Effect of African Catfish Mucilage Concentration on Stability of Nanoemulsion Using D-Optimal Mixture Design

Round 1

Reviewer 1 Report

The authors report the optimization of a nanoemulsion obtained with African Catfish mucilage. Besides, this mucilage is suggested as a natural stabilizer for applications in the food and biomedical industries. Although the overall process of optimization sounds properly designed, my main concerns are:

(1)  The introduction focuses on general aspects of emulsions and nanoemulsions. However, the motivation of using African Catfish mucilage should be more elaborated to attract the interest of the readers.  
 
(2)  Information on the chemical composition of the mucilage studied is lacking. Experimental support is needed, or literature references.

(3)  If the authors suggest this mucilage for food applications, its potential sensorial effects must be discussed.

Author Response

Responses

Form

(1).    We appreciate the due diligence of the reviewer and we agree with the reviewer’s comments on the introduction. The introduction has been revised to provide sufficient background and includes all relevant references. Please refer to paragraph four of the introduction in the revised thesis. The changes have been outlined in the comments section of this file.

 

(2)    We appreciate the due diligence of the reviewer and accept the reviewers’ observation that the results can be improved by adding the results of sensory evaluations. Adding the results of sensory evaluations would have supported the claim that the African catfish mucilage could be applied in food-grade applications. The line item in the applications section on food application has been deleted and now reads ‘African catfish mucus stabilized oil in water nanoemulsions can be used as vehicles for cosmetics, drug delivery, and personal care product which are applicable in biomedical and personal care industries. Also, the line item on the food application has been deleted from the abstract. The sentence now reads ‘therefore ACM could be used as a natural stabilizer in oil-in-water nanoemulsions that are applicable in biomedical and personal care industries’. The decision to remove the claim was taken based on the advice of the reviewer as the sensory evaluation of African catfish mucilage and its stabilised emulsions have been delayed as a result of the COVID-19 pandemic.

(3)       We appreciate the due diligence of the reviewer and we agree with the reviewer’s comment that the conclusion can be improved. The phrase food-grade emulsion product has been deleted to reflect the line of thought in the conclusions. The last sentence in the conclusion now reads ‘hence, the microstructural stability attained in the formulated stable nanoemulsions could be applied to incorporate functional components into cosmetics, personal care, and drug delivery products for health benefits as the African catfish mucilage can be applied as a natural emulsifier.

Concerns

The authors report the optimization of a nanoemulsion obtained with African Catfish mucilage. Besides, this mucilage is suggested as a natural stabilizer for applications in the food and biomedical industries. Although the overall process of optimization sounds properly designed, my main concerns are:

(1)  The introduction focuses on general aspects of emulsions and nanoemulsions. However, the motivation of using African Catfish mucilage should be more elaborated to attract the interest of the readers.  

We appreciate the due diligence and agree with the comments of the reviewer on the introduction and motivation for the use of African Catfish mucilage has been added to the introduction to address the reviewer’s concerns. Please refer to paragraph four in the revised thesis. The paragraph inserted is captured below as:

‘The mucilage secreted from the fish skin when fish is stressed is a waste that contains elements such as carbohydrates, enzymes, lipids, metabolites, proteins, and water [24, 28, 29]. Several authors have established that vital enzymes and proteins have been identified in fish skin mucilage which is responsible for its natural defense [28-30]. These include antimicrobial peptides (AMP), glycoproteins, immunoglobulin, lectins, lysozyme, mucins, complement proteins, proteases, transferrins, and various other antibacterial proteins and peptides [28-30]. The skin of fish contains goblet and paneth cells which play a focal role in defending the fish by producing humoral immune factors and mucins [28-30]. The humoral immune factors include antimicrobial peptides, cytokines, immune-globulins, and lectins [29, 30]. Mucins present in mucilage are glycoproteins joined to considerable amounts of high molecular weight sugars that play a vital role in fish defense [29, 30]. Glycoproteins are structurally intricate chemical compounds that comprise proteins and carbohydrates [31, 32]. Authors have reported that oligosaccharide chains of glycoproteins are covalently attached to the polypeptide side-chains and proteins secreted from fish are glycosylated [31, 32]. Authors have reported that stabilized oil in water emulsions have been formulated with amphiphilic mucilage such as hagfish or saliva mucins which have the potentials to form cross-linked stable structures [33]. These emulsions were also shown to adhere to pig mucus in the buccal cavity, which confirms that amphiphilic mucilage can be used to formulate mucoadhesive oil-in-water (O/W) emulsions [33]. Hagfish mucilage has been used as an emulsifier because of its mucoadhesive properties the authors concluded that hagfish mucilage could emulsify soy milk emulsions [34]. Authors have proposed that the mucilage from fish, insects, and pigs could be used as vehicles for cosmetics, drug delivery, and personal care product which are applicable in biomedical and personal care industries for animals and humans [35-37]. Hence, African catfish mucilage (ACM) could be a potential emulsifier’.

 

(2)    Information on the chemical composition of the mucilage studied is lacking. Experimental support is needed, or literature references.

