Effect of Drum-Drying Conditions on the Content of Bioactive Compounds of Broccoli Pulp

Round 1

Reviewer 1 Report

The strength of the paper is that there are no studies in literature on the effect of broccoli drum-drying on glucoraphanin and sulforaphane. The weakness of the paper are that (1) the measurement of the total phenolic compounds (Folin) is not exactly the most updated, and it does not give much information of the changing in the phenolic compound profile during drying, (2) the antioxidant activate measurement does not add anything to the results (it can be removed), (3) I think that the used ascorbic acid analytical method does not allow to distinguish ascorbic acid and the dehydroascorbic acid, so we cannot check the oxidation of the ascorbic acid (right it is not clear), (4) the discussion is lacking in some parts or it should go deeper (see specific comments later), a table/figure should be added: the authors should show the effect of each run on the compounds measured: initial content in each run the corresponding final content.

Specific comments.

60-74. “ No studies about drum drying of broccoli are available so far.” The authors should add what they expected to be different from all the other drying. The lack of studies does not necessarily mean that it is interesting to investigate. E.g., why the rotation frequency of the drums, drum surface temperature, and water content of the broccoli pulp should affect the glucosinolate and the break down products?

Table 1, the results should be shown in another table or figure in the R&D. M&M do should not show results.

85-86.”... in order to maximize the sulforaphane content, as suggested in literature [18]. This sentence should not be here, it should be only in the R&D .

Line10. Check this “80ºC”

Line19. Be consistent in the way you report the T : 30 °C, here the is a space between the number and the unit.

Paragraph 3.1. Final moisture content. Discussion with literature is missing.

Caption Figure 3. The abbreviation on the y axis should be reported.

There is no way the reader can see the effect of each run on the compounds measured. The authors should show those results toom: initial content in each run the corresponding final content.

Table 2. the authors added water in the pulp, to improve the adherence of the pulp. And then they mentioned that addition of compounds such as maltodextrin should also be a good option and that it should be investigated. The different moisture content/water content is a very important element of the thermal degradation of glucosinolate (see Effect of water content and temperature on inactivation kinetics of myrosinase in broccoli (Brassica oleracea var. italica, Oliviero et al. 2014; Impact of different drying trajectories on degradation of nutritional compounds in broccoli (Brassica oleracea var. italica), Jin et al. 2014) . the addition of water or of other compounds changes the water moisture and water activity and this can lead to a different thermal stability of glucosinolate (and ascorbic acid). This part should be better discussed.

Table 3. the SFN result is the average of all the runs (both initial and final)? It should be specified.

123-136 (pag.5). the authors should discuss the effect of the pre-treatment that they applied. Do the other studies shown in table 3 applied the same? Did the blanching affected the SFN retention? In a studies where gluoraphanin and sfr are analysed , it is crucial to discuss this part. The authors talk a bit about it in page 7 (line6-11), but they should also describe how it was done in the studies cited in table 3. Moreover, why SFR which is a volatile compound, did not decrease during drum drying? By inactivation of the myrosinase upon pre-treatment, we may assume no further formation of SFR, I was expecting a significant drop at the end of drying, especially when drum-drying a broken meshed structure such as a pulp.

3.4. Ascorbic acid content. The comparison with literature is missing.

Author Response

Dear Sir/Madam,

the authors thank your time for revising the manuscript and acknowledge the suggestions.

Reviewer 1

The strength of the paper is that there are no studies in literature on the effect of broccoli drum-drying on glucoraphanin and sulforaphane.

The weakness of the paper are that (1) the measurement of the total phenolic compounds (Folin) is not exactly the most updated, and it does not give much information of the changing in the phenolic compound profile during drying,

R: I apologize but I have to disagree with the reviewer. The Folin-Ciocalteu method is up-to-date, this is a classical method currently used by most authors. As an example I can mention the article from Gasinski et al. (2020) “Assessment of Volatiles and Polyphenol Content, Physicochemical Parameters and Antioxidant Activity in Beers with Dotted Hawthorn (Crataegus punctata)”, published on June 2020 in Foods (DOI: 10.3390/foods9060775).

