Encapsulation of Amyloglucosidase in Chitosan-SDS Coacervates as a Means to Control Starch Hydrolysis in Plant-Based Beverages
Round 1
Reviewer 1 Report
An overall good report and with the following changes to achieve publication.
The researchers did well to include investigations into the feedback inhibition, immobilisation yield and enzyme efficiency as part of the study.
Ultrasonic details needed. Exact time, temperature, intensity.
What volume of chitosan-amyloglucosidase solution was pipetted?
Did you observe any coagulation of the chitosan-amyloglucosidase solutionand how was this minimised?
".....solution colour from white to a cloudy transparent appearance" solution colour white is not well defined. Was the solution colour different or the cloudiness?
Line 241, add a space between numbers and units
Line 256, burst when subjected to pressure is not well defined. Was it a change in static pressure of the fluid that caused them to burst? or mechanical pressure, thermodynamic pressure? Or did you simply prod them?
The authors need to explain better why they specifically consider that maltose was more readily available in the encapsulated environment (line 535). There is more to it than simple diffusion, especially under the condition described in the methods.
The researchers did well to include investigations into the feedback inhibition, immobilisation yield and enzyme efficiency as part of the study.
Ultrasonic details needed. Exact time, temperature, intensity.
What volume of chitosan-amyloglucosidase solution was pipetted?
Did you observe any coagulation of the chitosan-amyloglucosidase solutionand how was this minimised?
".....solution colour from white to a cloudy transparent appearance" solution colour white is not well defined. Was the solution colour different or the cloudiness?
Line 241, add a space between numbers and units
Line 256, burst when subjected to pressure is not well defined. Was it a change in static pressure of the fluid that caused them to burst? or mechanical pressure, thermodynamic pressure? Or did you simply prod them?
The authors need to explain better why they specifically consider that maltose was more readily available in the encapsulated environment (line 535). There is more to it than simple diffusion, especially under the condition described in
the methods.
Author Response
The researchers did well to include investigations into the feedback inhibition, immobilisation yield and enzyme efficiency as part of the study.
We thank the reviewer for these kind observations
Ultrasonic details needed. Exact time, temperature, intensity.
Lines 145 to 147 now read: "The mixture was subjected to ultrasonication for 5 minutes at room temperature to eliminate air bubbles using an ultrasonic bath (Ultrawave U500H, Ultrawave Ltd., Cardiff, United Kingdom)."
What volume of chitosan-amyloglucosidase solution was pipetted?
The volume of that solution was 10ml, as specified line 143
Did you observe any coagulation of the chitosan-amyloglucosidase solutionand how was this minimised?
We did not observe any coagulation/turbidity in the chitosan-amyloglucosidase solution
".....solution colour from white to a cloudy transparent appearance" solution colour white is not well defined. Was the solution colour different or the cloudiness?
Starch solutions are known to change visually from white dispersions to slightly turbid solutions upon heating and solubilisation of the starch granules [38]. To remove ambiguity, we have deleted that sentence (lines 179-182 of original manuscript)
Line 241, add a space between numbers and units
We have added a space systematically between numbers and units
Line 256, burst when subjected to pressure is not well defined. Was it a change in static pressure of the fluid that caused them to burst? or mechanical pressure, thermodynamic pressure? Or did you simply prod them?
Thanks for this question. The capsules were physically burst by cutting them open with the aid of a scalpel in order to release the inner liquid core. This sentence (now line 254-256) now reads:" Upon further observation, the capsules were physically cut open with the aid of a scalpel and were found to be made of a liquid core surrounded by a gel-like membrane."
The authors need to explain better why they specifically consider that maltose was more readily available in the encapsulated environment (line 535). There is more to it than simple diffusion, especially under the condition described in the methods.
Thanks for this comment. We meant to say that Maltose, as the smaller substrate when compared to Starch, was diffusing more readily into the capsule and therefore was more readily hydrolysed. We have made this fact clearer lines 532-539
Reviewer 2 Report
The manuscript beverages-2592390, “Encapsulation of Amyloglucosidase in Chitosan-SDS coacervates as a means to control starch hydrolysis in plant-based beverages”, provides an interesting observation about immobilized amyloglucosidase using encapsulation technique. I think that experimental design and presentation quality of this manuscript is relatively fair. My comments are as follows:
Line 502-506:
‘Remarkably, encapsulation led to a decreased affinity between the enzyme and gelatinised corn starch, as evidenced by an increase in the KM value from 1030 mg/L to 1962 mg/L. Conversely, the KM value decreased from 4465 mg/L to 2980 g/L in the case of maltose, indicating a better interaction between maltose and amyloglucosidase after encapsulation.’
I think that this paragraph should be deleted. This decrease of Km values clearly occurred by shielding effect of chitosan-SDS membrane, not by changing the affinity of enzyme itself. This paragraph could induce readers’ misunderstanding. Authors’ discussion in line 513-555 is OK.
Line579-580:
‘Subsequently, no significant variations were observed in enzymatic activity or capsule weight.’
Indeed, there are no significant variations. However, residual acitivy was 76.2% after 28-days storage. Therefore, it was thought that a leak of amyloglucosidase from the capsule was continuing. I think that this result showed an instability of this immobilized amyloglucosidase. In addition, the leaking enzyme activity should be measured for confirming authors’ hypothesis. Have authors had such data?