We appreciate the due diligence and agree with the comments of the reviewer. Information on the chemical composition of the mucilage studied of African Catfish mucilage has been added to the introduction as literature references. Please refer to paragraph four which is captured below:

‘The literature claims that the catfish mucilage contains about 2.3 % ash, 1 0 % fat 2 0 % fibre, 7.4 % protein, 10.9 % moisture 76.3% carbohydrates [38]. The mucin extract from fish has been reported to have exceptional antimicrobial, lubricating, and wound healing properties A wavelength peak of 1136 cm⁻¹ was recorded in the raw infra-red spectra of African catfish mucilage which could be attributed to the anti-symmetric glycoside bond [19]. Several authors have established that the presence of the glycosidic bond in glycoproteins is the reason a glycan can covalently attach to polypeptide side-chains and form stable structures that are suitable for biological processes [39, 40]. It can be inferred that the raw spectrum of ACM has glycosidic linkages similar to other glycoproteins which can encapsulate emulsions and confer stability [19]. A study has proposed that it could be applied in stabilizing emulsions that could be used as vehicles for nutritious neutraceutical, cosmetic and drug-delivery systems for animals and humans [19]’.

 

(3) If the authors suggest this mucilage for food applications, its potential sensorial effects must be discussed.

We appreciate the due diligence of the reviewer and accept that the claim that the African catfish mucilage could be applied in food-grade applications is ambiguous. The line item on the food application has been deleted from the abstract. The sentence now reads ‘therefore ACM could be used as a natural stabilizer in oil-in-water nanoemulsions that are applicable in biomedical and personal care industries’. The decision to remove the claim was taken based on the advice of the reviewer as the sensory evaluation of African catfish mucilage and its stabilised emulsions have been delayed as a result of the COVID-19 pandemic.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript can be described as a quite comprehensive and descriptive work organised in a convenient way for the reader. The main papers on the topic are mentioned and discussed. Still, this paper requires minor English language typing mistakes (e.g., the extra letter ‘s’ after the word ‘industries’ to be deleted (row number 12); ‘oil-in-water' must be written with hyphens (row number 15); space to be added between ‘1-‘ and ‘to’, as well as between ‘3-‘ and ‘to’ (row number 97) and after the hyphen in ‘1-' and 'to’; the extra letter ‘t’ in the word ‘constant’ to be deleted (row number 171); comma to be added before the word ‘respectively’ (rows number 155, 166, 189), as well as after the word ‘hence’ (rows number 195, 207, 235, 282, 297, 407, 431, 433)). Also, I would suggest specifying what exact protease inhibitor cocktail was used (rows number 85-86). In addition, what was the supplier of sunflower oil, and specify the manufacturer of the MilliQ water machine used, please (rows number 93-94). While in the 2.4 section, can you specify the temperature for the DLS analysis of your samples (as I assume it was 25 degrees)? This article should be published after minor English language corrections. In general, it is well organised and written!

Author Response

Responses

Form

We appreciate the due diligence and the comments of the reviewer on the manuscript submitted. The English language typing mistakes have been corrected and the changes are reflected in the revised manuscript.

Comments

(1)      This manuscript can be described as a quite comprehensive and descriptive work organised in a convenient way for the reader. The main papers on the topic are mentioned and discussed.

The comment of the reviewer is highly appreciated.

(2)     The minor English language typing mistakes (e.g., the extra letter ‘s’ after the word ‘industries’ to be deleted (row number 12) have been corrected.

We agree with the comment of the reviewer and this line items has been corrected. Please refer to the revised manuscript.

(3)      ‘Oil-in-water' must be written with hyphens (row number 15);

We agree with the comment of the reviewer and this line item has been corrected. Please refer to the revised manuscript.

(3)     Space to be added between ‘1-‘ and ‘to’, as well as between ‘3-‘ and ‘to’ (row number 97) and after the hyphen in ‘1-' and 'to’; the extra letter ‘t’ in the word ‘constant’ to be deleted (row number 171);

We agree with the comment of the reviewer and this line item has been corrected. Please refer to the revised manuscript.

(4)       Comma to be added before the word ‘respectively’ (rows number 155, 166, 189),

We agree with the comment of the reviewer and this line item has been corrected. Please refer to the revised manuscript.

(5)     ‘Hence’ (rows number 195, 207, 235, 282, 297, 407, 431, 433)). Also, I would suggest specifying what exact protease inhibitor cocktail was used (rows number 85-86).

We agree with the comment of the reviewer and this line item has been corrected. Please refer to the revised manuscript.

(6)      Specify what exact protease inhibitor cocktail was used (rows number 85-86). In addition, what was the supplier of sunflower oil, and specify the manufacturer of the MilliQ water machine used, please (rows number 93-94).

We agree with the comment of the reviewer and the information has been inserted in the revised manuscript. The exact protease Inhibitor Cocktail (Sigma Aldrich P8340) has been specified. The mixture contained water-soluble protease inhibitors with broad specificity for the inhibition of serine, cysteine, and metalloproteases which is recommended for general applications. Sunflower seed oil from Helianthus annuus was procured from Merck, and Merck is the manufacturer of the equipment (Milli-Q^® IQ Water Purification System) used to generate MilliQ water.

(7)       While in the 2.4 section, can you specify the temperature for the DLS analysis of your samples (as I assume it was 25 degrees)? This article should be published after minor English language corrections

We agree with the comment of the reviewer and the information has been inserted in the revised manuscript. The information has been inserted and captured as ‘the ACM stabilized O/W-type nanoemulsions’ particle size was analyzed in three replicates at 25°C.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript has been significantly improved. I recommend it for publication.