Poultry Shelf-Life Enhancing Potential of Nanofibers and Nanoparticles Containing Porphyra dioica Extracts

Round 1

Reviewer 1 Report

Thank you for the opportunity to review this manuscript. My recommendation is "Major revision (nearly rejected)". Reboleira et al. herein reported the development of coating methods of a water-soluble extract from commercially available red macroalgae to films and evaluated their anti-microbial effects on foods as packages. However, I could not find any difference (or merit) for their coating technology. Therefore, the authors need to improve the manuscript before the decision.

Thank you very much for your timely review.

I will attempt to address both the merits of the technology applied and the materials used (P. dioica extract), highlighting the additions to the original manuscript.

The coatings were constructed with a purified natural extract, and with a majority compound with verified presence (porphyran)

A large volume of current research incorporating natural extracts in established technologies is focused on large scale screening of bioactivities and in the use of crude or roughly fractioned extracts. While this approach can lead to very promising natural additives, and should by no means be discredited, it often faces problems when dealing with large scale implementation and reproducibility of results. This is often due to the complex mixture of molecules and its dependency on external factors, growth conditions and seasonality of natural resources.

By working with a purified extract and confirming the presence of porphyran in our residues we can work with a resource that is more reproducible and trace its activity to the compound itself with greater confidence.

Changes to our description of the FTIR methodology were added to topic 2.2., as well as Introduction content addressing the use of purified natural extracts.

There were strong indicators of antimicrobial activity in electrospun fibers containing 1% P. dioica extract

While there was a lack of statistical differences with between coated and uncoated films in microbial counts, we decided that these results were worthy of publication when backed with the significant difference in the sensory evaluation of the coated samples against uncoated film and uncoated control samples.

Also, there were increases on initial microbial load, in part due to handling of the sample, and due to the non-sterile environment of the facility where our electrospinning device is located. Even with an actively antimicrobial coating, the dried coated surface and handling of the films until moment of testing can impart bacterial spores that increase the initial (t0) counts and inevitably shift the final measures of organisms above the 10⁵ threshold.

Further testing could even reveal successful maintenance of a microbial count below the required maximum for retail sale even after the required handling. An integrated packaging device that immediately applied the coated films to mechanically separated chicken breasts would likely achieve this as well.

These results were obtained in the only formulation that successfully incorporated these extracts into fibres

The use of P. dioica extract concentrations above 1% on our gelatin and PVA solutions lead to a disruption of the fibre structure, or the appearance of large and uniform beads in the fibre matrix. As we intended to test both sprayed and spun coatings, we worked with an electrospinning solution (B) with limited extract concentration.

Seeing a reduction in microbial counts with this extract indicates not only that the homogenous fibre structure is important to its effect, but also that we may yet see a higher inhibition in an optimized formulation that achieves >1% P. dioica extract concetrations. We are continuing to improve our understanding of our own electrospinning device and are working to find a way to achieve these fibres with higher concentrations of extract.

We have now added content on topic 2.3 addressing the spinning of solutions with higher amounts of extract, and on topic 3.2.1 addressing the possibily of using these solutions in the future.

Industry is yet to apply these innovations, and our research is industry-driven and focused on direct application 

While research regarding active packaging is currently rising in popularity, its application is still riddled with challenges. By working closely with industry partners, we have the oportunity of sharing research that is relevant to peers constrained by the challenges and limitations of industrial implementation.

We hope that these statements have assisted in clarifying the relevance of our research from the author's perspective. Thank you again for your time and attention.

Reviewer 2 Report

The manuscript deals with the application to poultry products of coatings of electrospun and electrosprayed P. dioica-porphyran- rich extract aiming at enhance the shelf-life and inhibit the microbial growth. The introduction is well organized and represents a good description of the state of the art (probably could be improved with some references, see attached .pdf). The experimental needs for a slight revision as regards the instrumental section, FTIR must be described in details and SEM improved. The results are interesting and worthy of publication. According to the comments above and to the suggestions reported in the attached file in order to remove some typos and mistakes, the manuscript can be accepted after a minor revision.

Thank you very much for your thorough and timely review.

I have addressed all the suggestions present in the pdf file you have attached in a revised and highlighted manuscript.

Thank you for your attention.

1. SEM: define abbreviation first time is used.

Scanning 23 Electron Microscopy (SEM)

2. 3,6-anhydro-a-L-galactose units, "a" please use correct greek symbol.

3,6-anhydro-α-L-galactose units

3. ref 23 I suggest to increase the reference for this statement considering you wrote about "purposes" maybe could be better do not limit to one reference. ie: Photoactivity of hierarchically nanostructured ZnO-PES fibre mats for water treatments; Ognibene, G., Cristaldi, D.A., Fiorenza, R., (...), Scirè, S., Fragalà, M.E. 2016 RSC Advances 6(49), pp. 42778-42785 Electrospinning of nanofibers: Reinventing the wheel? Li, D., Xia, Y. 2004 Advanced Materials 16(14), pp. 1151-1170.

Added.

4. ATR-FTIR description of the measurements and instrument are due.

Fourier transform infrared (FTIR) spectroscopy with 135 attenuated total reflectance (ATR), confirming the presence of porphyran, as per displayed in figure 136 2. (See Figure SM 1 in the Supporting Material for the full spectrum). FTIR analyses were carried out 137 using an Alpha-P Brucker FTIR-ATR spectrometer, in the range of 4000–400 cm−1, at a 4 cm−1 138 resolution with 64 scans. It was possible to observe the absorption bands typically assigned to the 139 sulphate ester moieties at ca. 1245 and 813 cm-1. The strong bands within the 1200-1000 cm-1 range 140 were tentatively assigned to secondary alcohol C-O stretching vibrations and galactose residue ring 141 vibrations, as reported for closely related galactans such as carrageenan and agarose. [39,40] The 142 existence of a weak band at ca. 932 cm-1 was attributed to the presence of 3,6-anhydrogalactose 143 residues in the polysaccharide chain. Additional bands could be observed at ca. 3360 and 1652 cm-1, 144 which were respectively assigned to hydroxyl and carbonyl stretching vibrations. The C-H stretching 145 vibrations were also observed as weak bands in the 3000-2800 range [36–38].