(2) the antioxidant activate measurement does not add anything to the results (it can be removed),

R: Unfortunately I disagree with reviewer; antioxidant activity must be assessed by at least three methods in order to detect any effect on it. In our work we used FRSA, FRAP, ascorbic acid and total phenolic compounds. The comparison of the tendencies of the measurements allows the validation of the effects on antioxidants. Besides, it is expected that the tendency shown by FRSA agrees with that of TPP, and that FRAP agrees with ascorbic acid, considering the type of chemical species detected by each method. This is discussed in the manuscript (section 3.5).

 (3) I think that the used ascorbic acid analytical method does not allow to distinguish ascorbic acid and the dehydroascorbic acid, so we cannot check the oxidation of the ascorbic acid (right it is not clear),

R: We agree, the method we used detects total ascorbic acid and does not distinguish between two oxidation states. However, Ullah et al. (2012; DOI: 10.5829/idosi.mejsr.2012.12.8.6675), who reported this method, also informed that: “This method was also compared with oxidation-reduction methods using standardized 2,6-Dichloroindophenol (DCP), to quantifying verifying ascorbic acid levels. We observed that the amounts of ascorbic acid reported by the two methods were mostly same and identical”. Therefore, we believe that the data as obtained in our work is representative of the actual content of ascorbic acid. 

(4) the discussion is lacking in some parts or it should go deeper (see specific comments later),

R: responses are given below.

a table/figure should be added: the authors should show the effect of each run on the compounds measured: initial content in each run the corresponding final content.

R: these values are given in Tables. Initial content (pulp before drying) properties are given in Table 2; final contents are given in Table 3.

Specific comments.

60-74. “ No studies about drum drying of broccoli are available so far.” The authors should add what they expected to be different from all the other drying. The lack of studies does not necessarily mean that it is interesting to investigate. E.g., why the rotation frequency of the drums, drum surface temperature, and water content of the broccoli pulp should affect the glucosinolate and the break down products?

R: a new reference was included (Quintero et al., 2020) and in the text the following sentences were added:

“The use of this technology would probably result in a lower cost process than the technologies explored until now, such as tray-, fluidized bed- and freeze- drying.” (Page 2, line 60-61)

“The effect of surface drum temperature and rotation frequency must be evaluated since some bioactive compounds of broccoli such as sulforaphane and ascorbic acid are highly thermo labile. Besides, increasing water content to the pulp could probably affect the content of hydrophilic compounds such as glucosinolates.” (page 2, line 75-79)

Table 1, the results should be shown in another table or figure in the R&D. M&M do should not show results.

R: Table 1 was separated in two tables, the first set in materials and methods, and the second in results and discussion. Also the position of the tables was changed.

85-86.”... in order to maximize the sulforaphane content, as suggested in literature [18]. This sentence should not be here, it should be only in the R&D .

R: I disagree with this observation since the sentence does not refer to a result or its interpretation but to the reason that supports the use of this pretreatment. Explanation was included in the results and discussion section.

Line10. Check this “80ºC”

R: this was corrected.

Line19. Be consistent in the way you report the T : 30 °C, here the is a space between the number and the unit.

R: this was corrected.

Paragraph 3.1. Final moisture content. Discussion with literature is missing.

R: discussion was included (page 8 line 13-16).

Caption Figure 3. The abbreviation on the y axis should be reported.

R: This was included in the caption.

There is no way the reader can see the effect of each run on the compounds measured. The authors should show those results toom: initial content in each run the corresponding final content.

R: the initial values are given in Table 2.

Table 2. the authors added water in the pulp, to improve the adherence of the pulp. And then they mentioned that addition of compounds such as maltodextrin should also be a good option and that it should be investigated. The different moisture content/water content is a very important element of the thermal degradation of glucosinolate (see Effect of water content and temperature on inactivation kinetics of myrosinase in broccoli (Brassica oleracea var. italica, Oliviero et al. 2014; Impact of different drying trajectories on degradation of nutritional compounds in broccoli (Brassica oleracea var. italica), Jin et al. 2014) . the addition of water or of other compounds changes the water moisture and water activity and this can lead to a different thermal stability of glucosinolate (and ascorbic acid). This part should be better discussed.

