: This sentence is difficult to understand and needed to be modified.

They have been modified and we hope they were now easier to understand by the reader.

Reviewer 2 Report

This manuscript aims to evaluate the effects of two modified atmosphere packaging approaches and two doses of UV-C irradiation, individually and in combinations, on the preservation of the quality of raspberries during storage at 6 °C. For this purpose, the authors analysed the respiratory activity, various relevant physical-chemical, microbiological, biochemical and nutritional parameters during 12 d of storage. Although the overall idea is not new, the issue may be interesting in this combination. Thus, the topic may fit the scopes of Agriculture.

The experiments and the analyses performed seem to be valid and the results are generally presented meaningfully. Nevertheless, the manuscript is unnecessarily long in some parts. Most of all, the manuscript is unfortunately not adequately written in all parts and contains weakness and incorrectness. Below and directly in the pdf of the manuscript I proposed many changes that might improve the text.

General comments:

Keywords: As electronic search engines focus on both Title and Keywords, it is better not to repeat words, already used in the title. This increases the chance to get listed.

Please either use “shelf-life” or “shelf life”. The latter is preferred.

The use of comma needs to be checked throughout the text.

According to ISHS (https://www.ishs.org/authors), cultivar names are to be placed within single quotation marks (e.g., Malus ×domestica ‘Fuji').

Please always replace the term “weight” by “mass”. Although still often used instead of mass (SI unit is “kg”), not only in everyday language, weight is a force (SI unit is N), derived from mass (Fw = m * g). See relevant books of physics or engineering, or, simply, http://en.wikipedia.org/wiki/Mass

SI requires that numerals be followed by proper SI units, e.g., 12 d, not 12 days (min for minute(s), h for hour(s), s for second(s)). Likewise, words should not be followed by unit abbreviations, e.g., twelve days, not twelve d.

Although still in use, the unit symbol “M” for “molar” substance concentration is obsolete and the equivalent concentration should also be given in mol L-1 according to SI rules.

Please check commas before “which”.

In correct scientific English, the plural of fruit is fruit. It is a singulare tantum. “Fruits” denotes various types of fruit, e.g., in fruits and vegetables.

Why do you think that it is always important to name the authors? Better concentrate on their findings!

Specific comments:

35-36 This is not correct. Loss of firmness is mainly due to water losses and rot due to microbial contaminations.

38 “The temperature must not fall below 0 °C in order to avoid chilling injury” This is incorrect. T < 0 °C induce frost injury NOT chilling. T < approx. 10 °C induce chilling injuries in chilling sensitive fruit.

39 Please be correct and precise; “high dehydration” does not depend on temperature but on the water vapour deficit (VPD), i.e., the water vapour partial pressure gradient between produce and air. In addition, “relative air humidity” is not the best parameter to control transpirational water losses. Again, it is only VPD!

40-41 Please be correct and precise. Here, you combine two different aspects that should be better introduced clearly separated in this chapter, i.e. reduction of water and mass losses and respiration (MAP, edible coating), and of microbial contamination (irradiation). You are aware that gamma irradiation is not accepted in every country?

42-43 “control fruit decay and maintain its quality during” This directly relates to “decay”! Better write “control decay and maintain quality of fruit during”.

43 “in sale. This may further enhance the positive effects of the aforementioned low temperature storage.” Please do not use “cool” here. While “cold” usually refers to very cold temperatures, approaching the freezing point, “cool” reflects temperatures much higher than the freezing point and close to “moderate” temperatures. Thus, “cool storage” is obvious not what you want to refer to.

45 “Irradiation is still considered an emerging technology”. I am not convinced. What should still “emerge”? Better provide meaningful references. See comment about gamma irradiation above. This may be extended in some degree to “irradiation” in general, probably except UV.

46 “Which “effectiveness for microbial inactivation.”? Please be precise and provide meaningful references.

48 “and leave no residue in food items”; I doubt, this depends on the type of radiation used and the type of interaction with the components of the produce surface tissues.

49 “drawbacks of e-beam and gamma-irradiation”. Please be precise.

Please shorten 45-49 or 40-44.

56 The true in-vivo “bioactivity” of carotenoids other than beta-carotene for eyesight, e.g., for cancer or heart disease prevention etc. as shown for some polyphenols still needs clinical verification.

56-57 Why should “oxidation of carotenes, vitamin C or polyphenols” result in “a darkening of the surface “? This is neither clear nor obvious, and the underlying mechanisms are much more divers and complex.

60-61 “exceeding CO₂ concentrations of 25% or fall below O₂ concentration of 1% “ Mechanisms of very high [CO₂] are not only linked to anaerobiosis.

273-279 Please rewrite, correct and improve (see pdf).

280-288 Please rewrite, correct and improve (see pdf). Please do not stop at the “other have found similar results level”. This does not really add. What’s about the changes in RR_CO2 and RR_O2 at high concentrations of O₂ or CO₂? Why did you measure until these pronounced concentration changes? At which CO₂/O₂ concentrations did the RR start to decline?

296-297 “In high transmission rate packages, the gas composition varied similarly for UV1 and UV2 treated fruit at higher CO₂ and lower O₂ concentrations, respectively, but differed from controls (Figure 2 A).” Please use A and B for Figure 2 to denote MAP1 and MAP2.

