Changes in Carbohydrates, Organic Acids, and Minerals at Different Development Stages of Hexachlamys edulis Fruit, a Wild South American Species with Horticultural Potential

Round 1

Reviewer 1 Report

Introduction

The final part starting from line 76 refers to general considerations on fruits that should be better contextualized. There is no need to make a list of compounds (like in lines 90 – 95), as it is not a treatise on chemistry. If the purpose of this work is to identify the moment in which the accumulation of some chemical components is optimal, the authors must refer to the factors that might influence this accumulation and the beneficial effects that these components could have.

Line 51: Suddenly, authors go from general into specifics, mentioning names of plants which are unknown for most people (açaí, Andean berries and camu camu)

Line 62: of ubajay leaves

Lines 69-71: This is a general consideration that would be better included among the potential of the species and not in this paragraph describing results obtained on this species.

Lines 113-114: Which fruits?

Material and methods

Lines 167-175: replace is with was, and are with were

Lines 179-183: Elements are not specified. Why macro and micro elements (N P K Ca Mg Na Fe Zn Cu Mn) have not been examined?

Results and discussion

The results and discussion are largely to be rewritten. The authors report numerous works on other fruit species (the most disparate), the results of which, however, do not help to understand or make sense of the values obtained in H. edulis. Indeed, in this work there is no consideration that helps to decide what is the optimal harvest time.

I don't think it is useful to the manuscript to mention the carbohydrates, organic acids and minerals of other fruits without there being a precise connection. Instead, I suggest referring only to comparable results or those that in some way can help defining specific ripening characteristics of this fruit.

Line 210: not clear

Lines 219-222: Adds nothing to the paper and difficult to follow (check was and are)

Lines 229-231: Check grammar

Lines 234-236: cherimoya??? (everyone knows this name?) What is the connection with the above reported results on H. edulis?

Lines 237-243: Already mentioned on page 5

Lines 244-247: What is the connection with the reported results on H. edulis?

Line 274: Reference 34 refers only to watermelon, everything else is missing.

Line 276-277: could? To which species and bibliographic reference does this sentence refer? I also believe that there is a very extensive bibliography on this topic.

Lines 277- 281: What is the connection with the reported results on H. edulis?

Lines 285-287: what’s the meaning in this context?

Line 299: replace is with was

Lines 301-303: in which fruits and which taste? If this reference is used to formulate a hypothesis, it must be very clear

Line 305: check states

Lines 314- 316: These values and increments are totally different

Lines 317- 320: What is the connection with the reported results on H. edulis?

Lines 333-337: What is the connection with the reported results on H. edulis?

Line 357: check attribute

Conclusions

In the paper no considerations are given that helps to decide what is the optimal harvest time of H. edulis.

I warmly recommend the authors to modify the discussion and the conclusions by inserting some references and considerations for the definition of the ideal moment for fruit harvesting, since this was the aim of the study.

Author Response

Reviewer 1

We thank to the reviewer 1 for the detailed suggestions made to improve the manuscript.

Introduction

The final part starting from line 76 refers to general considerations on fruits that should be better contextualized. The sentence “Nowadays, consumers are aware of the nutritional value of the new products they incorporate to the diet” was moved to the first paragraph.

There is no need to make a list of compounds (like in lines 90 – 95), as it is not a treatise on chemistry. If the purpose of this work is to identify the moment in which the accumulation of some chemical components is optimal, the authors must refer to the factors that might influence this accumulation and the beneficial effects that these components could have. The following sentences were removed “Moreover, dietary fiber is composed of homopolysaccharides, heteropolysaccharides, resistant starches, oligosaccharides, lignin, gums, and mucilages. Insoluble dietary fibers include plant cellulose, hemicellulose, and lignin material, and provide structural strength to plant material, whereas, gums, mucilages, pectins, β-glucan, galactomannans, and glucomannans are considered as soluble fibers and have gel formation capacity”.

