Sustainable Valorization of Halophytes from the Mediterranean Area: A Comprehensive Evaluation of Their Fatty Acid Profile and Implications for Human and Animal Nutrition

Round 1

Reviewer 1 Report

I have several concerns about this manuscript:

1.       The plants were all collected in June but is this the optimal time to harvest them? Are they all at their peak level of yield, nutritional value, texture, taste or flavour? I would be very surprised if this is the case. Accordingly, what is the usefulness of including data from the species that are not at their optimal state in June? This should be addressed in the manuscript.

2.       The total amount of FAME reported for these species (Figure 1) is very small. Even the maximum value is 8mg/g = 0.8% and the lowest is 1mg/g = 0.1%. I do not believe this can be correct, especially when it is on a dry weight basis. In my experience, most plants are ~1% lipid on a wet weight basis, so when you dry them the apparent lipid level increases by 4-5 fold (assuming 80-85% moisture). Is it possible the authors have miscalculated by a factor of 10? (they do cite 2 papers where the comparable values were 15-27mg/g).

3.       I am confused as to why Table 1 shows qualitative (%) data when the authors have the quantitative values from the calibration curves. The limited utility of quantitative data is exemplified by the claims made about the nutritional benefit of S. vera – it may have 36% ALA (1^st species column in Table 1 on page 5) but it has got the quantitatively lowest level of total FAME at 0.1% = so that is about 0.036 mg ALA/g DW. So a consumer would have to eat 27g of DW or >150g of wet weight S. vera to get 1 mg of ALA? That is not a good nutritional return!

4.       I would query the minimum limit of detection for FAME when the levels are so low. It is no co-incidence that the high number of FAME shown as n.d. for S. vera are because the machine is operating at the limit of detection. Given this, can the authors justify reporting these values to 2 decimal places?

5.       The conclusion (lines 199-200) about some of these species having “good nutritional value” needs to be modified since the data suggest the actual quantitative amount of omega-3 fatty acid is almost negligible. Also, ‘good nutritional value’ cannot be determined on one macro or micronutrient (omega-3 content) alone – what is the sodium content of the species? Do they contain any antinutritional (phytate) or toxic (cyanide) compounds?  

Author Response

Reviewer #1

I have several concerns about this manuscript:

Reviewer comment 1. The plants were all collected in June but is this the optimal time to harvest them? Are they all at their peak level of yield, nutritional value, texture, taste or flavour? I would be very surprised if this is the case. Accordingly, what is the usefulness of including data from the species that are not at their optimal state in June? This should be addressed in the manuscript.

 

Authors’ reply: The authors understand and are thankful for the reviewer comments. To the best of our knowledge, there is no information on the optimal time to harvest the species included in this study, regarding yield, nutritional value, texture, taste or flavour. Therefore, this study can be used as a reference for the fatty acids content for June. This information was added in the discussion of the manuscript (lines 213-217). Moreover, for promising selected species, work is in progress aiming the evaluation of the nutritional profile (including fatty acids) along the year, twice a season, in different locations.

 

Reviewer comment 2. The total amount of FAME reported for these species (Figure 1) is very small. Even the maximum value is 8mg/g = 0.8% and the lowest is 1mg/g = 0.1%. I do not believe this can be correct, especially when it is on a dry weight basis. In my experience, most plants are ~1% lipid on a wet weight basis, so when you dry them the apparent lipid level increases by 4-5 fold (assuming 80-85% moisture). Is it possible the authors have miscalculated by a factor of 10? (they do cite 2 papers where the comparable values were 15-27mg/g).

 

Authors’ reply: We agree that the FAME levels are very small. However, we have found other halophytes with even smaller levels, namely 0.152 mg/g for Spartina anglica, 0.192 mg/g for Salicornia europaea and 0.350 mg/g for Halimione portulacoides (Meziane et al., 1997). This information is in the manuscript (lines 104-107) as well as the following reference: Meziane, T.; Bodineau, L.; Retiere, C.; Thoumelin, G. 1997. The use of lipid markers to define sources of organic matter in sediment and food web of the intertidal salt-marsh-flat ecosystem of Mont-Saint-Michel Bay, France. J Sea Res 38: 47-58.

