The Efficacy of Plant Enzymes Bromelain and Papain as a Tool for Reducing Gluten Immunogenicity from Wheat Bran

Round 1

Reviewer 1 Report

Review of the work entitled: The efficacy of plant enzymes bromelain and papain as a tool for
reducing gluten immunogenicity from wheat bran. Manuscript ID: processes-1933872

 

The undertaken research topic is very important, however, since from 5% to more than 30% of persons in various social groups and age classes have allergies. Of this group, one-third suffer from food allergies. The phenomenon has an upward trend. In persons showing gluten intolerance, gliadins and glutenins cause IgE-mediated allergies and coeliac disease. Because wheat proteins play a significant role in the human diet, intensive research has been carried out to find the possibilities for eliminating or limiting alergenicity of it. In spite of much progress, this problem has not been solved yet. Therefore, the subject of research is justified. The manuscript is generally acceptable but needs improvement.

I have some general questions:

 

1.  Why did the Authors choose papain and bromelain for hydrolysis?  Despite many advantages, such as: low substrate specificity, general availability and showing therapeutic properties, they can be allergenic. For example bromelain has been shown to crossreact with the sera in about 28% of persons with IgE allergic response to honeybee venom.

3. Did the Authors consider the use of  proteolytic preparations like Protamex and Neutrase, which  are approved for use in food technology?

4. The proteolysis of proteins from gluten with the use bromelain and papain resulted in the reduction of allergenicity by almost half. In this context, have the Authors considered combined methods e.g. freezing, ultrasound associated freezing, or other physical treatment before proteolysis to improve hypoallergenic effect? Chemical modifications e.g. acetylation are also known. It may change molecular conformation of wheat proteins, and these methods in combination with proteolysis may be more effective than proteolysis only. Proteolysis may yield fragments less allergenic than the protein substrates. However, change in hydrolysis direction can also cause a separation of complete allergenic sequences and, in effect, an increase in hydrolysate allergenicity.

5. Such a deep progress in hydrolysis certainly affects the product's functional properties. Have the Authors thought to expand their research on this?

6. Deep hydrolysis leads to obtain short bitter peptides, which are unacceptable by consumers, did the Authors subject the hydrolysates to organoleptic analysis?

7. Do the Authors know of a different use for the bran hydrolyzate they obtained than for bread? During baking, high temperature causes undesirable reactions.  Heated polysaccharides can bind amino acids via the Maillard reaction, favouring increased allergenicity of the complex.

8. My most important question is related to the scope of the research. Why the Authors did not analyze the peptide sequence by LC-MS? Especially when performing RP-HPLC separations of the hydrolysates, it was possible to collect fractions. Knowing the sequence of peptides would greatly enhance the scientific quality.

 

 

Detailed comments:

 

Abstract:

Lines 24-25. The type of chromatography should be reported.

 Keywords:

In my opinion, instead “plant enzyme” should be immunogenicity

Introduction:

A brief description of the gluten proteins should be added (2-3 sentences).

Some parts of the text in the introduction general and require more details. For example: Lines 58-60: “Most immunogenic peptides are composed of at least nine amino acids and contain celiac toxic motifs of four or five amino acid sequences related to celiac toxicity and IgE-mediated allergies in food products”.  Please provide examples of “toxic peptides” such as: Gln-Gln-Gln-Pro-Pro- this is actually the shortest sequence necessary for IgE binding.

Materials and Methods:

Sentences 107-111 should be transferred from Materials and Methods to Results

The description of conditions of enzymatic hydrolysis should be rewritten.  The sentence Linies  (119-120): “To determine the optimal pH for protease activity, enzymatic hydrolysis was performed by suspending the enzymes in buffers of pH 4, 5, 6, 7, and 8” please change to: “To determine the optimal pH for protease activity, enzymatic hydrolysis was performed by suspending the enzymes in buffers of pH 4, 5, 6, 7, 8 and water”.  Next sentences:  “In parallel, enzymatic hydrolysis was performed with enzymes without the addition of any buffers to the substrates, in which case their initial pH was close to neutral, and changes during enzymatic hydrolysis were observed. “should be deleted.

How was the hydrolysis reaction stopped?

The sentence (Lines 133-134): “After each hour of hydrolysis, the samples were shaken with a vortex and the pH  was measured using a pH meter (PP–15, Sartorius AG, Germany).” Should be removed from the  Physico-chemical analysis of the composition of wheat bran. This is description of hydrolysates.

Please explain, why  protease activity for wheat bran substrates after 4 h  reaction was determined?

In my opinion, should be measured the progress of hydrolysis. The progress of hydrolysis can be measured  for example by analyzing the increase in TCA soluble peptides, the increase in free amino groups concentration , or electrophoretically.

Gluten content  instead  mg/kg should be expressed in  mg/g

Results:

The sentences (204-205) should be rewritten: “The data showed that the hydrolysis conditions of wheat bran had a significant effect on the soluble protein content of the substrates and decreased the gluten content”. It should be: “The data showed that the hydrolysis conditions of wheat bran had a significant effect on the soluble protein  and gluten content .” The words” substrate” and “hydrolyzate” should not be used interchangeably. After adding the enzyme and carrying out the reaction, we have a hydrolyzate (degradation products). Same example:  lines 208-209 “….. was observed between the control sample and the substrates with enzymes….”, instead “substrates with enzymes” should be hydrolysates.

Line 225: “3.1.2. Soluble protein content in wheat bran substrates” probably should be Soluble protein content in wheat bran hydrolysates” the same comment to methodology.

Next, instead “3.1.3. Decrease in gluten content in wheat bran substrates” should be:” Decrease in gluten content in wheat bran hydrolysates”.

Line 230: (see Figure 3) and Line 240 (see Table 1), please delete “see”.

Figure 6 presented HPLC chromatogram of wheat bran control sample extract after incubation of 4 h  at 45 °C, therefore it is not clear why "hydrolysed peptides" is shown in the figure. Please show the entire chromatogram of the selected hydrolysate with retention times between 0 and 60 minutes.

Figure 7 and Figure 8: Please at the little add description for “A” and “B”

How do we know what are the retention times for  types of gliadins? Whether any standard was analyzed?

Discussion:

Lines 374-384:  “level of hydrolysis “ is the same as degree of hydrolysis, this means the amount of hydrolyzed peptide bonds in the protein, so I think it would be safer to use gluten content.

Comments for author File: Comments.docx

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

First of all I would like to congratulate you on a very interesting topic. Although after reading the manuscript I have some comments, questions and suggestions.

1. Materials and Methods

Lines 116-117

…were added to the others at the enzyme-to-substrate ratios [E/S] 0.25, 0.5, 116 and 0.75% (7.5, 15, and 22.5 U/1 g of substrate),

the enzyme-to-substrate ratios [E/S] suggested to change the protein content according to the substrate, such as  (U/1 g of substrate proein),

Lines113

Add operations after enzymatic hydrolysis, such as inactivation, drying.

2. Results

Lines 296

 

Complementary qualitative and quantitative analysis of each peak area.

 

Best regards!

Author Response

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Author Response File: Author Response.pdf