Line 51: Suddenly, authors go from general into specifics, mentioning names of plants which are unknown for most people (açaí, Andean berries and camu camu). This phrase was improved as follow: “So, in some underutilized fruits such as Euterpe oleracea, Andean berries and Myrciaria dubia the positive effect of the bioactive substances on the antioxidant status and oxidative stress in humans was verified”.

Line 62: of ubajay leaves. The word “leaves” was added.

Lines 69-71: This is a general consideration that would be better included among the potential of the species and not in this paragraph describing results obtained on this species. The sentence “Consumption of carotenoid compounds has positive health impacts, including defense against cancer, cardiovascular disease, age-related disorders, and oxidative stress, as well as protection against macular degeneration [9].” was moved above.

Lines 113-114: Which fruits? Due to this sentence refers to a great number of wild fruits based on the reading of the bibliography on this topic, it was rewritten as follow: “At this time, knowledge about the quality and potential use of wild fruits throughout the world is still scarce. Their biochemical analysis could lighten information to breeders, producers and consumers”.

The objective was redefined as follow: “The aim of this work was to study the patterns of accumulation of carbohydrates, organic acids and minerals during different stages of H. edulis fruit development, for its evaluation as a source of health-promoting compounds, which is necessary in order to be included into the Argentine Food Code. Also, the obtained results will allow deciding the optimal time for consumption to get better flavor and a good contribution of the nutrients evaluated”. We consider that this definition is more in agreement with the obtained results and its discussion.

Materials and methods

Lines 167-175: replace is with was, and are with were. These changes were made.

Lines 179-183: Elements are not specified. Why macro and micro elements (N P K Ca Mg Na Fe Zn Cu Mn) have not been examined? The following sentence was added “The minerals Mg, K and Mn were quantified”. Mg and K were selected due to they are important minerals in the electrolytic balance which gives highlighted properties in populations of children, adults and athletes, while Mn for its antioxidant activity.

Results and Discussion

The results and discussion are largely to be rewritten. The authors report numerous works on other fruit species (the most disparate), the results of which, however, do not help to understand or make sense of the values obtained in H. edulis. Indeed, in this work there is no consideration that helps to decide what is the optimal harvest time. I don't think it is useful to the manuscript to mention the carbohydrates, organic acids and minerals of other fruits without there being a precise connection. Instead, I suggest referring only to comparable results or those that in some way can help defining specific ripening characteristics of this fruit. Special attention was put in the connection among the references and the text. We think that it is useful to compare the carbohydrates, organic acids and minerals with other fruit species in order to consider H. edulis fruits a good source of health- promoting compounds. Also, considerations were made about the optimal time for consumption.

Line 210: not clear. The sentence “Total organic acids analysed accounted for up to 22.7% of the dry weight of the ripe flesh of fruits” was deleted.

Lines 219-222: Adds nothing to the paper and difficult to follow (check was and are). The objective of the manuscript was adjusted to emphasize the study of the evolution of carbohydrates, organic acids and minerals during the development stages to decide the most convenient consumption time for a better amount of health-promoting compounds and good flavor. Therefore, comparison with other fruits contextualized the information about ubajay to choose whether it is (or not) a good source of the studied compounds.

Lines 229-231: Check grammar. The sentence “Total organic acids analysed could not explain the levels of total titratable acidity due to both variables did not present a significant correlation (r=-0.439, p=0.1537)” was modified as follow: “In H. edulis fruits, total titratable acidity kept high during unripe, medium ripe and ripe stages, and decreased in overripe stage (1.8, 1.6, 1.8 and 1.4%, respectively) [8], following a different behaviour with respect the total organic acids, fact that explained the non-significant correlation found between total titratable acidity and the total organic acids (r=-0.439, p=0.1537).”

Lines 234-236: cherimoya??? (everyone knows this name?) What is the connection with the above reported results on H. edulis?. The sentence “In “cherimoya”, the increase in acidity during ripening is related to the accumulation of malic acid [25,26]”, was modified as follow: “In the subtropical fruit Annona cherimola, a significant increase in organic acid levels during ripening occurred as in H. edulis, and the increase in acidity was related to the accumulation of malic acid [25,26]”. This change connects the results obtained in H. edulis with those found in other subtropical fruit, which presented a similar pattern of malic acid accumulation.