299-300 This means that UV-C radiation is disadvantageous for shelf life?

300-303 “In low gas transmission packages (MAP2; Figure 2 B), O₂ and higher CO₂ concentrations were generally lower and higher, respectively although the relative changes of these gases were similarly.”

318-320 “shelf life. Given are means (± SD; n = 5) of controls and of raspberry samples, irradiated with UV-C (UV1: 2 kJ m-2; UV2: 4 kJ m-2) and packed with films of two gas transmission rates (A) MAP1 and (B) MAP2.”

348-349 “CO₂ concentrations (10% or 16%) inside the packages, “

353-354 Please rewrite, this is incorrect the way it is written. It is not the transmission rate.

354-355 “total yeast counts of UV2MAP2 samples increased by only 0.3 log unit compared to controls”

364-365 “microbial loads of treated samples closely reflect those of controls “

380-382 “However, UV-C treatments did obviously not affect the color of the samples [5].”

382-384 “Lightness of raspberries packaged in MAP2, UV1, UV1MAP1 and UV2MAP1 were higher after simulated shelf life, which may increase customer acceptance.” You did not measure acceptance!

387-389 “decreased in samples of all batches but varied significantly among samples on each day of analysis. This was especially valid for…”

389-390 Please rewrite and improve.

390-391 Please rewrite and improve. Not clear what this means.

392-393 This sentence is a bit odd. “, raspberries treated with UV1 maintained higher TSS during the entire the shelf life.” Or similar.

394-403 Please try to improve these sentences.

404-412 Please try to improve these sentences.

404-405 Why should a maturity index reflect the stage of senescence? These are two different parameters. Who called it MI?

413-542 Please try to improve the sentences.

532-536 This sentences is odd and does not add at all.

536-537 This sentences does not add.

Comments for author File: Comments.pdf

Author Response

Reviewers 2 suggest that minor to moderate language changes might be required along the manuscript. Thus, the version revised by the authors has been checked by a native English colleague to improve language and style.

REVIEW REPORT 2

General comments:

1) Keywords: As electronic search engines focus on both Title and Keywords, it is better not to repeat words, already used in the title. This increases the chance to get listed.

Agreeing with the suggestion some of the keywords have been changed.

2) Please either use “shelf-life” or “shelf life”. The latter is preferred.

In agreement with the reviewer, there was a lack of consistency with this term. Thus, in the revised version “shelf life” has been used in the manuscript instead of “shelf-life”.

3) The use of comma needs to be checked throughout the text.

Punctuation has been revised.

4) According to ISHS (https://www.ishs.org/authors), cultivar names are to be placed within single quotation marks (e.g., Malus ×domestica ‘Fuji').

Single quotes have been added around cultivar epithets to adequate them to taxonomic rules.

5) Please always replace the term “weight” by “mass”. Although still often used instead of mass (SI unit is “kg”), not only in everyday language, weight is a force (SI unit is N), derived from mass (Fw = m * g). See relevant books of physics or engineering, or, simply, http://en.wikipedia.org/wiki/Mass

We kindly appreciate such piece of knowledge. Nowadays the term “weight loss” is used more than often in a wide range of disciplines including food science and agriculture. Moreover, “weight” is extensively accepted to name the magnitude mass in renowned scientific journals of different areas of knowledge. However, considering your recommendation, that “mass” is the correct denomination of the magnitude we measured and that it is also used in academia, the term “mass” has been used in the manuscript instead of “weight”.

6) SI requires that numerals be followed by proper SI units, e.g., 12 d, not 12 days (min for minute(s), h for hour(s), s for second(s)). Likewise, words should not be followed by unit abbreviations, e.g., twelve days, not twelve d.

These format mistakes have been corrected in the revised version of the manuscript.

7) Although still in use, the unit symbol “M” for “molar” substance concentration is obsolete and the equivalent concentration should also be given in mol L-1 according to SI rules.

Thank you. The authors have considered your suggestion and the units have been changed along the manuscript to fit SI standards.

8) Please check commas before “which”.

The manuscript has been revised to fit punctuation rules.

9) In correct scientific English, the plural of fruit is fruit. It is a singulare tantum. “Fruits” denotes various types of fruit, e.g., in fruits and vegetables.

This mistake has been corrected when required.

10) Why do you think that it is always important to name the authors? Better concentrate on their findings!

It is not always important, sometimes it is just required due to the way the sentence is structured. However, we agree with the reviewer that there is an excessive use of that formula. Thus, the name of the authors has been removed from the manuscript in some occasions.

Specific comments:

- 35-36 This is not correct. Loss of firmness is mainly due to water losses and rot due to microbial contaminations.

It is true, so the sentence has been changed in the manuscript.

- 38 “The temperature must not fall below 0 °C in order to avoid chilling injury” This is incorrect. T < 0 °C induce frost injury NOT chilling. T < approx. 10 °C induce chilling injuries in chilling sensitive fruit.

Sorry for the mistake, we have already corrected it. Raspberries, like other berries crops, are chilling tolerant and can be stored at temperatures close to 0 °C. However, when decreasing the temperature and maintained between -0.5 and -1.0 °C fruit may start to freeze.