R: we agree with reviewer regarding the effect of moisture content on enzyme kinetics. We revised the suggested literature and the food matrixes are different. Oliviero et al (2014) used re-hydrated broccoli at different moisture contents (10 to 90%) and measured myrosinase inactivation at temperatures between 40 and 70°C, and found a correlation between moisture content and inactivation kinetic constant suggesting that low moisture content implies a more stable enzyme. The authors did not measure thermal degradation of glucosinolates but their enzymatic degradation, i.e. myrosinase inactivation. In our work the food matrix was more like a solution than a porous solid (as in the case of Oliviero et al), and therefore diffusional impediment is lower and results are difficult to compare. Besides, an increase from 91% (which is the moisture content of fresh and blanched broccoli) to 93.3% or 93.8% moisture content in the broccoli pulps (as shown in Table 2) should not result in significant difference in myrosinase activity or stability attributable to moisture content. Prove of that is the not significant effect of W:P ratio on glucoraphanin content. Besides, Olivero et al. (2014) concludes that broccoli with 67% and 90% moisture content showed similar initial inactivation rate constants, therefore it would be expected that at 93% moisture inactivation would remain the same. This observation can be extended to ascorbic acid, its stability and solubility within this range of moisture content most likely remain the same. 

Table 3. the SFN result is the average of all the runs (both initial and final)? It should be specified.

R: Table 4 (former table 3) was modified according to the suggestion. SFN values are the content in the material before drying (initial) and after drying (final) in a specific drying experience. These values were taken from literature; in those articles one can find the exact drying conditions in each case, based on the final moisture content. For this reason the final moisture content in each case is reported.

• Mahn, A.; Martin, C.; Reyes, A.; Saavedra, A. Evolution of sulforaphane content in sulforaphane-rich broccoli during tray drying. J Food Eng 2016, 186, 27 – 33.
• Mahn, A.; Román, J.; Reyes, A. Efecto de la liofilización de brócoli pre-procesado sobre la cinética de secado y el de sulforafano. Información Tecnológica 2016, 27, 95 – 106.
• Mahn, A.; Pérez, C.; Reyes, A. Effect of drying conditions in a pulsed fluidized bed dryer on the sulforaphane content of broccoli. Int J Food Biosyst Eng 2017, 5, 39-44.

123-136 (pag.5). the authors should discuss the effect of the pre-treatment that they applied. Do the other studies shown in table 3 applied the same?

R: in the cited literature authors used the same blanching conditions. This was indicated in caption to table 4.

Did the blanching affected the SFN retention? In a studies where gluoraphanin and sfr are analysed , it is crucial to discuss this part. The authors talk a bit about it in page 7 (line6-11), but they should also describe how it was done in the studies cited in table 3.

R: We think that this aspect was not clear in the text. The blanching conditions that we use since 2014 are the optimum, in order to maximize SFN content. For this reason in this work we used the same blanching pretreatment: in order to compare and to be sure that SFN content was maximized in the raw material before drying. This was indicated in caption of Table 4 and also in section 3.2 (9 line 36-39 and page 10 line 52). More details can be found in our previous works: Pérez et al. (2014; DOI http://0-dx-doi-org.brum.beds.ac.uk/10.1016/j.foodchem.2013.08.053); Mahn et al. (2016; doi:      10.1016/j.jfoodeng.2016.04.007); Mahn and Pérez (2016; DOI: 10.1007/s13197-016-2386-6); Mahn et al. (2018; DOI: 10.1007/s13197-018-3395-4), among others.

Moreover, why SFR which is a volatile compound, did not decrease during drum drying?

R: sulforaphane is not a volatile compound; it is thermo labile and one of its decomposition products (a thiourea derivative) is highly volatile (Mahn et al. 2016; doi:10.1016/j.jfoodeng.2016.04.007). Thermal degradation of SFN occurs at 40°C or higher, then we also expected to observe a drastic reduction in its content, however results indicate something different. This may be related to the short time of exposition to temperature. This was discussed in the text (section 3.2., page 9 line 46-50).

By inactivation of the myrosinase upon pre-treatment, we may assume no further formation of SFR, I was expecting a significant drop at the end of drying, especially when drum-drying a broken meshed structure such as a pulp.

R: Pre-treatment preserves myrosinase activity; even at 80°C myrosinase remains active, and we used 60°C in blanching. Other enzymes (epithiospeficier and nitrile-specifier proteins) are inactivated at 50°C thus avoiding the formation of hydrolysis products different form SFN. The pretreatment was designed to maximize SFN formation, resulting in SFN-rich broccoli. This was clarified in the text (section 3.2)

3.4. Ascorbic acid content. The comparison with literature is missing.

R: the results were compared with literature (page 12, line 31-33). References were included.