Lines 237-243: Already mentioned on page 5. The following paragraph was deleted “The organic acid concentration in H. edulis fruit showed different behavior during the development stages according to its nature, i.e. succinic acid concentration was high in unripe fruit, decreased in medium ripe and ripe fruit and increase again in overripe fruits. However, malic acid increased to attain a maximum concentration in ripe fruits, to then decrease in overripe fruits, while quinic acid, was minimum in unripe fruits to then keep invariable. The same behavior was observed when the sum of the organic acids analysed was evaluated. The levels of quinic acid were studied in stone fruits at different stages of development [31]. It was found that quinic acid (g^–1 FW) was higher in peach and plum than in apricot. Interestingly, although the quinic content of these fruits does not change, the increase in the mass of the flesh reduces the concentration of quinic acid through development [31]”.

Lines 244-247: What is the connection with the reported results on H. edulis? The following phrase was eliminated: “It was found that quinic acid (g^–1 FW) was higher in peach and plum than in apricot. Interestingly, although the quinic content of these fruits does not change, the increase in the mass of the flesh reduces the concentration of quinic acid through development [31].

Line 274: Reference 34 refers only to watermelon, everything else is missing. We rewrote the sentence as follows: Accumulation of sucrose, glucose, and fructose during ripening was found in sweet fruits such as Citrullus lanatus (watermelons) and Phoenix dactylifera (date) [34, 35].

Line 276-277: could? To which species and bibliographic reference does this sentence refer? I also believe that there is a very extensive bibliography on this topic. The sentence "In addition, starch degradation could contribute to the increase in glucose content as it was demonstrated for Malus domestica Borkh. cv. Gala (apples) and Actinidia deliciosa (kiwifruit)" was added with its references.

Lines 277- 281: What is the connection with the reported results on H. edulis? This test was deleted.

Lines 285-287: what’s the meaning in this context? The contents of carbohydrates in H. edulis fruits were expressed in terms of fresh weight to be compared with other underexploited fruits from India.

Line 299: replace is with was. It was corrected.

Lines 301-303: in which fruits and which taste? If this reference is used to formulate a hypothesis, it must be very clear. The reference refers to grapes, where the sweet taste is related to lower glucose/fructose ratio.

Line 305: check states. It was corrected.

Lines 314- 316: These values and increments are totally different. Besides the specific numbers we wanted to highlight that as in H. edulis soluble dietary fiber increased and insoluble dietary fiber decreased. We rewrote the sentence to gain clarity.

Lines 317- 320: What is the connection with the reported results on H. edulis?. This phrase was deleted.

Lines 333-337: What is the connection with the reported results on H. edulis? We wanted to highlight the similarity in the patterns of dietary fiber accumulation and the amount of total fiber between H. edulis and tropical and underutilized fruits. This comparison provides information to decide how significant for consumers the ubajay fruit could be as a source of fiber.

Line 357: check attribute. Attribute was changes by “attributes”.

Conclusions

In the paper no considerations are given that helps to decide what is the optimal harvest time of H. edulis. The conclusions were completely rewritten, including how the obtained results allow as to define the optimal time for consumption.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

Reviewer 2

We thank to the reviewer 2 for the detailed suggestions made to improve the manuscript.

Introduction

- Line 87: Intrinsic and extrinsic factors determine the content of sugars, as well as their transport, metabolism, accumulation and the relationship between them. I would use the words internal and external factors instead of Intrinsic and extrinsic. The words Intrinsic and extrinsic were replaced by “internal and external”.

- Line 101: Fruit palatibility to be consumed raw or used in fruit products is influenced by the presence of organic acids in their fleshy parts. I would use the words taste or flavor instead of palatability. The word “palatability” was replaced by “flavor”.