- 39 Please be correct and precise; “high dehydration” does not depend on temperature but on the water vapour deficit (VPD), i.e., the water vapour partial pressure gradient between produce and air. In addition, “relative air humidity” is not the best parameter to control transpirational water losses. Again, it is only VPD!

Ok. Fruit transpiration and consequently dehydration is product of the difference between water vapor pressure of the fruit (saturated) and that of the external atmosphere. As you said, it is call VPD and dehydration depends on it. In that sentence we related high dehydration with temperature because when the difference of temperature of the fruit and that of the air is high is when the VPD is also higher, so dehydration occurs. However, the sentence has been corrected to be more precise.

Moreover, VPD controls transpiration of the fruit, but to the best of our knowledge the most common way to control VPD in warehouses is increasing relative air humidity and equilibrate the water vapor partial pressure between produce and air.

- 40-41 Please be correct and precise. Here, you combine two different aspects that should be better introduced clearly separated in this chapter, i.e. reduction of water and mass losses and respiration (MAP, edible coating), and of microbial contamination (irradiation). You are aware that gamma irradiation is not accepted in every country?

We do not understand why it would be necessary to separate these aspects. The sentence only intends to introduce a range of postharvest technologies including those that were assayed, and that are or could be applied to preserve fruit quality. There is no denying that there are much more technologies aiming this, and that their principles and mechanisms of action differ. However, the final objective of all of them is that which the sentence in line 40-41 claims.

Finally, we are completely aware that ionising radiation is not allowed in every country and at no time was our intention to led the reader believe it is a widespread practice in food industry. Where allowed, its application it is restricted to certain food products and without detriment of the good farming and manufacturing practices to ensure the food safety. For example, in our case, the Food and Drug Administration (FDA) accept treating fresh fruit (such as raspberries) with ionising radiation. However, although the European Food Safety Authority does not perceive any potential risk in irradiating foodstuffs, European commission does not allow irradiating fruits, and its application is restricted to certain products such as dried herbs and spices.

Finally, we also consider that “…have been applied to control fruit decay…” (line 42) could led to misunderstanding. Thus, the sentence has been changed in the manuscript as follows:

“… have been studied to control decay and …”

- 42-43 “control fruit decay and maintain its quality during” This directly relates to “decay”! Better write “control decay and maintain quality of fruit during”.

Thank you for the correction, we have changed the sentence as suggested.

- 43 “in sale. This may further enhance the positive effects of the aforementioned low temperature storage.” Please do not use “cool” here. While “cold” usually refers to very cold temperatures, approaching the freezing point, “cool” reflects temperatures much higher than the freezing point and close to “moderate” temperatures. Thus, “cool storage” is obvious not what you want to refer to.

Sentence in lines 44-45 has been modified by the one proposed by the reviewer because the original one did not reflect exactly what authors wanted to express.

- 45 “Irradiation is still considered an emerging technology”. I am not convinced. What should still “emerge”? Better provide meaningful references. See comment about gamma irradiation above. This may be extended in some degree to “irradiation” in general, probably except UV.

Probably it is nothing to emerge, but we wanted to introduce irradiation as an alternative technology. Thus, the sentence has been modified.

“Irradiation is still considered an alternative technology…”

- 46 “Which “effectiveness for microbial inactivation.”? Please be precise and provide meaningful references.

The references have been added to the text.

- 48 “and leave no residue in food items”; I doubt, this depends on the type of radiation used and the type of interaction with the components of the produce surface tissues.

We agree with the reviewer. We are not experts on ionizing radiation (e-beam, X-ray or gamma-ray), that is what we talk about in that sentence. Thus, it is not correct to assert that no harmful residues are generated after the treatment. We just pointed that based on the reports on food irradiation of different food safety agencies, and in the claims of researchers who has worked with these technologies. Moreover, chemical composition of irradiated foodstuff may change because of the interaction between radiation and food constituents. However, we have not been focused on that kind of publications, so it is not possible to state that food do not present any residue after being irradiated.

Considering the aforementioned, the authors have removed “…and leave no residue in food items…” from the sentence in line 48.

- 49 “drawbacks of e-beam and gamma-irradiation”. Please be precise.

The sentence has been changed.

- Please shorten 45-49 or 40-44.

Some modifications have been made. They can be seen in the revised version of the manuscript.

- 56 The true in-vivo “bioactivity” of carotenoids other than beta-carotene for eyesight, e.g., for cancer or heart disease prevention etc. as shown for some polyphenols still needs clinical verification.

We agree with you. This statement is questionable because clinical studies are needed to claim that they are bioactive compounds. Thus, “bioactive” has been removed of the manuscript considering that the importance of the sentence lies in the “…oxidation of the compounds such as…”.

- 56-57 Why should “oxidation of carotenes, vitamin C or polyphenols” result in “a darkening of the surface “? This is neither clear nor obvious, and the underlying mechanisms are much more divers and complex.

