Improved Extraction of High Value-Added Polyphenols from Pomegranate Peel by Solid-State Fermentation

Round 1

Reviewer 1 Report

The manuscript deals with the extraction of high value-added polyphenols from pomegranate peels by solid-state fermentation. With the purpose of assessing the effect of the main variables of the process, the authors propose two experimental designs, a Box Hunter & Hunter followed by a Central Composite Design.

The topic is suitable to be published in Fermentation. The experimental section and the results are very interesting, but they discussion section should be improved. In my opinion, it could be published in Fermentation after some revisions:

Comments:

1.     The state of the art of the manuscript topic is not clear. Please, improve it.

2.     It is not clear the contribution of this study in the field. Please, add it in the introduction section.

3.     Could the dehydration step influence in the recovery of phenolics? Was this stage previously optimized? Is this stage necessary?

4.     Please, provide additional information about the experimental conditions of the kinetic showed in Figure 1

5.     The KCl has not been in included as variable in the CCD, why?

6.     Please, improve the results section, comparing the results (TPC and AA) obtained with the proposed method with that reported in literature for the recovery of phenolics from pomegranate (traditional and emerging technologies).

7.     Please, include the multiple regression of the AA

Author Response

Response to Reviewer 1

Dear reviewer, many thanks for your valuable comments, we did our best to attend your suggestions 

1. 1. The state of the art of the manuscript topic is not clear. Please, improve it.

Response: Amends to the introduction were included.

It was:

Pomegranate intake is mainly in juice, jellies, jams and liquors; however, their biological activity is highly appreciated in the food, cosmetic and pharmacy industries [Ascacio-Valdés, et al. 2011].  According to Kazemi et al. [2016] one ton of fresh pomegranate generates 669 kg of by-products and those include 78 % peel and 22 % seeds.  Although, the fast increase of pomegranate cultivation limits the precise calculation of the worldwide production, in 2021 based on the 8,636.2 tons of pomegranate harvested in 2021 for Mexico, 6,736.2 tons of pomegranate peels are available for the highly valued biomolecules extraction [https://nube.siap.gob.mx/cierreagricola/]. Organic solvents are commonly used for the extraction of vegetal biomolecules; however, their use is associated with environmental pollution and with toxicological safety concerns. Implementation of emerging technologies [i.e. supercritical fluid, microwave, electric field, pressurized liquid] for biomolecules extraction improves extraction yields; however, the requirement of polar solvents and expensive extractive equipment strongly limits its applications [Makris et al. 2007; Martins et al. 2011; Cano-Lamadrid et al. 2022].

In contrast, fermentative or enzymatic methods for the extraction of biomolecules from agricultural by-products have been successfully used [De la Torre et al. 2019; Buenrostro-Figueroa et al. 2017; Robledo et al. 2008]. Optimization methods based on one-factor-at-a-time are expensive, time-consuming and interactions between processes variables are not considered [Saffarzadeh-Matin and Khosrowshahi, 2017].  Experimental designs for the study of several variables at a time, as well as their interactions are highly desirable.

Polyphenols are molecules that present different biological and therapeutic activities; several studies have reported their health effects. Bioactive agents such as antioxidant, anti-proliferative/anticarcinogenic, antifungal, antimicrobial, fat-lowering, anti-Inflammatory, anti-aging neuroprotective, antibacterial, hypocholesterolemic, renoprotective, hepatoprotective, hypoglycaemic activity, antihyperglycemic, antihypertensive activities, among others, have been attributed to polyphenols [Reguengo et al. 2022]. Value-added polyphenols “bound or insoluble” remain attached to the agroindustry by-products [Roasa et al., 2022] and those can be successfully extracted by fermentative processes. Solid state fermentation (SSF) consists on the growth of a specific microorganism in a fermentable substrate in absence or nearly absence of free water. Due to its simplicity, SSF is preferred for the valorization of the agroindustry by-products [Orzua et al., 2009].