- Line 111: There are several preharvest and postharvest factors that influence the contents of minerals in horticultural crops. Please add some of the factors and references for this. Some preharvest and postharvest factors were added, as well as three more references.

The objective was redefined as follow: “The aim of this work was to study the patterns of accumulation of carbohydrates, organic acids and minerals during different stages of H. edulis fruit development, for its evaluation as a source of health-promoting compounds, which is necessary in order to be included into the Argentine Food Code. Also, the obtained results will allow deciding the optimal time for consumption to get better flavor and a good contribution of the nutrients evaluated”. We consider that this definition is more in agreement with the obtained results and its discussion.

Materials and methods

Lines 121-129: In the Chapter 2.1. Plant material and growing conditions there is no data about environmental growing conditions (air temperature and precipitation during examined growing season). Also, in this part should be add how many days passed between each harvest stage. Data on growing conditions was added as follow: “Mean air daily temperatures in October, November and December 2018, months when the flowering and fruit growth and ripening took place, were 17.3, 20.9 and 22.0 ºC, respectively. Rainfall was 200, 81 and 157 mm along the mentioned months, respectively. Also, information about days after full bloom was added in relation to how many days passed between each harvest day: “Stage 1: Unripe, fruits with all green skin (fruits with 21 days after full bloom: dafb); Stage 2: Medium ripe, fruits with green and yellow skin (fruits with 35 dafb); Stage 3: Ripe, fruits with all yellow skin (fruits with 42 dafb); and Stage 4: Overripe, fruits with yellow and brown skin (fruits with 49 dafb)”.

- Lines 176-178: It is not clear what the Figure 1 presents in the chapter. Is it part of the results or what? Figure 1 (Figure 2 in the revised manuscript) presents Total sugars and Total organic acids, and it is part of the Results section. This Figure was moved to Results and Discussion, above the Table 2.

- Line 179: In the Results you present some data about correlation? It should be mentioned and explained in the methods. Mention about data correlation was included in Materials and methods as follows: “Pearson's correlation analysis was used to examine the relationship between total titratable acidity with each organic acid and with total acids to understand how these variables were related”.

- Table 1: Align Table. Table 1 was aligned.

Results and Discussion

Lines 223-225: In citrus fruits, quinic acid was the major organic acid at the beginning of the development; later, citric acid was predominant in acidic varieties, while in less sour types, malic acid overtooked it. I would use the words acidic types instead of sour types. Sour types were changed by acidic types.

- Lines 229-231: Total organic acids analysed could not explain the levels of total titratable acidity due to both variables did not present a significant correlation (r=-0.439, p=0.1537). You only mention the correlation coefficients in the part of the results related to acids, and we do not see these data in the tables. Also, data on total acids should be added to the table. Due to only a few correlations were statistically significant, we decided to include these ones in the text. Data on Total acids were shown on Fig. 1. This is the reason due to these data were not included in Table 1.

- Table 2: Align and add data of total sugars as well as data about sugar/acids ratio. In the text, you often mention the taste of fruits, and the taste depends on the ratio of sugar and acids. Table 2 was aligned. Total acids as well as total sugars were shown in Fig. 1. The ratio between these two variables were not included in the original manuscript; however, according to the suggestion made by the reviewer 2, the ratio was calculated and included in Table 2.

- Table 3 and 4: Align Tables. Tables 3 and 4 were aligned.

Conclusions

The conclusions should be more related to the title and purpose of your paper. The conclusions were completely rewritten, including how the obtained results allow as to define the optimal time for consumption.

Author Response File: Author Response.docx

Reviewer 3 Report

The presented article is on an interesting topic, rather appropriately processed, however it is necessary to make certain corrections. It is also a pity that vitamin determination was not carried out for this minor fruit species.

Abstract

Throw away where the fruit was grown, it is stated in the methodology.

Delete and replace the abbreviation DW with the whole word (dry weight). This abbreviation is only explained in the text of the methodology.