We apologize for the mistake. Although it may be a relation between these two concepts, it was not our intention to correlate them directly, so the sentence has been modified in the manuscript. The authors are aware that is difficult to explain biochemical changes in one sentence due to its complexity. We just wanted to point out the importance to use appropriate UV-C dosages, which depends on the fruit because excessive dosages may lead to undesirable changes in fruit quality. We suggested, based on the literature revised, that these biochemical changes may influence fruit color. Finally, we hope there were not be any inconvenience in citing literally the references we have consulted, which results are at the same time contradictory. Thus, it is not possible to support clear effects or correlations.

- Gonzalez-Barrio et al. (2005) [17]:

“PPO is considered to be the key enzyme in the melanogenesis pathway (10), the posible role of POD in melanin formation has been questioned due to the low hydrogen peroxide content of vegetable tissues. However, the generation of hydrogen peroxide in the oxidation of some phenolics catalyzed by PPO could indicate a posible synergistic action between both PPO and POD, which suggests the involvement of POD in browning processes (11)”.

“Oxidation of phenolic compounds catalyzed by oxidative enzymes such as PPO or POD can lead to the formation of quinones that can polymerize to form melanins (7). Therefore, possible changes in the levels of phenolics upon UV-C treatment might be associated with the observed browning and, thus, we investigated the phenolic composition (flavonols, hydroxycinnamic acids derivatives, flavan-3-ols, and total phenols) of control and treated grapes throughout the storage period”

“However, UV-C irradiation can also promote browning in some white table grapes, such as in var. Superior. Nevertheless, it should be stressed that the browning does not occur in all of the UV-C-irradiated white grape varieties. (…). Therefore, the UV-C-induced browning in white table grapes does not seem to be a “universal” undesirable side effect, but it seems to depend on the features of the variety irradiated (skin thickness, chlorophyll content, etc.).”.

- 60-61 “exceeding CO2 concentrations of 25% or fall below O2 concentration of 1% “ Mechanisms of very high [CO2] are not only linked to anaerobiosis.

The authors agree with the commentary and the sentence has been changed as suggested.

Only high CO₂ concentrations do not cause anaerobiosis (lack of O₂), but it promotes fermentative metabolic pathways, which is what we are suggesting. We have been careful and tell that “…may induce physiological changes…”. The review by Mathooko et al. (1996) explains that high CO₂concentrations may promote fermentative pathways in different fruits and vegetables, although high O₂ concentration. For instance, we literally cite and extract from the review, “an atmosphere rich in carbon dioxide in the presence of 20% (atmospheric) oxygen did not significantly increase acetaldehyde and ethanol concentrations in avocados, in contrast to its effects on 'Bartlett' pears (Ke et al., 1990; 1993b; 1994), strawberries (Ke et al., 1991), lettuce (Mateos et al., 1993) and fig fruit (Colelli et al., 1991)”. Moreover, we expect to explain, at some extent, the changes in color due to high CO₂ concentration. It is thought that CO₂ treatments inhibit the postharvest accumulation of anthocyanins (Gil et al., 1997; Holcroft et al., 1998) by modulating anthocyanin biosynthesis, degradation, or both. Thus, elevated CO₂ concentration during storage of strawberry fruit was inversely correlated with anthocyanin biosynthesis both in internal and external tissues (Holcroft & Kader, 1999).

- Holcroft, D.M., Gil, M.I., Kader, A.A. Effect of carbon dioxide on anthocyanins, phenylalanine ammonia lyase and glucosyltransferase in the arils of stored pomegranates. Journal of the American Society of Horticultural Science 1998, 123, 136-140.

- Holcroft, D.M, Kader, A.A. Carbon Dioxide–induced Changes in Color and Anthocyanin Synthesis of Stored Strawberry Fruit. HortScience 1999, 34 (7), 1244-1248.

- Gil, M.I., Holcroft, D.M., Kader, A.A. Changes in strawberry anthocyanins and other polyphenols in response to carbon dioxide treatments. Journal of agricultural and food chemistry 1997, 45, 1662-1667.

- Mathooko, F.M. Regulation of respiratory metabolism in fruits and vegetables by carbon dioxide. Postharvest biology and technology 1996, 9 247-264.

- 273-279 Please rewrite, correct and improve (see pdf).

The sentence has been rewritten to shortened it and make it easier to understand.

- 280-288 Please rewrite, correct and improve (see pdf). Please do not stop at the “other have found similar results level”. This does not really add. What’s about the changes in RR_CO2 and RR_O2 at high concentrations of O₂ or CO₂? Why did you measure until these pronounced concentration changes? At which CO₂/O₂ concentrations did the RR start to decline?

We did not consider necessary to explain in detail all the information that could be extracted from figure 1, because we focused the article in the application of UV-C to preserve fruit quality rather than in its effects over respiration. It is true that RR is determinant in postharvest quality, and obviously respiration curves provide much more information than that included in the manuscript. However, it was not our intention to detail it. Nevertheless, the paragraph has been modified expecting that it will be more clear.

- 296-297 “In high transmission rate packages, the gas composition varied similarly for UV1 and UV2 treated fruit at higher CO₂ and lower O₂ concentrations, respectively, but differed from controls (Figure 2 A).” Please use A and B for Figure 2 to denote MAP1 and MAP2.