Considering the hasty increase in the consumption of pomegranate, the development of optimized eco-friendly processes to increase the extraction of high added value com-pounds from pomegranate peels is highly desirable. Therefore, this work is aimed to increase the extract concentration of Total Polyphenol Compounds (TPC) with AA from pomegranate peels by the environmental process of SSF, furthermore the identification of the extracted high-valuable phenolic compounds is presented. Accordingly, this work is aimed to increase the extract concentration of Total Polyphenol Compounds (TPC) with AA from pomegranate peels by SSF, furthermore the identification of the extracted high-valuable phenolic compounds is presented.

Was modified to:

Pomegranate intake is mainly in juice, jellies, jams and liquors; however, their bio-logical activity is highly appreciated in the food, cosmetic and pharmacy industries [Ascacio-Valdés, et al. 2011].  According to Kazemi et al. [2016] one ton of fresh pomegranate generates 669 kg of by-products and those include 78 % peel and 22 % seeds.  Although, the fast increase of pomegranate cultivation limits the precise calculation of the worldwide production, in 2021 based on the 8,636.2 tons of pomegranate harvested in 2021 for Mexico, 6,736.2 tons of pomegranate peels are available for the highly valued biomolecules extraction [https://nube.siap.gob.mx/cierreagricola/]. Organic solvents are commonly used for the extraction of vegetal biomolecules; however, their use is associated with environmental pollution and with toxicological safety concerns. Implementation of emerging technologies [i.e. supercritical fluid, microwave, electric field, pressurized liquid] for biomolecules extraction improves extraction yields; however, the requirement of polar solvents and expensive extractive equipment strongly limits its applications [Makris et al. 2007; Martins et al. 2011; Cano-Lamadrid et al. 2022].

In contrast, fermentative or enzymatic methods for the extraction of biomolecules from agricultural by-products have been successfully used [De la Torre et al. 2019; Buenrostro-Figueroa et al. 2017; Robledo et al. 2008]. Optimization methods based on one-factor-at-a-time are expensive, time-consuming and interactions between processes variables are not considered [Saffarzadeh-Matin and Khosrowshahi, 2017].  Experimental designs for the study of several variables at a time, as well as their interactions are highly desirable.

Polyphenols are molecules that present different biological and therapeutic activities; several studies have reported their health effects. Bioactive agents such as antioxidant, antiproliferative/anticarcinogenic, antifungal, antimicrobial, fat-lowering, antiInflammatory, antiaging neuroprotective, antibacterial, hypocholesterolemic, renoprotective, hepatoprotective, hypoglycaemic activity, antihyperglycemic, antihypertensive activities, among others, have been attributed to polyphenols [Reguengo et al. 2022]. Value-added polyphenols “bound or in-soluble” remain attached to the agroindustry by-products [Roasa et al., 2022] and those can be successfully extracted by fermentative processes. Solid state fermentation (SSF) consists on the growth of a specific microorganism in a fermentable substrate in absence or nearly absence of free water. Due to its simplicity, SSF is preferred for the valorization of the agroindustry by-products [Orzua et al., 2009].

Considering the hasty increase in the consumption of pomegranate, the development of optimized eco-friendly processes to increase the extraction of high added value com-pounds from pomegranate peels is highly desirable. Therefore, this work is aimed to increase the extract concentration of Total Polyphenol Compounds (TPC) with AA from pomegranate peels by the environmental process of SSF, furthermore the identification of the extracted high-valuable phenolic compounds is presented. Accordingly, this this work is aimed to increase the extract concentration of Total Polyphenol Compounds (TPC) with AA from pomegranate peels by SSF, furthermore the identification of the extracted high-valuable phenolic compounds is presented.

2. It is not clear the contribution of this study in the field. Please, add it in the introduction section.

Response: The following paragraph to highlight the contribution of this manuscript was added.

 “Considering the hasty increase in the consumption of pomegranate, the development of optimized eco-friendly processes to increase the extraction of high-added value compounds from pomegranate peels is highly desirable. Therefore, this work is aimed to increase the extract concentration of Total Polyphenol Compounds (TPC) with AA from pomegranate peels by the environmental process of SSF, furthermore the identification of the extracted high-valuable phenolic compounds is presented.”