Reviewer 2 Report

Effect of Drum Drying Conditions on the Content of Bioactive Compounds of Broccoli Pulp

The research work studies the stabilization of broccoli to improve their shell life and to keep their healthy properties to the maximum during their conservation. In particular they study the potentiality of drum drying and the effect of the rotation frequency, drum surface temperature, and water content on the final characteristic of dried broccoli.  

The topic is within the scope of the Journal, the manuscript is interesting and well-written, the study is well-analyzed.

Minor revision is required.

the abstract

line 20 please substitute “…dependent of…” with “….dependent on….”

The introduction.

Line 62 Please add some references to confirm the assertions

The materials and method

Line 87 during the treatment in Thermomix at 4500 RPM the temperature was controlled? the samples were put in ice? Please clarify.

Line 99 “in each experimental run…” how many run experimental runs? Please specify.

In the paragraph of Sulforaphane content (materials and method) “Quantification was made by comparison…” please add “Quantification was made by the method of external standard comparing the results with a sulforaphane…”

In the paragraph of Glucoraspnanin content (materials and method)

The same write above, please add “Quantification was made by the method of external standard comparing the results with a sinigrin…”

In the paragraph of Ascorbic acid content (materials and method)

Please add “Ascorbic acid content was assessed by HPLC according to the method reported in literature”

Results and discussion

Please replace “…(free radical scavenging ability and ferric reducing ability)…with the abbreviations (FRSA and FRAP)

Please invert the numbering of the table 1 and 2. In particular table 1 should be the one with the content of bioactive compounds of the pulps before drying and table 2 should be the one with Experimental design and responses. Because the content of table 2 is important to understand data in table 1. So along the test write “Table 2  shows results obtained from each experimental condition. The significant differences between the final product and the humid broccoli pulps are indicated with asterisks. The properties of the broccoli pulps are given in Table 1.”

Tables

Please invert the numbering of tables 1 and 2, as written above.

Table 1: in the legend of actual table 1 please add the definition of GAE (GAE = gallic acid equivalents)

Table 2: in the legend of the actual table 2 please remove the acronym MC = moisture content that doesn’t appear in the table and add the  acronyms of GAE, TE and W:P ratio (GAE = gallic acid equivalents; TE = Trolox equivalents and W: P = water to pulp ratio). Please replace “wb = humid base with wb = wet basis”

Table 3: in the legend of table 3 please replace “dw means dry weight and wb means wet basis” with  “dw = dry weight; wb = wet basis”, please add the acronym “SFN = sulforaphane” TPC = total polyphenols” , “SFN = sulforaphane” and “GAE = gallic acid equivalents”

Figures

Figure 1: Please remove the comma in the legend of figure 1. “Double-drum dryer (a) operation scheme and (b) operation images” instead of  “Double-drum dryer (a) operation scheme, and (b) operation images”

Figure 2: please write “Images of the dehydrated product obtained in different conditions, according to Table 2. (a) run 1, (b) run 2, (c) run 3, (d) run 4, (e) run 5, and (f) run 6.” instead of “Images of the dehydrated product obtained in different conditions, according to Table 1. (a) Run 1, (b) run 2, (c) run 3, (d) run 4, (e) run 5, and (f) run 6.”

Figure 3: please write “Standardized statistical effects of the experimental factors on the responses (Pareto charts). (a) Moisture content, (b) sulforaphane, (c) glucoraphanin, (d) phenolic compounds, (e) ascorbic acid, (f) FRSA = free radical scavenging ability, and (g) FRAP = ferric reducing ability.

Please specify in the legend of this figure the meaning of each acronym reported in the figure. It is important for the clarity and complete understanding of the figure itself.

Author Response

Dear Sir/Madam,

the authors thank your time for revising the manuscript and acknowledge the suggestions.

Reviewer 2

The research work studies the stabilization of broccoli to improve their shell life and to keep their healthy properties to the maximum during their conservation. In particular they study the potentiality of drum drying and the effect of the rotation frequency, drum surface temperature, and water content on the final characteristic of dried broccoli.  

The topic is within the scope of the Journal, the manuscript is interesting and well-written, the study is well-analyzed.