The authors have followed the recommendation.

- 299-300 This means that UV-C radiation is disadvantageous for shelf life?

We have tried not to make a direct assertion and we have introduced “may” in the sentence.

As most technologies, UV-C effects may act over different mechanisms, that is why it is difficult to claim that some treatments are advantageous or disadvantageous for fruit quality. Statements must be based in results, so with that sentence we just intend to explained what we observed, based in another scientific research. The effects of UV-C are now (after the peer review) much better discussed along the manuscript and also in our rebuttal to the referees.

In general terms, but being as precise as possible in this summary, UV-C radiation is a potent oxidative agent, which generates toxic molecular species such as ROS. That is why this technology is used for microorganisms’ inactivation. In plants or fruits, these phytotoxic effects promote the production of certain compounds as a defence mechanism. In our opinion, it has also been well documented in the manuscript and rebuttal.

Finally, we are not able to claim if UV-C radiation is advantageous or disadvantageous for fruit shelf life. We have discussed our results and in our opinion this technology may have pros and drawbacks for fruit quality, but combining it with MAP could be an alternative to preserve raspberries.

Thank you for the recommendation. We also consider that the sentence is now easier to understand.

- 318-320 “shelf life. Given are means (± SD; n = 5) of controls and of raspberry samples, irradiated with UV-C (UV1: 2 kJ m^-2; UV2: 4 kJ m^-2) and packed with films of two gas transmission rates (A) MAP1 and (B) MAP2.”

The caption has been changed as suggested.

- 348-349 “CO₂ concentrations (10% or 16%) inside the packages, “

We have considered the correction.

- 353-354 Please rewrite, this is incorrect the way it is written. It is not the transmission rate.

The sentence has been rewritten as follows:

“Total yeast counts also increased two-fold during storage (data not shown), probably related to lower O2 and higher CO2 concentrations due to the film transmission rate.”.

- 354-355 “total yeast counts of UV2MAP2 samples increased by only 0.3 log unit compared to controls”

The sentence has been improved, considering the reviewer suggestion.

- 364-365 “microbial loads of treated samples closely reflect those of controls “

The sentence has been shortened as suggested.

- 380-382 “However, UV-C treatments did obviously not affect the color of the samples [5].”

Thank you. The sentence has been shortened as suggested.

- 382-384 “Lightness of raspberries packaged in MAP2, UV1, UV1MAP1 and UV2MAP1 were higher after simulated shelf life, which may increase customer acceptance.” You did not measure acceptance!

The sentence has been modified as suggested. The reference “[27]” was added at the end of the sentence to support that brighter fruit are preferred by consumers instead of darker ones.

We did not measure acceptance, but it is known, and has been previously stated by other authors (already cited in the manuscript), that colour is one of the most important attributes influencing visual appearance and thus, consumer acceptance [5,27].

- 387-389 “decreased in samples of all batches but varied significantly among samples on each day of analysis. This was especially valid for…”

The sentence has been modified as suggested.

- 389-390 Please rewrite and improve.

It has been revised and changed in the manuscript as follows:

“This was especially valid for UV2, MAP2, UV2MAP1 and UV2MAP2 treated raspberries, which TSS content sharply diminished from day 4.”

- 390-391 Please rewrite and improve. Not clear what this means.

The authors agree with the reviewer that it was a confusing sentence. Considering that the sentence did not add much information, the sentence has been removed from the manuscript.

- 392-393 This sentence is a bit odd. “, raspberries treated with UV1 maintained higher TSS during the entire the shelf life.” Or similar.

The sentence has been corrected.

“However, raspberries treated with UV1 maintained higher TSS content until the end of their shelf life.”

- 394-403 Please try to improve these sentences.

These sentences have been shortened and combined to make them easier to read.

“Significant differences were observed in pH of the samples on each day of analysis, and also during fruit shelf life. However, these changes were not attributed to the effect of the applied treatments and they were the result of the variability of the samples [63,64].”

- 404-412 Please try to improve these sentences.

These sentences have been rewritten.

“The ratio TSS:TA is called maturity index (MI). It may give a general idea of the degree of fruit senescence because it has been inversely correlated with other quality parameters such as fruit firmness [65,66]. From day 8, UV treated raspberries showed higher MI than MAP or UVMAP samples, but lower than controls. This may indicate that fruit irradiated with UV-C had a shorter period of storage and in sale. On the other hand, several factors influence the changes in TSS and TA during postharvest preservation, being not possible to determine the influence of UV-C radiation [67,68].”

- 404-405 Why should a maturity index reflect the stage of senescence? These are two different parameters. Who called it MI?

It is known that TSS and TA, which contribute to fruit flavour, change during storage due to physiological alterations so, as well as fruit firmness, they would determine the aptitude of the fruit to be commercialized. Senescence induces fruit softening as a result of middle lamella degradation, and also makes fruit more susceptible to the infection of microorganisms, having to be discarded. Consequently, we think that MI could be inversely correlated with the time the fruit could be stored and commercialized.

We have changed the sentence as follows: “The ratio TSS:TA is called maturity index (MI). It may give a general idea of the degree of fruit senescence because it has been inversely correlated with other quality parameters such as fruit firmness [65,66].”.