3. 3. Could the dehydration step influence in the recovery of phenolics? Was this stage previously optimized? Is this stage necessary?

Response: Yes, the dehydration step may influence the recovery of polyphenols and AA, however the effect will depend on the drying method, temperature and time, chemical composition of the by-products (mainly cellulose and lignocellulose content), among others.  In the present work, the drying process was not optimized however, the drying optimization step will be considered in future works as dehydrating of the material is an important step for handling and transportation, and preventing rotting of material due to high moisture content.

4. Please, provide additional information about the experimental conditions of the kinetic showed in Figure 1

Response: Information of experimental conditions for the kinetics showed in Figure 1, is described in section “2.4 Solid-state fermentation (SSF)“

Certainly, discussion will be clearer if it is pointed out in the paragraph 3.2 then, it was modified as follows:

It was:

3.2 Kinetics of TPC extraction and AA 

Kinetics of metabolite production provide a quick-sign of the suitability of the substrate and culture conditions on the obtention of the desired metabolites. Kinetics of TPC release and AA by Aspergillus niger GH1 is shown in Fig. 1. Despite that the TPC release starts since the beginning of the process, the higher increase occurs from 24 – 36 h, attaining the value of 106.56 mgGAE/gdm at 36 h. The similar pattern was observed for AA showing the highest activity (7.95 mg GAE/gdm) at 36 h.   

Was modified to:

Kinetics of metabolite production provide a quick-sign of the suitability of the substrate and culture conditions on the obtention of the desired metabolites. Kinetics of TPC release and AA (as described in sec. 2.4) by Aspergillus niger GH1 is shown in Fig. 1. Despite that the TPC release starts since the beginning of the process, the higher increase occurs from 24 – 36 h, attaining the value of 106.56 mgGAE/gdm at 36 h. The similar pattern was observed for AA showing the highest activity (7.95 mg GAE/gdm) at 36 h.   

5. The KCl has not been in included as variable in the CCD, why?

Response: For de CCD experimental design, the factors whose values positively exceeded the dotted line (Figure 2) have a significant effect (a=0.05) on TPC extraction. In the case of MgSO₄, KH₂PO₄ and temperature variables, a positive effect as their level value increases was observed, then the response value also increases, therefore these variables were considered for the CCD. 

 In contrast, moisture and KCl have a negative effect on the response variable, thus any increase in those variables will cause a decrease on the TPC extraction therefore, the initial moisture and KCl lower levels were set.

6. Please, improve the results section, comparing the results (TPC and AA) obtained with the proposed method with that reported in literature for the recovery of phenolics from pomegranate (traditional and emerging technologies).

Response: Thanks for the comment, definitely the comparison with other extractive methods would be interesting however, such comparison is not the scope of this manuscript, at the moment, the research group is working in a review manuscript which compares the yields obtained by different extractive methods, pointed out the economic and technological advantages and disadvantages of the different technologies.

7. Please, include the multiple regression of the AA

Response: This work aimed to increase the concentration of TPC. For this reason, only a multiple linear regression was obtained for the release of TPC. Data from the extraction kinetics of TPC and AA by A. niger GH1 were used to estimate the Pearson correlation coefficient and show the relationship between TPC and AA. The value obtained was 0.86 (p˂0.01), indicating a significant positive correlation, that is, if the TPC value increases, the AA also increases. Therefore, only the TPC value was considered to optimize and validate the SSF model.

Even though the following paragraph was modified in the manuscript:

It was:

3.2. Kinetics of TPC extraction and AA

Kinetics of metabolite production provide a quick-sign of the suitability of the substrate and culture conditions on the obtention of the desired metabolites. Kinetics of TPC release and AA by Aspergillus niger GH1is shown in Fig. 1. Despite that the TPC release starts since the beginning of the process, the higher increase occurs from 24 – 36 h, attain-ing the value of 106.56 mgGAE/gdm at 36 h. The similar pattern was observed for AA showing the highest activity (7.95 mg GAE/gdm) at 36 h. 

