Effect of Asparaginase Enzyme in the Reduction of Asparagine in Green Coffee

Round  1

Reviewer 1 Report

The occurrence of acrylamide in food is a risk factor of toxic and cancerous effects, therefore it is worth considering any method of reducing the concentration of this substance in food products. For this reason, the study presented in the article is interesting and can be published. However, some fragments require supplementation or re-editing.
Page 1, line 25: Chlorogenic acids belongs to the family of phenolic acids so “and” can not be used. I suggest  change it to “such as”.

Page 1, line 28: It is worth quoting the publication here by G. Budryn, E. Nebesny, J. Oracz:: Correlation between the stability of chlorogenic acids, antioxidant activity and acrylamide content in coffee beans roasted in different conditions International Journal of Food Properties, 2015, 2,  290-302   

Page 1, line 30: Ochratoxin is produced by mold  at low temperature. Roasting causes partial decomposition of ochratoxin and certainly does not contribute to the formation.
References:
The numbering of references in the list does not match the numbering in the text because several publications are shredded into several lines and each line starts with the next number.
Several publications are written using a capital letter at the beginning of each word in the title, and only the first word of the title should be capitalized.

Author Response

Response to reviewer 1:

Page 1, line 25: Chlorogenic acids belongs to the family of phenolic acids so “and” can not be used. I suggest  change it to “such as[AC1] ”.
Page 1, line 28: It is worth quoting the publication here by G. Budryn, E. Nebesny, J. Oracz:: Correlation between the stability of chlorogenic acids, antioxidant activity and acrylamide content in coffee beans roasted in different conditions International Journal of Food Properties, 2015, 2,  290-302[AC2]    
Page 1, line 30: Ochratoxin is produced by mold  at low temperature. Roasting causes partial decomposition of ochratoxin and certainly does not contribute to the formation[AC3] .
References:
The numbering of references in the list does not match the numbering in the text because several publications are shredded into several lines and each line starts with the next number[AC4] ‍.
Several publications are written using a capital letter at the beginning of each word in the title, and only the first word of the title should be capitalized[AC5] .  

[AC1] The text was adjusted with “such as”

[AC2]There is no way to put a reference in the abstract. However, the interesting reference suggested by the reviewer was added in the discussion section with the number 34.

Other treatments can be associated to the enzymatic ones can be investigated, as dry processing or moist air velocity, to minimize the degradation of polyphenols and the antioxidant activity and reduce relatively the formation of acrylamide in coffee beans [34], finding a balance for the quality and safety of the coffee beverage.

[AC3] It may not have been clear in the text, but what we wanted to say  that high roast temperatures can reduce  the ochratoxin A levels in coffee. It was excluded from the text.

[AC4]All references were adjusted according mdpi rules.

[AC5]It was ajusted.

Reviewer 2 Report

The paper describes an interesting topic, the reduction of asparagine in coffee, which intends to reduce the acrylamide formation during roasting.

There are some deficits of the paper requiring major revisions:

-          Acrylamide is not analysed, so the influence on its content remains questionable. Perhaps the remaining asparagine is still sufficient to form acrylamide in similar contents? It must be considered that acrylamide is found only in contents around 200 µg/kg, so that only a minor amount of the asparagine present needs to react with sugars.

-          No method validation has been conducted

-          The statistical treatment of data is insufficient (see below)

-          The method description is inadequate and does not allow replication of results

-          English and Grammar need to be corrected by a native speaker

Some detailed comments

Line 3: aspagine?

Line 19 and throughout: should conilon read “robusta”, which is internationally preferred (are use the botanically correct canephora)

Line 26: the compounds are only partially and not completely destroyed

Line 29: cite the following study for claim of “other carcinogenic substances”: https://www.mdpi.com/2305-6304/7/1/1/htm

Line 30: lake?

Line 30: I never heard that ochratoxin is formed during roasting. Please check and revise.

Line 30: delete “complete”. The degradation is NOT complete.

Line 31: “Loss of characteristics” should read “change of characteristics”. Some people prefer black roasted coffee, e.g. Neapolitan espresso.

Line 33: delete “and neurotoxic”. The IARC only deals with carcinogen evaluation.

Line 34: what are “training routes”?

Line 28, 31 & 44: full stopa missing

Line 70: do you mean berries (cherries) or beans?

Section 2.3.: be much more specific. Specify all concentrations of all solutions.

Line 78: dilution is unclear. Was this diluted in deionized water or something else? Also specify the exact volumina.

Line 78: how can the pH be ensured with deionized water? Did you use a buffer?

Line 80: explain ASNU

Line 81: 60% is unclear (how much enzyme solution at which concentration was added to what amount of coffee)?

Line 83: how were the concentrations prepared from the 3500 solution?

Line 84: specify details of grinding, which mill?

Section 2.5.: unclear? Is this for preparation for HPLC analysis or something else. The other amino acids are not mentioned anymore in the text. This is very confusing.