Several Authors used the ratio TSS/TA and called it maturity index (MI), some references are listed below:

Olmo M., Nadas A., García J.M. Nondestructive Methods to Evaluate Maturity Level of Oranges. Journal of science 2008. https://doi.org/10.1111/j.1365-2621.2000.tb16008.x

Blanda, G., Rodriguez-Roque, Maria J., Comandini, P., Flores-Cordova, M.A., Salas-Salazar, N.A., Cruz-Alvarez, O., Soto-Caballero, M.C. Phenolic profile and physicochemical characterization of quince (Cydonia oblonga Mill.) fruits at different maturity index. Notulae botanicae horti agrobotanici Cluj-Napoca 2020, 48 (4), 2306-2315.

Mayorga-Martínez, A.A., Olvera-Trejo, D., Elías-Zúñiga, A., Parra-Saldívar, R., Chuck-Hernández, C. Non-destructive Assessment of Guava (Psidium guajava L.) Maturity and Firmness Based on Mechanical Vibration Response. Food and bioprocess technology 2016, 9, 1471–1480.

Sdiri, S., Bermejo, A., Aleza, P., Navarro, P., Salvador, A. Phenolic composition, organic acids, sugars, vitamin C and antioxidant activity in the juice of two new triploid late-season mandarins. Food Research International 2012, 49 (1) 462-468.

Teerachaichayut, S., Thanh Ho, H. Non-destructive prediction of total soluble solids, titratable acidity and maturity index of limes by near infrared hyperspectral imaging. Postharvest biology and technology 2017, 133 20-25.

- 413-542 Please try to improve the sentences.

We have made changes in these paragraphs trying to improve the discussion. Some explanations are listed below.

Lines 428-430 and line 436: We do believe that, at some extent, there may be a relation between the stimulation of PAL, which triggers the biosynthesis of phenolic compounds, and the maintenance of fruit firmness, based on the results obtained by different authors. In our case, it was a reasonable explanation to discuss the results that we obtained.

In the next lines we are going to present the results from other studies that led us to the aforementioned explanation. Fruit firmness decreased during storage, but strawberries treated with UV-C radiation underwent lower firmness decrease [26], and, in this study, PAL activity was promoted especially just after UV-C treatment. Moreover, other authors claimed that the activation of PAL is linked with UV-C radiation [63,64,66,70,72]. As we mentioned in lines 422-423, there is no denying that UV-C radiation inhibits cell wall degrading enzymes as reported Bu et al. [69] and Ortiz-Araque et al. (2019), by downregulating the expression of genes involved in cell wall degradation (Pombo et al., 2009). Thus, it may also be important to explain the maintenance of fruit firmness after UV-C treatments. However, it has been also demonstrated that the accumulation of phenolic compounds and the formation of lignin-like polymers strengthen the cell wall [72]. These compounds resulted from phenylpropanoid pathway so its relation with PAL is obvious. The possibility of cell wall reinforcement of the epicarp and mesocarp cells has been supported by ultrastructural examination, and induced resistance against B. cinerea was also suggested [72]. We suggested that this reinforcement made cell walls less prone to degradation, which is closely related with fruit softening. Finally, PAL activity, as well as, the anthocyanin, flavonoid and phenol concentration of phenylalanine-treated plums increased (Sogvar et al., 2020). Besides the reduction of chilling injury incidence, they also observed less weight losses and higher fruit firmness in treated samples. Moreover, chilling injury is assessed by electrolyte leakage and MAD content, being the former a reliable indicator of cell membrane damage (Sogvar et al., 2020).

- Ortiz-Araque, L.C., Ortiz, C.M., Darré, M., Rodoni, L.M., Civello, P.M., Vicente, A.R. Role of UV-C irradiation scheme on cell wall disassembly and surface mechanical properties in strawberry fruit. Postharvest Biology and Technology 2019, 150, 122-128.

- Pombo, M.A., Dotto, M.C., Martinez, G.A., Civello, P.M. UV-C irradiation delays strawberry fruit softening and modifies the expression of genes involved in cell wall degradation. Postharvest Biology and Technology 2009, 51(2), 141-148.

- Sogvar, O.M., Rabiei, V., Razavi, F., Gohari, G. Phenylalanine Alleviates Postharvest Chilling Injury of Plum Fruit by Modulating Antioxidant System and Enhancing the Accumulation of Phenolic Compounds. Food technology and biotechnology 2020, 58 (4), 433-444.

Lines 437-444: The authors agree with the reviewer that this paragraph was not well explained. Some modifications have been made, involving remove sentence in lines 437-438 because do not add much information. Finally, the paragraph results as follows:

“Mass loss of controls and UV treated raspberries reached 6.9% and 8.1% (Figure 4), since macroperforated trays were not able to reduce water vapor exchange through the package decreasing their attractiveness and making them no suitable for commercialization. Weight loss (1.0%) was satisfactorily reduced in MAP and UVMAP samples due to the limited water vapor transmission rate of the MAP packages. Similar values were obtained by Haffner et al. [24] after 7 d of storage”

- 532-536 This sentences is odd and does not add at all.

The sentence has been modified.