Was modified to:

3.2. Kinetics of TPC extraction and AA

Kinetics of metabolite production provide a quick-sign of the suitability of the sub-strate and culture conditions on the obtention of the desired metabolites. Kinetics of TPC release and AA (as described in sec. 2.4) by Aspergillus niger GH1is shown in Fig. 1. De-spite that the TPC release starts since the beginning of the process, the higher increase oc-curs from 24 – 36 h, attaining the value of 106.56 mgGAE/gdm at 36 h. The similar pattern was observed for AA showing the highest activity (7.95 mg GAE/gdm) at 36 h. Data from the extraction kinetics of TPC and AA by A. niger GH1 were used to estimate the Pearson correlation coefficient and show the relationship between TPC and AA. The value obtained was 0.86 (p˂0.01), indicating a significant positive correlation, that is, if the TPC value increases, the AA also increases. For this reason, only the TPC value was considered to optimize and validate the SSF model.

Author Response File: Author Response.pdf

Reviewer 2 Report

Hi dear Editorial board and the respected authors

This article "Improved extraction of high value-added polyphenols from 2 pomegranate peels by solid-state fermentation” was revised and has a novelty and I recommend it for publication after consideration of the following comments.

Title: If you can rewrite and make it more interesting for readers.

Abstract:

·       The type of statistical design used in this research should be mentioned.

·       There is no need to be mentioned all the mineral solution amounts in abstract.

Keywords: Keyword phrases are long. Please note that these words must be searchable for other researchers.

Introduction:

·       Describe about Value Added Polyphenols in one paragraph.

·       Describe about Solid-state fermentation in one paragraph.

Materials: It is OK.

Methodology:

·       Some methods do not have references. Please mention them “for Line 86-94 and Line 116-133.

“Results:

·       Kinetics of TPC extraction and AA Table 1: Describe more about AA pattern.

·       Fig 2: Why are you numbering next to the vertical column variables?

·        

Discussion:

Discussion text must be improved and in some cases it is very weak and maybe there is no discussion at all.

Conclusions:

 There is no conclusion in this article.

References: It is OK.

The article has many flaws in express and concept of English, it is suggested to be revised in a scientific and native way.

Comments for author File: Comments.pdf

The article has many flaws in express and concept of English, it is suggested to be revised in a scientific and native way.

Author Response

Response to Reviewed 2

Dear reviewer, many thanks for your valuable comments, we did our best to attend your suggestions.    

Title: If you can rewrite and make it more interesting for readers.

Response: Thank you for the suggestion, we rehearsal different titles however, we prefer to keep the title as it is, as the keywords and discussion were focused as the actual title.

Abstract:  The type of statistical design used in this research should be mentioned.

Response: The two statistic designs used are mentioned in the abstract:

“A Box Hunter & Hunter (BHH) followed by Central Composite Design (CCD) were performed to assess the effect of the process variables on TPC release”

  There is no need to be mentioned all the mineral solution amounts in abstract.

Response: We prefer to write them down because they are the optimized components

Keywords: Keyword phrases are long. Please note that these words must be searchable for other researchers.

Response: The keywords phrases were shortened

It was:

pomegranate peels; polyphenols improved extraction; high value-added molecules identification; solid-state fermentation

Modified to:

pomegranate peels; polyphenols; improved extraction; high value-added; molecules identification; solid-state fermentation

Introduction: Describe about Value Added Polyphenols in one paragraph.

Response: The following paragraph was added:

Polyphenols are molecules that present different biological and therapeutic activities; several studies have reported their health effects. Bioactive agents such as antioxidant, anti-proliferative/anticarcinogenic, antifungal, antimicrobial, fat-lowering, anti-Inflammatory, anti-aging neuroprotective, antibacterial, hypocholesterolemic, renoprotective, hepatoprotective, hypoglycaemic activity, antihyperglycemic, antihypertensive activities, among others, have been attributed to polyphenols [Reguengo et al. 2022]. Value-added polyphenols “bound or insoluble” remain attached to the agroindustry by-products [Roasa et al., 2022] and those can be successfully extracted by fermentative processes.