Percentage representation of acids: either indicate for all harvest stages or remove. In general, I suggest deleting, it is not clear from the text what - which acid was up to 100%.

Line 34 - should be properly oscillated.

Introduction

Considering that it is not a common commercial crop, it is necessary to add the family and a brief biological-botanical description.

Line - 51 what is camu camu, you mean Myrciaria dubia, use proper names do not slang.

Line - 77 should be organoleptic or organoleptically.

Line - 101 is supposed to be palatability.

Methodology

Specify the harvesting methodology, were the fruits harvested when the colour change of the fruit was achieved, or after a week of colouring?

Due to the lack of clarity in the harvesting methodology, fruits at different levels of harvest maturity could be presented as figures.

The period of coloration of the skin of the fruit varies in length, and during this period biochemical changes in the composition of the fruit gradually occur.

It is necessary to precisely specify the harvest date, it is suitable, for example, in the days after flowering, etc.

Line 146 is an extra dot after were…

Line 183 AAAnalyst 200 – shouldn't it be AAnalyst 200 ?

Results

Line 198-212 – just as in the abstract, the percentages of the total acid content of medium ripe and ripe fruit are not given, somewhere it is stated somewhere not...to be added.

Overall, I consider the part devoted to the determination of acids and total TTA to be confusing. Here, units of mg/g and percentages are mixed one over the other without a continuous link.

They recommend reworking so that it is clear which acids and in what percentage scale they are represented, ideally a graph.

Line 192 - should be depending.

Line 208 - should be fruits.

Line 235 - stated 2x the

Line 242 – should be behaviour.

Line 295 – should be differentiate.

Line 319 – should be cellulolytic.

All tables: what is the meaning of the last empty line of the table stating Mean±SE?

Conclusions

The conclusion needs to be strengthened more by scientific results. not what was measured but what was found and what it leads to...

There are quite a few typos throughout the text and the English should be improved.

Author Response

Reviewer 3

We thank to the reviewer 3 for the detailed suggestions made to improve the manuscript.

Abstract

Throw away where the fruit was grown, it is stated in the methodology. “Fruits were harvested from H. edulis plants growing at the experimental field of the University of Morón (Moreno, Buenos Aires, 34°35'4.98" SL, 58°48'52.09" WL, 14 m.a.s.l.).” This phrase was removed.

Delete and replace the abbreviation DW with the whole word (dry weight). This abbreviation is only explained in the text of the methodology. DW was replaced by dry weight along the manuscript.

Percentage representation of acids: either indicate for all harvest stages or remove. In general, I suggest deleting, it is not clear from the text what - which acid was up to 100%. Percentage representation of acids were removed from all the text.

Line 34 - should be properly oscillated. This phrase was corrected.

Introduction

Considering that it is not a common commercial crop, it is necessary to add the family and a brief biological-botanical description. The phrase “Hexachlamys edulis (O. Berg) Kausel & D. Legrand, “ubajay”, is a fruit tree species native from South America” was modified as follow: “Hexachlamys edulis (O. Berg) Kausel & D. Legrand, “ubajay”, is an underutilized species native from South America belonging to the Myrtaceae family. Also, the following sentence was included: “H. edulis is a fruit tree species with yellow globose drupes referred as sweet-sour to very acidic, pleasant, and with a quickly overripening. Fruit set and ripening occur during spring to early summer in South America.”

Line - 51 what is camu camu, you mean Myrciaria dubia, use proper names do not slang. The scientific names of açaí (Euterpe oleracea) and camu camu (Myrciaria dubia) were added.

Line - 77 should be organoleptic or organoleptically. The word “organoleptical” was changed by “organoleptic”.

Line - 101 is supposed to be palatability. Certainly, the correct word was “palatability”. However, according to the suggestion made reviewer 2 “Fruit palatability” was changed by “fruit flavor”.