- 536-537 This sentences does not add.

The sentence has been rewritten.

OTHER COMMENTS TO REVIWER 2 BASED ON THE ANNOTATIONS MADE IN THE PDF DOCUMENT

We appreciate the comments and highlighted sentences in the manuscript, because we consider they improve the quality of the text. Changes can be check in the revised version of the manuscript but some of them deserved an explanation so they are listed below:

- Line 33: We think that the increase in the consumption of berry fruits is related with their appearance but also with their nutritional properties. Nutritional value of these fruits has been studied in depth but it is nowadays when consumers are aware that their lifestyle may improve eating certain products.

We are also aware that this statement must be interpreted carefully because further clinical studies are required to conclude the effect of certain compounds in the organism.

- The terms “display cases” and “display cabinets” are synonyms. Although, the former may present lower frequency of use, according to Collin’s Dictionary, it is still widely used. Thus, we consider that is not necessary to change the term “display case” in the manuscript.

- Line 76: We are completely aware that, although closely related, technology and technique are not the same. We do really mean technology and we consider it is used adequately.

- Line 79: “aisle” is a long narrow space between the rows of shelves of a store or supermarket. An English native speaker checked the manuscript and the sentence is not hard to understand.

- Line 149: The sentence has been corrected. We only want to provide an average value of O₂ RR and CO₂ RR. We use an O₂ range where O₂ RR and CO₂ RR seem to be constant.

- Line 181: “cut into” instead of “split”. Yes, it was what we meaned, so it has been corrected.

- Line 278: The authors have removed this sentence from the manuscript because and explanation has been added in the next paragraph (lines 280-288).

- Line 305: As explained before, high CO2 concentrations may also promote fermentative pathways, inducing physiological changes, even when O₂ concentrations are far from anaerobiosis. See: Mathooko, F.M. Regulation of respiratory metabolism in fruits and vegetables by carbon dioxide. Postharvest biology and technology 1996, 9 247-264.

- Lines 315-317: We briefly presented there the results of microbial loads in order to point out their possible effect in the evolution of the gas composition inside the packages.

- Line 359: “This is relatively irregular…”. The sentence refers to the surface of the raspberries. It is irregular because of the causes explained in that sentence (359-360), which in our opinion is quite clear. Raspberries do not present a smooth surface hindering UV-C radiation effect.

- Line 360: “Styles” are structures part of the pistil of flowers.

In raspberries they still remain as a protection of the fruit, giving that hairy appearance, which contributes to the characteristic surface of raspberries.

- Line 430: “cellular barriers”. Sorry, we mean that these compounds are structural barriers. The sentence has been modified.

- Line 430: It is true. Thus, “(lignin and suberin)” has been removed and we only talked about “lignin-like polymers”.

Line 488-489: When giving results in fresh mass basis the concentration of the solutes proportionally increase as the water content of the fruit is reduced because they concentrate in the sample. Giving the results in dry mass basis is an alternative, but it would not be the real concentration of compounds when the fruit were eaten (in this case, fresh).

Line 507: It was not a comparison between fruit and leaves. They were two examples of the correlation between phenolic compounds and antioxidant activity in UV-C treated products.

Round 2

Reviewer 1 Report

This manuscript was improved after revision

Author Response

The authors are grateful for the revision and truly think that many parts of the manuscript have been upgraded after fitting the comments in revision round 1. The corrections have clarified and shortened the text, making it easier to read.

Reviewer 2 Report

In their revision, the authors successfully improved the very long manuscript. Nevertheless, some points, still, need to be rewritten and improved further, as stated below and directly in the pdf of the paper.

16-17 “(MAP) with transmission rates (TR) for O₂ and CO₂ of 1805 mL d-1 and 1570 mL d-1 (MAP1), and 902 mL d-1 and 785 mL d-1 (MAP2), respectively.” I feel that this is clearer. Please also note that “SI does not permit the use of intervening or modifying words among the terms in units; e.g., FM for fresh mass is not permitted, or mg / 100 g. Explain when first needed in your materials and methods section that results are expressed on a fresh mass basis, and use for example g kg-1 or mol kg-1 in the remainder of your manuscript”.

2/26-27 “CO₂ concentrations exceeding 25% or O₂ concentration below 1% may “

Chapter 3.1 Just a proposal; how about replacing “O2 and CO2 respiration rates” by “O2 and CO2 exchange rates”. This flexible but correct term is frequently used in plant physiology, and it is exactly that what you measured

6/33 The critical O2 concentration is known to be at least 2% or even less. So, you propose, it is the combination of very high cCO2 and low cO2 that initiate the switch. This is not fully clear from the sentence.

6/34 “reached, rates of O2 uptake decreases progressively, while those of CO2 release began to exceed O2 uptake, which may indicate the switch to….”

8/29 “with TRO2 of 902 mL d-1 and TRCO2 785 mL d-1.”

8/32-33 “low O2 (x% to y%) and moderate concentrations of CO2 (v% to w%)” Low and moderate are imprecise! Better give examples.