Describe about Solid-state fermentation in one paragraph

Response: The following paragraph was added:

Solid state fermentation (SSF) consists on the growth of a specific microorganism in a fermentable substrate in absence or nearly absence of free water. Due to its simplicity, SSF is preferred for the valorization of the agroindustry by-products [Orzua et al., 2009].

Materials: It is OK.

Methodology: Some methods do not have references. Please mention them “for Line 86-94 and Line 116-133.

Response: for Line 86-94

Reference was added

It was:

Lyophilized Aspergillus niger GH1(ENA-KP273835) fungal spores were suspended in sterile water, cultivates in PDA-plates (30 °C, 5 days). For inoculum preparation, fungal spores were harvested (Tween-80, 0.01 % v/v) and counted in a Neubauer chamber.

Modified to:

Lyophilized Aspergillus niger GH1(ENA-KP273835) fungal spores were suspended in sterile water, cultivates in PDA-plates (30 °C, 5 days). For inoculum preparation, fungal spores were harvested (Tween-80, 0.01 % v/v) and counted in a Neubauer chamber (De la Cruz et al., 2014).

Response for Line 116-133.

Reference was indicated however, the word “Briefly” was added to point out the reference.

It was:

Antioxidant activity of the extracts was evaluated based on the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich®) free radical, as described by Meléndez et al. [2014]. Reaction mixture, consisting of 7 µL of extract and 193 µL of 60 µM DPPH in absolute methanol, were analysed on a BioTek® Microplate reader (ELx808™, Vermont, USA) with absorbance filters for a wavelength of 520 nm. Decolouring process was recorded during 30 min of reaction. Antioxidant activity was calculated on a base of gallic acid (Sigma-Aldrich®) standard curve (0-200 µg/mL) and expressed as mgGAE/gdm. Control samples were prepared with methanol (100 µL); distilled water (100 µL) was used for equipment calibration. Samples were analysed in triplicates.

Modified to:

Antioxidant activity of the extracts was evaluated based on the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich®) free radical, as described by Meléndez et al. [2014]. Briefly, reaction mixture, consisting of 7 µL of extract and 193 µL of 60 µM DPPH in absolute methanol, were analysed on a BioTek® Microplate reader (ELx808™, Vermont, USA) with absorbance filters for a wavelength of 520 nm. Decolouring process was recorded during 30 min of reaction. Antioxidant activity was calculated on a base of gallic acid (Sigma-Aldrich®) standard curve (0-200 µg/mL) and expressed as mgGAE/gdm. Control samples were prepared with methanol (100 µL); distilled water (100 µL) was used for equipment calibration. Samples were analysed in triplicates.

Fig 2: Why are you numbering next to the vertical column variables?

Response: The Pareto chart (Fig 2) describes the absolute values of standardized effects, from the largest to the smallest. This chart also plots a reference line to indicate which effects are statistically significant. This reference line depends on the significance level (α). A vertical column is displayed, which indicates the minimum size of statistically significant effects, considering the current model and choosing the error term using the statistical significance criterion. Also, this value could be positive or negative, indicating if the factor helps to increase or not the values of the response´s variable.

Discussion: Discussion text must be improved and, in some cases, it is very weak and maybe there is no discussion at all.