5. Scanning Electron Microscopy: used voltage and working distance are due. Ok I know that these information are reported in the figure but I suggest to write them also here.

SEM, using a Vega3 Tescan 168 (Tescan, Czechia), with 15 kV of voltage and a working distance ranging from 15.80 to 15.87 mm.

Reviewer 3 Report

The work presented lack of reliable data and statements, significant modifications are required.

1.How about the adhesion of the coating on PP after the electrostatic spinning？Need to be dried?

The choice polyvinyl alcohol (PVA) as structural polymer for this study was due to its common use a water-soluble binding agent. We have not found any study using PVA in electrospinning applications that reported complications with adhesion, so long as the fibres were successfully formed. In the final tested conditions, we did not verify any issues with adhesion or drying, as the coating was firmly set and dried on the PP film by the end of each cycle. Working in a humidity-controlled environment ensured that these binding capabilities remained mostly unchanged throughout production.

2. How to ensure uniformity and thickness of such coating?

In the early stages of this study we performed extensive testing of the electrospinning conditions until a uniform coating was achieved with our desired solutions. These tests consisted of gradual shifts in applied voltage, needle tip to to collector distance, flow rates and polymer concentrations. The conditions achieving uniform spray or fibers were then registered and tested with the addition of P. dioica extract. Conditions that successfully spun the solutions that included extract were verified for uniformity using SEM. We decided against publishing these tests as they were largely based on previously reported conditions for the spinning of these polymers, only adjustments being made to accommodate our own custom equipment that is regularly undergoing modifications.

As our work with electrospinning progresses, and we aim towards increasing the scope of these tests, we plan on performing more extensive optimization of the physical properties of these coatings on new electrospinning equipment.

3. There is no gelatin in A, and it is mentioned in the article that gelatin has an effect on the quality of spinning. Then, is it possible that in the case of high concentration of both P.dioica extract and gelatin, the effect will be better than that of B?

The use of P. dioica extract concentrations above 1% on our gelatin and PVA solutions lead to a disruption of the fibre structure, or the appearance of large and uniform beads in the fibre matrix. As we intended to test both sprayed and spun coatings, we decided to move on with the use of coatings with disparate algae extract concentrations, indeed expecting a higher antimicrobial effect on the solution with more extract.

The results seem to indicate that having uniform fibres instead of sprayed particles has an even more important effect. As such, it is indeed probable that fibres spun with higher concentrations of P. dioica extract show higher inhibition of microbial growth. Unfortunately, and as stated, we could not achieve higher concentrations of this extracts and maintain uniform fibres. We are continuing to improve our understanding of our own electrospinning device and are working to find a way to achieve these fibres with higher concentrations of extract.

We have now added content on topic 2.3 addressing the spinning of solutions with higher amounts of extract, and on topic 3.2.1 addressing the possibily of using these solutions in the future.

4. What are the advantages of other reported substances?

I'm afraid I do not understand what substances you are referring to.

If you are addressing other substances in the electrospinning solutions, the only substance that could have an effect on tested parameters is the acetic acid used to dissolve the gelatin used in coating B. This compound is volatile and is reported to evaporate throughout the spinning process, leaving but trace amounts in the coating. 

The use of gelatin was required for the formation of nanofibers and is commonly paired with other synthetic polymers, including polyethylene oxide (PEO), as well as PVA, in electrospinning for biomedical use. It can, in certain applications, enhance material properties of coatings and scaffolds, and it proved essential to the formation of nanofibers in our work.

We have added some of these insights in our Introduction, (lines 91 to 96) with the proper citations. Do not hesitate to contact us if this does not adequately respond to your question.

Round 2

Reviewer 1 Report

I could not track the changes because there is no highlighting text in the text file. Therefore, I am unable to recommend anything.

Thank you again for your attention.

You will find the changes mentioned in our previous reply highlighted in this current attachment.
My apologies for this oversight.

Reviewer 3 Report

Authors answered my questions but the manuscript still should be revised: 

1.What is the width of the fibrous structure in the SEM images of Fig.4? Please addressed in discussion or caption (zoomed inserts are suggested). 

Information regarding the width and thickness of the coatings was added to section 3.1, highlighted in the annexed file. 

2. About Q4: What are the advantages of other reported substances? The substances is like  P. dioica you used in this article rather than other substances in the electrospinning solutions.

More information about natural extracts and purified natural extracts (such as the P. dioica extracts used here) used as additives in electrospinning was added to the Introduction. It should also be noted that the Introduction already contained a segment regarding the use of synthetic additives in electrospinning for shelf-life enhancing effects. These were stated as effective but with significant drawbacks regarding safety and sustainability.

Also, some new comments on how the growth inhibition of our enriched coatings compares with published literature was added to section 3.2.1.

All of these changes are also highlighted in the annexed file.

3. Figure SM should not appeared in main file, please remove it.

Revised.

4. line 149, unit was missed. 

Both of these were noted and changed accordingly. Thank you again for your thorough review.

Round 3

Reviewer 1 Report

Line 298: Some text might be missing. Please check carefully and correct that.

The requested changes are included in the manuscript attached to your previous report (Round 2).