8/33-34 “may intensify the respiration activity in raspberries and, thus, the variation in the…”

14/39-41 “that the phenolic and anthocyanin contents and the antioxidant capacity increased in ‘Grandeur’ raspberries packaged with different”

16/53-58 "The combination of UV-C radiation and MAP but not their separate application improves the quality maintenance of raspberries. This, however, is only valid if a suitable MAP film with optimized O2/CO2 permeability effectively controls gas composition and, thus, fruit respiration within the packages. This, combined with a pretreatment of 4 kJ m-2 of UV-C radiation, successfully prolongs the shelf life and maintains high contents of bioactive compounds in fruit stored at 6 °C for up to 8 d."

Comments for author File: Comments.pdf

Author Response

The authors are grateful for the revision and truly think that many parts of the manuscript have been upgraded after fitting the comments in revision round 1. Below you can find the answers to your comments in revision round 2. We have considered these suggestions, making some changes in the manuscript, which are marked up using the Microsoft word track changes tool.

REVIEW REPORT 2

16-17 “(MAP) with transmission rates (TR) for O2 and CO2 of 1805 mL d-1 and 1570 mL d-1 (MAP1), and 902 mL d-1 and 785 mL d-1 (MAP2), respectively.” I feel that this is clearer. Please also note that “SI does not permit the use of intervening or modifying words among the terms in units; e.g., FM for fresh mass is not permitted, or mg / 100 g. Explain when first needed in your materials and methods section that results are expressed on a fresh mass basis, and use for example g kg-1 or mol kg-1 in the remainder of your manuscript”.

Thank you. We also think that the suggested sentence is clearer, so we have changed it in the manuscript. We have also corrected some units along the text to fit SI standard, as the reviewer suggested.

2/26-27 “CO2 concentrations exceeding 25% or O2 concentration below 1% may “

Thank you for the suggestion, the sentence has been changed in the manuscript.

We do appreciate the proposal and recognize that different terms are used in other scientific areas. However, “respiration rate” is commonly used in postharvest technology, and we think that it is accepted by other colleagues when talking about fruit respiration activity. Thus, we consider that, in this case, it is better to maintain the term used in the manuscript to be consistent with the main topic of the paper.

Yes, it is what we proposed, but maybe we have been imprecise when considering as critical higher concentrations than 2%, so the term “critical” has been removed from the sentence. Finally, we expect that the sentence will be clearer after the changes proposed by the reviewer in the next comment.

6/34 “reached, rates of O2 uptake decreases progressively, while those of CO2 release began to exceed O2 uptake, which may indicate the switch to….”

Thank you for the suggestion. We have considered the correction and the sentence has been modified in the manuscript.

8/29 “with TRO2 of 902 mL d-1 and TRCO2 785 mL d-1.”

It has been corrected in the manuscript.

8/32-33 “low O2 (x% to y%) and moderate concentrations of CO2 (v% to w%)” Low and moderate are imprecise! Better give examples.

The authors consider low O₂ concentrations below 5%, and in ULO controlled atmospheres, O₂ decreases below 1%, so 1-5% will be the range. However, in the references cited in the paper ([58,59]), higher concentrations of O₂ were evaluated in one of the cases. Thus, besides giving the examples, we have considered to substitute the term “low” by “reduced”. Moreover, moderate CO₂ concentrations will be between 10-15%, although some colleagues related it directly to 10% CO₂ concentration.

8/33-34 “may intensify the respiration activity in raspberries and, thus, the variation in the…”

The sentence has been modified as suggested.

14/39-41 “that the phenolic and anthocyanin contents and the antioxidant capacity increased in ‘Grandeur’ raspberries packaged with different”

The sentence has been also corrected in the text.

The authors kindly appreciate the suggestion to modify this paragraph in the conclusions section. We consider that now it is clearer, so it has been changed in the manuscript.

Other comments from the pdf file

The corrections are listed by line number according to the pdf.

- Line 245/247: “display cases” has been changed in the manuscript for “display cabinets” to avoid confusions in these sentences.

- Lines 918/922/1701/1718: plurals have been corrected.

- Line 931: The sentence has been modified as suggested

- Line 937: “not” has been added.

- Subheading in 3.2 section has been modified

- Line 1019: “differences” instead of “different evolution”

- Line 1036: has been changed by “respiration activity”.

- Line 1046: “by” has been added.

- Line 1131: The sentence has been changed as suggested

- Line 1133: Besides the explanation, in the previous round we also answered that, apart from the obvious aggregates, raspberries present a “hairy” surface due to the presence of styles. There is no doubt for us that raspberries present such characteristic, and, as far as we concerned, styles are responsible. However, we appreciate the experience of the reviewer and considering the insistence, probably due to a wider knowledge, we have removed “the styles” from the sentence.

- Line 1274: Sentence has been modified.

- Some prepositions have been changed along the manuscript (lines 1283, 1285. 1933)

- Line 1290: The sentence has been change as suggested

- Lines 1722-1723: Sorry, it is clear for us, as stated in the corresponding references. Thus, the sentence has been changed.

- Lines 1723-1724: The sentence has been rewritten, expecting that the structure has been improved.

- Line 2035: The sentence has been change as suggested

- Besides paragraph in lines 2053-2058, conclusions section has been further modified. The changes can be checked in the manuscript.