Section 2.6.: please add validation/method performance data

Line 101: which gradient, specify

Lines 102-103 and 105-106: these sentences report exactly the same details

All figures: resolution is insufficient

Lines 114&116: please provide underlying statistics for “best” result. Considering the SD all treatment times look almost similar. There appears to be no signficiant difference between 15, 45 and 60 minutes?

Line 164: this might be true for other products but has not been proven for coffee

Line 168: I really doubt the “different optimal times”. See comment above.

Conclusion section: cost should be discussed. Is this treatment economically viable at all?

Author Response

Reviewer 2
The paper describes an interesting topic, the reduction of asparagine in coffee, which intends to reduce the acrylamide formation during roasting.
There are some deficits of the paper requiring major revisions:
-          Acrylamide is not analysed, so the influence on its content remains questionable. Perhaps the remaining asparagine is still sufficient to form acrylamide in similar contents? It must be considered that acrylamide is found only in contents around 200 µg/kg, so that only a minor amount of the asparagine present needs to react with sugars[AC1] .

-          No method validation has been conducted
-          The statistical treatment of data is insufficient (see below)
-          The method description is inadequate and does not allow replication of results
-          English and Grammar need to be corrected by a native speaker
Some detailed comments
Line 3: aspagine[AC2] ?
Line 19 and throughout: should conilon read “robusta”, which is internationally preferred (are use the botanically correct canephora[AC3] )
Line 26: the compounds are only partially and not completely destroyed[AC4] 
Line 29: cite the following study for claim of “other carcinogenic substances”: https://www.mdpi.com/2305-6304/7/1/1/htm[AC5] 
Line[AC6]  30: lake?
Line 30: I never heard that ochratoxin is formed during roasting. Please check and revise.[AC7] 
Line 30: delete “complete”. The degradation is NOT complete[AC8] .
Line[AC9]  31: “Loss of characteristics” should read “change of characteristics”. Some people prefer black roasted coffee, e.g. Neapolitan espresso.
Line[AC10]  33: delete “and neurotoxic”. The IARC only deals with carcinogen evaluation.
Line[AC11]  34: what are “training routes”?
Line 28, 31 & 44: full stopa missing[AC12] 
Line 70: do you mean berries (cherries) or beans[AC13] ?
Section[AC14]  2.3.: be much more specific. Specify all concentrations of all solutions.
Line[AC15]  78: dilution is unclear. Was this diluted in deionized water or something else? Also specify the exact volumina.
Line 78: how can the pH be ensured with deionized water? Did you use a buffer[AC16] ?
Line 80: explain ASNU[AC17] 
Line 83: how were the concentrations prepared from the 3500 solution[AC18] ?
Line 84: specify details of grinding, which mill?
Section 2.5.: unclear? Is this for preparation for HPLC analysis or something else. The other amino acids are not mentioned anymore in the text. This is very confusing.
Section 2.6.: please add validation/method performance data
Line 101: which gradient, specify
Lines 102-103 and 105-106: these sentences report exactly the same details
All figures: resolution is insufficient[AC19] 
Lines 114&116: please provide underlying statistics for “best” result. Considering the SD all treatment times look almost similar. There appears to be no signficiant difference between 15, 45 and 60 minutes?
Line 164: this might be true for other products but has not been proven for coffee
Conclusion section: cost should be discussed. Is this treatment economically viable at all[AC20] ?

RESPONSES:

 [AC1]The probable reduction of acrylamide would not occur in 100%, as there would be an amount of free asparagine that would react with the reducing sugars during the maillard reaction, which does not stop.

 [AC2] Adjusted

 [AC3] It was modified in all text for  C. arabica and C. canephora

 [AC4]ok

 [AC5] The reference was added and  numbered [7].

 [AC6]It was excluded  as well the ochratoxin paragraph

 [AC7] It was excluded

 [AC8]ok

  [AC9]ok

 [AC10]ok

 [AC11]changed for only routes

 [AC12]ok

 [AC13]ok

 [AC14]ok, It was adjusted and detailed according methodology used at Embrapa Lab. 

 [AC15] It was described that it was used deionized water as well its  volume.

 [AC16] It was used a buffer. Included in the text.

 [AC17]units of enzyme measurement

 [AC18]Todas essas marcações em verde estão descritas no texto e são apresentadas na metodologia, não entendi os questionamentos.

 [AC19] The image resolution was improved in attached files containing such images

 [AC20] It was added in the final text.

Round  2

Reviewer 2 Report

Many of my points were not sufficiently answered. At least, a section regarding the limitations of the research should be added:

1. Acrylamide is not analysed, so everything about mitigation is speculation.
2. No method validation has been conducted.

Otherwise, several of my points above lack a proper response/rebuttal.

Author Response

·         A paragraph with 5 lines highlighted in text were added in materials and methods section. It was also included a new Table (Table 1), justifying the question point 2 of the reviewer "2. No method validation has been conducted".

Some parameters were provided by the company concerning the reaction conditions of the enzyme Acrylaway™ as ideal temperature, pH, among others (data not published). The Table 1 shows the HPLC parameters used for extraction, amino acid profile and asparagine analysis, following methodology previously used at Embrapa Food technology Labs.

 Table 1 – Parameters for enzyme reactions conditions