 

Reviewer comment 3. I am confused as to why Table 1 shows qualitative (%) data when the authors have the quantitative values from the calibration curves. The limited utility of quantitative data is exemplified by the claims made about the nutritional benefit of S. vera – it may have 36% ALA (1^st species column in Table 1 on page 5) but it has got the quantitatively lowest level of total FAME at 0.1% = so that is about 0.036 mg ALA/g DW. So a consumer would have to eat 27g of DW or >150g of wet weight S. vera to get 1 mg of ALA? That is not a good nutritional return!

 

Authors’ reply: We consider that giving both qualitative and quantitative data allows for the readers to have a better knowledge on the FA contents of target halophytes, precisely because they can relate the amounts of each detected FA with the FAME percentage. We agree that it is not an optimal nutritional return. However, we must keep in mind that the use of such plants, in fresh or as a matrix for the extraction of such FA, could never be considered as the sole source of any nutrient, including ALA. We have added the word “additional” source of ingredients to the discussion of the manuscript (line 197) to clarify this.

 

Reviewer comment 4. I would query the minimum limit of detection for FAME when the levels are so low. It is no co-incidence that the high number of FAME shown as n.d. for S. vera are because the machine is operating at the limit of detection. Given this, can the authors justify reporting these values to 2 decimal places?

 

Authors’ reply: Changes were made in the tables to address this comment. We replaced “n.d.” (not detected) to “*” (below the detection limit) in all the tables presented.

 

Reviewer comment 5. The conclusion (lines 199-200) about some of these species having “good nutritional value” needs to be modified since the data suggest the actual quantitative amount of omega-3 fatty acid is almost negligible. Also, ‘good nutritional value’ cannot be determined on one macro or micronutrient (omega-3 content) alone – what is the sodium content of the species? Do they contain any antinutritional (phytate) or toxic (cyanide) compounds?  

 

Authors’ reply: The authors agree with the reviewer comment and have included in the manuscript information regarding the presence of antinutritional components in halophytes biomass (lines 204-208). Regarding the “good nutritional value” classification, in this work, the classification of the nutritional profile was based not only on the omega-3 contents but also on the PUFA/SFA and n-6/n-3 ratios. The purpose of this work was to make the first approach to establish the nutritional profile of target species, focusing on fatty acids. Further nutritional evaluations are in progress, including samples from different areas of collection and seasons, to determine other relevant nutrients, proximate composition, minerals (including sodium), and antinutritional and toxic elements (e.g. tannins, phytate, saponins).

Author Response File: Author Response.docx

Reviewer 2 Report

The MS entitled “ Sustainable valorisation of halophytes from the  Mediterranean area: A comprehensive evaluation of their fatty acid profile and implications for human and animal nutrition” deals with a topic of interests and fits into the scope of the journal.

The article technically sounds fine, it is well organized, starting from the abstract till conclusions.

BUT the great weakness of this study is that it is based on a unique sampling of plant material. A repetition of sampling during different seasons of the year in the same locations, or sampling of each species from several areas would have provided more reliable results. So, I strongly recommend that at least authors compare their findings with previous published reports. I also consider that the reference list is rather short, not only in respect to similar studies, but also in relation to the Introduction, for instance when defining halophytes several important reviews are not mentioned (e.g. Flower and Colmer, 2008).

Statistic analysis is rather poor. A cluster or Principal Component Analysis would improve the quality of the Discussion.

As a minor aspect, scientific names and authorship of taxa should be revised (e.g. Crithmum not Chrithmum), and only valid names (not synonyms) should be used.

Author Response

Reviewer #2

The MS entitled “Sustainable valorisation of halophytes from the Mediterranean area: A comprehensive evaluation of their fatty acid profile and implications for human and animal nutrition” deals with a topic of interests and fits into the scope of the journal.

The article technically sounds fine, it is well organized, starting from the abstract till conclusions.

Reviewer comment 1. BUT the great weakness of this study is that it is based on a unique sampling of plant material. A repetition of sampling during different seasons of the year in the same location or sampling of each species from several areas would have provided more reliable results. So, I strongly recommend that at least authors compare their findings with previous published reports. I also consider that the reference list is rather short, not only in respect to similar studies but also in relation to the Introduction, for instance when defining halophytes several important reviews are not mentioned (e.g. Flower and Colmer, 2008).

 

Authors’ reply: We agree with the reviewer. This was, in fact, the first approach. Work is in progress aiming to evaluate the FA on samples collected in different sites of Portugal (Algarve and Lisbon), along different seasons during the year. There is limited available information on the FA contents of halophytic species but we increased our reference list in relation to the Introduction and Discussion. References added after revision include:

Abd El-Hack ME, Samak DH, Noreldin AE, Arif M, Yaqoob HS, Swelum AA. (2018) Towards saving freshwater: halophytes as unconventional feedstuffs in livestock feed: a review. Environ Sci Pollut Res Int. 25:14397-14406.