The objective was redefined as follow: “The aim of this work was to study the patterns of accumulation of carbohydrates, organic acids and minerals during different stages of H. edulis fruit development, for its evaluation as a source of health-promoting compounds, which is necessary in order to be included into the Argentine Food Code. Also, the obtained results will allow deciding the optimal time for consumption to get better flavor and a good contribution of the nutrients evaluated”. We consider that this definition is more in agreement with the obtained results and its discussion.

Materials and methods

Specify the harvesting methodology, were the fruits harvested when the colour change of the fruit was achieved, or after a week of colouring? We think that with the inclusion of this phrase the harvest methodology was clarified: “Stage 1: Unripe, fruits with all green skin (fruits with 21 days after full bloom: dafb); Stage 2: Medium ripe, fruits with green and yellow skin (fruits with 35 dafb); Stage 3: Ripe, fruits with all yellow skin (fruits with 42 dafb); and Stage 4: Overripe, fruits with yellow and brown skin (fruits with 49 dafb)”.

Due to the lack of clarity in the harvesting methodology, fruits at different levels of harvest maturity could be presented as figures. A new Figure (Fig. 1) was included showing the fruits at the four development stages.

It is necessary to precisely specify the harvest date, it is suitable, for example, in the days after flowering, etc. Information about days after full bloom was added in relation to how many days passed between each harvest day: “Stage 1: Unripe, fruits with all green skin (fruits with 21 days after full bloom: dafb); Stage 2: Medium ripe, fruits with green and yellow skin (fruits with 35 dafb); Stage 3: Ripe, fruits with all yellow skin (fruits with 42 dafb); and Stage 4: Overripe, fruits with yellow and brown skin (fruits with 49 dafb)”.

Line 146 is an extra dot after were…The extra dot was deleted.

Line 183 AAAnalyst 200 – shouldn't it be AAnalyst 200? AAAnalyst 200 was changed by AAnalyst 200.

Results and Discussion

Line 198-212 – just as in the abstract, the percentages of the total acid content of medium ripe and ripe fruit are not given, somewhere it is stated somewhere not...to be added. As was mentioned in the Abstract section, percentage representations of acids were removed from all the text.

Overall, I consider the part devoted to the determination of acids and total TTA to be confusing. Here, units of mg/g and percentages are mixed one over the other without a continuous link. This paragraph was rewritten to clarify, as follow: “In H. edulis fruits, total titratable acidity kept high during unripe, medium ripe and ripe stages, and decreased in overripe stage (1.8, 1.6, 1.8 and 1.4%, respectively) [8], following a different behaviour with respect the total organic acids, fact that explained the non-significant correlation found between these both variables (r=-0.439, p=0.1537). The only acid that decreased in overripe stage compared with ripe stage was malic acid showing a positive and significant correlation with total acids (r=0.930, p<0.001), while quinic acid also presented a positive and significant correlation with total acids (r=0.855, p<0.001). In both cases, these acids explained the increase in total acid concentration throughout fruit stages.”

They recommend reworking so that it is clear which acids and in what percentage scale they are represented, ideally a graph. Percentage representation of acids were removed from all the text.

Line 192 - should be depending. The word “dependending” was corrected by “depending”.

Line 208 - should be fruits. The word “fruis” was corrected by “fruits”.

Line 235 - stated 2x the. It was corrected.

Line 242 – should be behaviour. It was corrected.

Line 295 – should be differentiate. It was corrected.

Line 319 – should be cellulolytic. It was corrected.

All tables: what is the meaning of the last empty line of the table stating Mean±SE? The last line of all the tables was removed. Also, “Values represent means ±S.E.” was changed by “Values represent means ± standard error” in the Table legends.

Conclusions

The conclusion needs to be strengthened more by scientific results. not what was measured but what was found and what it leads to... The conclusions were completely rewritten, including how the obtained results allow as to define the optimal time for consumption.

There are quite a few typos throughout the text and the English should be improved. The typos throughout the text were revised. The English was improved.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript has been greatly improved, although I suggest checking the English language and style.

Reviewer 3 Report

I believe that the corrections were made correctly and the overall quality of the submitted article was increased.

I agree with the corrections to the text of the article.