Response: We appreciate your comment

The manuscript´s main sections, were organized according to the authors´ guide (Tittle, abstract, keywords, introduction, methodology, results, discussion including conclusion, and references). We present a discussion and conclusion´ section accordingly to each results sub index, however, no sub indexing is allowed in the discussion-conclusion section, nevertheless, we wrote the section following the results sub index order. Then, in order to attend you comment, the discussion by each result index is annotated:

3.1. Physicochemical characterization of pomegranate peels

The results of this section were discussed as follows:

Culture media is a mixture of nutrients that, in adequate concentrations and under optimal physical conditions, allow the growth and metabolic processes of the desirer micro- organisms. Obtained results (Tab. 3) are similar to those reported by Bhol et al.  [2016] in fat (2.37 ± 0.15 %) and protein (8.03 ± 0.21). However, differences were observed in ash (0.67 ± 0.02 %), fibre (4.80 ± 0.10 %) and carbohydrates (46.21 ± 0.11 %) content, while C/N value (65) was lower than the reported by Ben-Ali et al. (2017). A carbon-to-nitrogen (C/N) ratio is the relationship between the mass of carbon to the mass of nitrogen present in any substance, the C/N ratio is highly important for the regulation of the metabolic pathway either to biomass or to secondary metabolites production.  Then, the C/N ratio must be established according to the product of main interest [Lopez-Flores et al., 2016]. Carbon and nitrogen content and consequently the C/N ratio can be adjusted by adding any source of carbon or nitrogen; however, any excess of these compounds might result toxic and affect fungal growth and enzymes production then, enzymatic breakdown of the cell wall of the substrate and subsequent polyphenols release is reduced [Rajarathnam et al. 1989]. The difference in the values of the chemical components is due to factors related to the variety of pomegranate used, the geographic location of the crop, the irrigation conditions and other environmental and technological factors. 

The water absorption index (WAI) and critical humidity point (CHP) are physicochemical properties with a relevant importance in materials to be used as a substrate-support (S-S) in SSF.  WAI is related to hydroxyl groups present on substrate fibre, which allows additional water-interaction throughout hydrogen bonding [Martins et al. 2017], then the WAI value indicates the amount of water that can be absorbed by the S-S. The best materials for SSF are those with high WAI, since the moisture content of these materials can be modified to required values either for the microorganism growth or for a bioprocess convenience. Pomegranate by-products presented a WAI of 4.38 g/g; similar WAI values were reported for creosote bush leaves [Orzua, et al. 2009], candelilla stalks [Ascacio-Valdés et al. 2010; Buenrostro-Figueroa et al. 2014] and grape by-products (Martínez-Ávila et al. 2012), all of them reported as good S-S for SSF.  The CHP is the amount of water linked to the support macromolecules and represents the water that cannot be used for the microbe for their metabolic processes. High CHP value represents a high amount of water bounded to the material, which can select the type of strain able to grow over the substrate, then materials with low CHP are preferred in SSF [Martins et al. 2017]. The CHP value obtained for pomegranate by-products was 10.13 %, this value is lower than those reported for agroindustry by-products such coconut husk 16 %, orange peel 40 %, lemon peel 28 %, apple pomace 35 % and grape 53 % (Buenrostro-Figueroa et al. 2014; Martínez-Avila et al. 2012; Orzua et al. 2009]. Based on the physicochemical characterization, pomegranate by-products are suitable to be used as SS for SSF. Physicochemical characterization suggested that pomegranate by-products possessed the required characteristics for its potential use as substrate-support for SSF.

Yellow high lined correspond to the conclusion of the section.

3.2. Kinetics of TPC extraction and AA

The results of this section were discussed as follows:

Kinetics of metabolite production provide a quick-look of the microbial growth, suitability of the substrate and culture conditions, maximal production time and the process yield. Lower TPC extraction values when using conventional methods or commercial enzymes for have been reported (Coetzee et al. 2012). In contrast, in SSF, different enzymes such amylases, pectinases, xylanases, proteases, β-glucosidase, tannase and ellagitannase are simultaneously produced then, the sum of the different enzymatic activities increases the release of phenolic compounds as the result of the breakdown of the links between polyphenols moieties and other macromolecules, then the amount of TPC release and AA is increased in a short time process [Ascacio-Valdés et al.   2014; Santos da Silveira et al. 2019]. Accordingly, to the TPC kinetics, time was set at 36 for the following experiments.