Flowers TJ, Colmer TD. (2008) Salinity tolerance in halophytes. New Phytol. 179:945-63.

Ryckebosch, E., Muylaert, K. and Foubert, I. (2012). Optimization of an analytical procedure for extraction of lipids from microalgae. J Am Oil Chem Soc 89: 189-198.

Inácio M.; Pereira, V.; Pinto, M. (2008) The Soil Geochemical Atlas of Portugal: Overview and applications. Journal of Geochemical Exploration. 98: 22-33.

Meziane, T.; Bodineau, L.; Retiere, C.; Thoumelin, G. (1997) The use of lipid markers to define sources of organic matter in sediment and food web of the intertidal salt-marsh-flat ecosystem of Mont-Saint-Michel Bay, France. J Sea Res 38: 47-58.

 

Reviewer comment 2. Statistic analysis is rather poor. A cluster or Principal Component Analysis would improve the quality of the Discussion.

 

Authors’ reply: The authors understand this comment but consider that a PCA is beyond the scope of this study.

 

Reviewer comment 3. As a minor aspect, scientific names and authorship of taxa should be revised (e.g. Crithmum not Chrithmum), and only valid names (not synonyms) should be used.

 

Authors’ reply: The paper was revised and corrected accordingly, as suggested.

 

Author Response File: Author Response.docx

Reviewer 3 Report

In general, the manuscript is well developed and provides new and interesting information and thus, it could be suitable for publication.

However, I have some suggestions to improve the Manuscript. Introduction can be improved by including more details of PUFA relevance. Also, I suggest to include information about anti-nutritional factors, this concept is pretty important when the intention is to use the halophytes for food and feed. There are several studies that show toxicity results in halophytes due to several secondary metabolites in the plants, for instance content of saponins in some halophytes makes the oil unlikely for edible. However, the rate of toxicity is affected by several factors such as rate of ingestion, type, and rate of microbial transformation of such metabolites.

In line 74 authors mention that samples were milled. It is important to specify the milling procedure because depending on the process, different oil yield can be obtained. Also, it will be useful if authors can include information about extractions methods already reported since FA content may depend on the extraction method which could be done for future studies and for large-scale processing.

I suggest some discussion on how environmental and soil conditions can be influencing the content of fatty acids. Environmental characteristics of the selected regions may be included.

Author Response

Reviewer #3

In general, the manuscript is well developed and provides new and interesting information and thus, it could be suitable for publication.

However, I have some suggestions to improve the Manuscript.

Reviewer comment 1. Introduction can be improved by including more details of PUFA relevance. Also, I suggest to include information about anti-nutritional factors, this concept is pretty important when the intention is to use the halophytes for food and feed. There are several studies that show toxicity results in halophytes due to several secondary metabolites in the plants, for instance content of saponins in some halophytes makes the oil unlikely for edible. However, the rate of toxicity is affected by several factors such as rate of ingestion, type, and rate of microbial transformation of such metabolites.

 

Authors’ reply: Introduction was improved by adding suggested information (lines 55-57). The following reference was added: Wu D, Lewis ED, Pae M, Meydani SN. (2019) Nutritional Modulation of Immune Function: Analysis of Evidence, Mechanisms, and Clinical Relevance. Front Immunol. 15:3160. Also, information about anti-nutritional factors and possible toxicity was added to the manuscript discussion (lines 204-208), as suggested.

Reviewer comment 2. In line 74 authors mention that samples were milled. It is important to specify the milling procedure because depending on the process, different oil yield can be obtained. Also, it will be useful if authors can include information about extractions methods already reported since FA content may depend on the extraction method which could be done for future studies and for large-scale processing.

 

Authors’ reply: Indeed, different extraction methods will obtain different yields. Changes were made to address this comment (lines 109-112).

 

Reviewer comment 3. I suggest some discussion on how environmental and soil conditions can be influencing the content of fatty acids. Environmental characteristics of the selected regions may be included.

 

Authors’ reply: The authors appreciate the reviewer’s comment and have addressed to it accordingly. Environmental characteristics of the selected regions were included (lines 208-217), as suggested.

 

 

 

 

 

Author Response File: Author Response.docx