Minor revision is required.

the abstract

line 20 please substitute “…dependent of…” with “….dependent on….”

R: This was corrected. (page 1 Line 20)

The introduction.

Line 62 Please add some references to confirm the assertions

R: Assertions in lines 61 – 65 were based in a classic book that is referenced in line 65. More references were added in lines 70 – 75.

The materials and method

Line 87 during the treatment in Thermomix at 4500 RPM the temperature was controlled? the samples were put in ice? Please clarify.

R: Temperature was controlled at 40°C, after 60 s the pulp was mixed with water. This is mentioned in the text (page 2 line 92).

Line 99 “in each experimental run…” how many run experimental runs? Please specify.

R: there were 12 experimental runs. Redaction was modified for clarity (line 96 and 103-104). A mistake was detected in Materials and methods section 2.1; the correct amount of pulp is 15 kg.

In the paragraph of Sulforaphane content (materials and method) “Quantification was made by comparison…” please add “Quantification was made by the method of external standard comparing the results with a sulforaphane…”

R: this sentence was included (Line 145-147)

In the paragraph of Glucoraspnanin content (materials and method)

The same write above, please add “Quantification was made by the method of external standard comparing the results with a sinigrin…”

R: this sentence was included (Line 160-162)

In the paragraph of Ascorbic acid content (materials and method)

Please add “Ascorbic acid content was assessed by HPLC according to the method reported in literature”

R: This was corrected (line 173)

Results and discussion

Please replace “…(free radical scavenging ability and ferric reducing ability)…with the abbreviations (FRSA and FRAP)

R: This was replaced.

Please invert the numbering of the table 1 and 2. In particular table 1 should be the one with the content of bioactive compounds of the pulps before drying and table 2 should be the one with Experimental design and responses. Because the content of table 2 is important to understand data in table 1. So along the test write “Table 2  shows results obtained from each experimental condition. The significant differences between the final product and the humid broccoli pulps are indicated with asterisks. The properties of the broccoli pulps are given in Table 1.”

R: table 1 was divided into 2 tables, (as suggested by reviewer 1), the first was located on materials and methods and the second was moved to results and discussion. The properties of broccoli pulp were given in a table between the both described above. Then:

Table 1: experimental design

Table 2: properties of broccoli pulps

Table 3: responses of the experimental runs.

Tables

Please invert the numbering of tables 1 and 2, as written above.

R: please see previous response.

Table 1: in the legend of actual table 1 please add the definition of GAE (GAE = gallic acid equivalents)

R: it was added.

Table 2: in the legend of the actual table 2 please remove the acronym MC = moisture content that doesn’t appear in the table and add the  acronyms of GAE, TE and W:P ratio (GAE = gallic acid equivalents; TE = Trolox equivalents and W: P = water to pulp ratio). Please replace “wb = humid base with wb = wet basis”

R: corrections were included.

Table 3: in the legend of table 3 please replace “dw means dry weight and wb means wet basis” with  “dw = dry weight; wb = wet basis”, please add the acronym “SFN = sulforaphane” TPC = total polyphenols” , “SFN = sulforaphane” and “GAE = gallic acid equivalents”

R: this was corrected.

Figures

Figure 1: Please remove the comma in the legend of figure 1. “Double-drum dryer (a) operation scheme and (b) operation images” instead of  “Double-drum dryer (a) operation scheme, and (b) operation images”

R: this was removed.

Figure 2: please write “Images of the dehydrated product obtained in different conditions, according to Table 2. (a) run 1, (b) run 2, (c) run 3, (d) run 4, (e) run 5, and (f) run 6.” instead of “Images of the dehydrated product obtained in different conditions, according to Table 1. (a) Run 1, (b) run 2, (c) run 3, (d) run 4, (e) run 5, and (f) run 6.”

R: this was revised according to the modifications of the tables described above.

Figure 3: please write “Standardized statistical effects of the experimental factors on the responses (Pareto charts). (a) Moisture content, (b) sulforaphane, (c) glucoraphanin, (d) phenolic compounds, (e) ascorbic acid, (f) FRSA = free radical scavenging ability, and (g) FRAP = ferric reducing ability.

R: this was corrected.

Please specify in the legend of this figure the meaning of each acronym reported in the figure. It is important for the clarity and complete understanding of the figure itself.

R: this was included.

Round 2

Reviewer 1 Report

no comments