Yellow high lined correspond to the conclusion of the section.

3.3. Significant factors for TPC recovery by SSF   

The results of this section were discussed as follows:

Positive effect of MgSO4 on the TPC release is explained on the fact that magnesium is related to the growth of hyphae in A. niger, the increase in sporulation rate ensures an efficient enzyme synthesis, increasing the nutrients availability and in consequence, the microbial biomass proliferation (Jamal et al. 2011). Sepúlveda et al. [2012] reported that the increase in MgSO4 levels promotes a major ellagic acid accumulation from pomegranate husk powder by A. niger GH1 in SSF. Temperature directly affects the fungal metabolism; consequently, it may affect either the microbial growth or the enzymes production rate, thus impacting the TPC release. Most studies related to growth and enzyme production for A. niger GH1 are performed at 30 °C [De la Cruz et al. 2014; Lopez-Trujillo et al. 2017]. In this study, temperature had a positive effect (Fig. 2), showing good TPC release from 30 °C. Different microbial species have different needs of specific moisture content to sup- port their growth and metabolites production, in this study, moisture had a negative effect on the TPC release. Therefore, fermentation processes require a close control of water con- tent as it affects the adequate nutrients and oxygen transport; a small deviation from the optimal moisture values may decrease the production of enzymes and consequently affects the release of the product of interest [Beniwal et al. 2013].  

The addition of KCl exhibited a negative effect on the TPC release (Tab. 1 and Fig. 2).  Potassium ions may trigger the conformational transition when binding to a distant protein enzyme site promoting suitable conformational changes in the active site [Vašák and Schnabl, 2016]. However, experimental results (Tab. 1, Fig. 2) show, that the concentration of KCl used was high (3.04 g/L) that the possible positive effect of the K+ was reversed causing a decrease in the enzymatic activity for the TPC release, then KCl was set at its lower level (1.52 g/L). Then, the variables of MgSO4, temperature and moisture were further considered in the CCD to optimize the TPC release from pomegranate by-products by SSF. 

Yellow high lined correspond to the conclusion of the section.

3.4. Optimization of the culture conditions for release of TPC

The results of this section were discussed as follows:

Based on above results, the optimized process conditions (CCD) defined satisfactorily the TPC release by SSF at 36-h process.  Robledo et al. (2008) reported TPC recovery of 6.3 and 4.6 mg/gdm from pomegranate peels with A. niger GH1 and A. niger PSH respectively. Ascacio-Valdés et al. [2014] reported TPC production of 42.02 mg/g for the fungal biodegradation of punicalin previously recovered and purified from pomegranate peels used as carbon source. The TPC released from pomegranate in the present work is 24 – 80 % higher from values previous reported [Robledo et al. 2008; Ascacio et al. 2014]. In addition, optimal SSF conditions provided an increase of 5.81-fold in the AA of the extract (46.40±0.04 mgGAE/gdm) compared with the value before the optimization (7.98±0.06 mgGAE/gdm) at 36-h process.  The increase in AA is attributed to the amount and type of the released phenolic compounds. The obtained results show the suitability of SSF to obtain TPC with AA from by-products over of the chemical synthesis or by the use of commercial enzymes.

Yellow high lined correspond to the conclusion of the section.

3.5. Identification of phenolic

The results of this section were discussed as follows:

Identification of phenolic compounds (Fig. 4) starts with a compound signal (com-pound 1) at m/z 191 and matched to 2-Hydroxypropane-1,2,3-tricarboxylic acid or citric acid. Citric acid has been reported as the main organic acid found in pomegranate wine, juice and peels [Kalaycıoğlu and Erim 2017; Pande and Akoh 2009]. Compounds 2 and 3 have a molecular ion at m/z 781 at two elution times (17.8 and 21.38 min) corresponding to 4,6-gallagyl-glucoside or punicalin isomers (namely α and β anomers), both molecules are considered intermediate compounds during ellagitannins biodegradation [Aguilera-Carbó et al. 2008; Ascacio-Valdés et al. 2014]. Furthermore, compounds 4 and 5 (m/z 1083) were identified as punicalagin (2,3-HHDP-4,6-gallagylglucoside) isomers (30.9 and 32.95 min of elution time), the main phenolic compound found in pomegranate [Amyrgialaki et al. 2014; Fischer et al. 2011]. Punicalagin is considered a key precursor in pomegranate ellagitannins degradation and it is determinant molecule for the induction of fungal ellagitannase production by SSF [Ascacio-Valdés et al. 2016]. Compounds 6 (m/z 801.2) and 7([M-H]- m/z 633) were identified as digalloyl-HHDP-gluconic acid (puniglu-conin) and galloyl-HHDP-(hexoside or corilagin) respectively, both hydrolysable tannins previously found in pomegranate juice [Gómez-Caravacan et al. 2013] and seeds [Am-bigaipalan et al., 2016]. Finally, compound 8 corresponds to 2,3,7,8-tetrahydrxy-chromen [5,4,3-cde] chromene-5, 10-dione or ellagic acid (m/z 300.9).  There are no reports about the identification of phenolic compounds obtained from solid fermented pomegranate by-products; however, Ascacio-Valdés et al. [2016] suggested the complete biodegradation pathway of ellagitannins by SSF of ellagitannins previously extracted from pomegranate by-products by A. niger GH1. The same authors reported that punicalin, gallagic and el-lagic acids were obtained from punicalagin, identifying the intermediate molecules and immediate precursor of ellagic acid. In this study, gallic acid was not detected at the final process time (36 h). Fischer et al. [2011] reported the identification and quantification of phenolic compounds from pomegranate peel, mesocarp, aril and differently produced juices however, they did not report citric acid, punicalin isomers (α and β) nor puni-calagin.  Li et al. [2015] reported the gallic acid, punicalagin- α, punicalagin-β, catechin, chlorogenic acid, epicatechin, rutin, and ellagic acid as the eight characteristics chemical fingerprint of polyphenols extracted from pomegranate peel, but they don’t find punicalin (α and β), citric acid punigluconin nor galloyl HHDP hexoside.  According to Gumienna et al. [2016], differences among the formed bioactive compounds are explained by reac-tions of polymerization, condensation, oxidation, hydrolysis, enzyme activity and mole-cules interactions. Furthermore, different phenolic profiles may be obtained depending on the microbial strain (fungi, yeast or bacteria), and the enzymes that they may produce, even when using the same substrate and fermentation process. 

The identified polyphenol molecules have different biological activities with a wide number of possible applications in the food, pharmacy and cosmetics industries [Ascacio-Valdés et al. 2011] and when obtained from by-products are considered as high-added value product [Holic et al. 2018].  Then, bearing in mind that all pomegran-ate peels were treated under the described process, it could be obtained up to 248 kg of TPC per ton dm of pomegranate peels, and considering that in Mexico (2021) 5937.28 tons dm of pomegranate peels, then 1,472,445.44 kg of valuable TPC may be obtained from dry pomegranate peels.  Considering the commercial price of the ellagic acid and punicalagin is 94 USD/50 mg and 494.70 USD/10 mg respectively (Sigma-Aldrich®), the SSF extraction process may result quite profitable for industrial interest. The improved biotechnological extraction process has a foremost impact on the recovery of high-value molecules from pomegranate peels, providing higher TPC and AA values in a short-time process.  The recovered molecules are of a great interest in the food, pharmacy and cosmetic industries, and at the same time a diversification in the use of agroindustry by-products is obtained thus approaching the highly desired circular economy model.

Yellow high lined correspond to the conclusion of the section.

Conclusions:  There is no conclusion in this article.

Response: As follow the authors´ guide, conclusions were incorporated into the discussion section. However, the conclusion paragraphs were high lined (yellow) answer above.

References: It is OK.

The article has many flaws in express and concept of English, it is suggested to be revised in a scientific and native way.

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