Minor corrections are required.
Author Response
The manuscript beverages-2592390, “Encapsulation of Amyloglucosidase in Chitosan-SDS coacervates as a means to control starch hydrolysis in plant-based beverages”, provides an interesting observation about immobilized amyloglucosidase using encapsulation technique. I think that experimental design and presentation quality of this manuscript is relatively fair.
We thank the reviewer for these kind observations
My comments are as follows:
Line 502-506:
‘Remarkably, encapsulation led to a decreased affinity between the enzyme and gelatinised corn starch, as evidenced by an increase in the KM value from 1030 mg/L to 1962 mg/L. Conversely, the KM value decreased from 4465 mg/L to 2980 g/L in the case of maltose, indicating a better interaction between maltose and amyloglucosidase after encapsulation.’
I think that this paragraph should be deleted. This decrease of Km values clearly occurred by shielding effect of chitosan-SDS membrane, not by changing the affinity of enzyme itself. This paragraph could induce readers’ misunderstanding. Authors’ discussion in line 513-555 is OK.
Thanks for this comment. We have changed this paragraph to only account for changes in KM values, and deleted all mention of changes in enzyme affinity for the substrate in lines 500-504, lines 534-536, line 547 and lines 567-568.
Line579-580:
‘Subsequently, no significant variations were observed in enzymatic activity or capsule weight.’
Indeed, there are no significant variations. However, residual acitivy was 76.2% after 28-days storage. Therefore, it was thought that a leak of amyloglucosidase from the capsule was continuing. I think that this result showed an instability of this immobilized amyloglucosidase. In addition, the leaking enzyme activity should be measured for confirming authors’ hypothesis. Have authors had such data?
This is a very pertinent comment but we have unfortunately no direct observation of a leakage of the enzyme out of the capsule. We have never been able to measure any residual enzymatic activity outside of the capsule. Instead, our current thinking is that the enzyme progressively and very slowly tends to interact with the capsule membrane and is no longer able to hydrolyse substrates (either because of physical entrapment of the enzyme into the membrane coacervate gel, or because of a possible SDS-induced denaturation of the enzyme inside the capsule). But this is just supposition at this stage and we would need to study this further to come to a definite conclusion
Reviewer 3 Report
Journal: Beverages (ISSN 2306-5710)
Manuscript ID: beverages-2592390
Title: Encapsulation of Amyloglucosidase in Chitosan-SDS coacervates as a means to control starch hydrolysis in plant-based beverages
The study describes the immobilization of an amyloglucosidase using chitosan-SDS coacervates.
In addition to the immobilization efficiency, the authors evaluated its storage stability and studied kinetic parameters. Although there are some disadvantages, the authors point out the advantages of potential applications compared to free enzymes.
The efficiency of immobilization of amyloglucosidases using chitosan-SDS coacervates was highlighted as a new strategy for immobilization and conversion of corn starch or maltose into glucose. The authors present the results as successful, although the conversion rates and the total products achieved were still not comparable with the free enzymatic form.
The study is well designed, and the manuscript is generally well written. However, some points can be improved:
1. The study also proposed the use of SDS as an additional surfactant to overcome mechanical limitations and promote stable pH ranges. However, the use or the influence of SDS was not highlighted or evaluated enough in terms of how it affected encapsulation, pH stabilization, or enzyme-substrate interaction. This can be further discussed. At the same time, what is the novelty of using SDS based on? Please explain what is intended to be new compared to previous methods.
2. The immobilization efficiency was not fully achieved as when the enzyme is in its free state. It was found that encapsulation reduced the enzymatic reaction rate, much more for starch than for maltose. Did the authors evaluate how the exposure of the enzyme's catalytic site to the chitosan surface is available or well distributed along chitosan material? Did the size of the substrate interfere with its accessibility to the catalytic sites in immobilized amyloglucosidases?
3. On page 12, line 518, the authors comment that the interaction between chitosan and SDS probably formed a membrane, challenging the diffusion of corn starch and resulting in a visible lag phase in the activity curve. The authors could additionally discuss what happened after the lag phase which allowed the access of the substrate to the catalytic sites of the immobilized enzymes. Since an exponential phase was anyway achieved.
4. The authors discuss that enzymes can probably hydrolyze starch better than maltose. As both have glycosidic bonds to hydrolyze, how could this preference for starch be justified? The same was explained on page 10 line 477, that maltose presented less divisional difficulties in entering the pores of the capsule. However, it was even less hydrolyzed and converted into glucose.
Other small corrections:
Please correct or remove texts marked in red and already deleted texts throughout the manuscript.
Page 3 line 132 . Aspergillus niger in italic.
Page 3 line 134. Check paragraph.
Page 3 line 143. Space between value and unity 240mg. This should be corrected throughout the manuscript.
Page 3 line 144. Enzyme not capitalized.
Page 4 line 182. Please review the expression: “maltose substrate solution's solubilization”
Page 5 line 241. Please write Glucose not capitalized.
Page 6 line 282. Check long Paragraph (spacing after).
Page 12 line 489. V0 – 0 as subscript. The same with Vm and Km.
Author Response
We thank the reviewer for their questions and suggestions.
Detailed responses are included in the attached file and changes to the manuscript are in red to facilitate reviewing.
Author Response File: 
Author Response.docx
