Valorization of Green Arabica Coffee Coproducts for Mannanase Production and Carbohydrate Recovery

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title: Valorization of green Arabica coffee coproducts for mannanase production and carbohydrate recovery

-In this study, it was investigated: coffee press cake as a mannan-rich carbohydrate source for submerged fermentation with Aspergillus niger, aiming at the induction of 105 endo-β-mannanases and β-mannosidases production.
-Is every agricultural waste suitable for enzyme production? Are some agricultural wastes suitable for enzyme production because they are loaded with pesticides?
-Does Arabica coffee press cake contain sufficient carbohydrates for the cultivation of Aspergillus niger (CFAM 1234) and mannanase production, and is it economical for enzyme production?
-How did you determine the physiological environment for enzyme production?

-Details should be provided on how you performed potassium phosphate buffering.
-What is the percentage of yeast extract? What is the carrier substance in the extract? When applied to the hydrolysis of healthy and defective coffee beans, the enzyme preparation can recover approximately 25% mannose, demonstrating its potential as a mannose source. However, is the waste product (coffee cake) economically feasible for this purpose?

-Is mannanase production from coffee cake more suitable than the plant or its byproduct, which is the best mannanase source? A comparative discussion is needed.

-To what extent did the oil remaining after the oil in ground coffee is removed by pressing contribute to the production of mannanase? This is because peroxidation and lipase activity caused the material to freeze.

Comments on the Quality of English Language

should be improved

Author Response

We would like to thank you very much for taking the time to review this manuscript. Please find detailed responses below.

 

Comments 1: Is every agricultural waste suitable for enzyme production? Are some agricultural wastes suitable for enzyme production because they are loaded with pesticides?

Response: No, not all agro-industrial residues are suitable as a carbon or nitrogen source for fungal growth and enzyme production. One of the most important factors is the chemical composition of the residue, which can induce or inhibit fungal growth and/or enzyme production. For example, we used coffee, which is rich in mannan, a polysaccharide that serves as a carbon source for fungal growth and for inducing mannanases production. The chemical monosaccharides composition is presented in Table 7 (acid hydrolysis). In another study by the group, we used palm fruit seeds, also rich in mannan, to achieve the same result; however, fungal growth was inhibited, without mannanases production, due to the presence of a high concentration of phenolic compounds. To achieve similar results, a pretreatment was necessary to extract the phenolic compounds. Furthermore, the type of carbon source present will directly influence the outcome. For example, sources rich in starch may allow fungal growth, but will not promote the induction of mannanases production. Another example, in the present study, the presence of lipids in green coffee beans negatively affects enzymatic activity compared to coffee cake, as discussed for Figure 1.

Other parameters should also be considered, such as moisture and nutrient balance, particle size and structure, recalcitrance, and the presence of inhibitory compounds such as pesticides. Thus, although most agricultural wastes are potentially useful, sometimes they need pretreatment, detoxification, or supplementation to make them effective substrates. Fortunately, this was not the case for the waste used in this study, which was a carbon source with superior performance in the production of mannanases when compared to commercial mannan.

 

Comments 2: Does Arabica coffee press cake contain sufficient carbohydrates for the cultivation of Aspergillus niger (CFAM 1234) and mannanase production, and is it economical for enzyme production?

Response: Thank you for the question. Although we have not conducted a detailed economic evaluation study of the use of Arabica coffee press cake as a carbon source for A. niger cultivation and mannanases production, we would like to emphasize that our study compared the performance of this residue with that obtained using a commercial mannan source. Locust bean gum (LBG) from Ceratonia siliqua seeds is a high-cost commercial galactomannan (approximately R$1,308.00 per 100g; Sigma-Aldrich, 2025). When comparing the results obtained with coffee press cake and with LBG as carbon source (Figure 1), it becomes evident that this agro-industrial residue has great potential to reduce mannanases production costs through this biotechnological approach.

This discussion can be found below Figure 1: “The medium containing only LBG, a commercially available galactomannan, resulted in lower mannanases activity compared to media containing coffee cake or green coffee beans. These results indicate that coffee beans are an excellent substrate for mannanases production. Furthermore, since coffee cake is an industrial waste product, its use in the enzyme production process could be highly cost-effective”.

Currently, with only bench-scale cultivation data, we cannot perform a detailed and accurate economic evaluation. We can only present the potential impact on costs. Looking ahead to this work, we intend to scale up production using bioreactors, which will allow for a thorough economic analysis of the process.

 

Comments 3: How did you determine the physiological environment for enzyme production?

Response: To determine the physiological environment favorable to enzyme production, the carbon and nitrogen load of the medium was evaluated using elemental analysis (CHNSO) and the C/N ratio (Table 4), since the balance between these nutrients directly influences microbial metabolism, modulating the expression of enzymes involved in the assimilation of the mannanases-inducing source. The parameters of pH, temperature, and rotation of the culture media were also investigated through scientific literature, as described in topic 3.1.1: “Cultivations were conducted at 30 °C and 200 rpm, conditions commonly reported as optimal for Aspergillus [44–47]. The pH was adjusted to 4.8, based on previous studies on mannanases production from Aspergillus [36–40].”.

 

Comments 4: Details should be provided on how you performed potassium phosphate buffering.

Response: The following paragraph was added to section 2.2.1: “Potassium phosphate buffer was prepared by mixing 9.55 g of KH2PO4 with 0.24 g of K2HPO4, dissolving it in 800 mL of distilled water, and adjusting the pH to 5.5 with KOH or HCl. The final solution was brought to a volume of 1 L using a volumetric flask.”

 

Comments 5: What is the percentage of yeast extract? What is the carrier substance in the extract?

Response: Yeast extract is a commercially available powdered product (approximately R$337.00 per 250 g; Sigma-Aldrich, 2025) obtained from the autolysis of yeast cells, typically Saccharomyces cerevisiae. During this process, the yeast’s own enzymes degrade intracellular macromolecules, releasing soluble components into the medium. After autolysis, the insoluble cell wall material is removed, and the soluble fraction is concentrated and dried to obtain the final powder.

The product is widely used as the main source of nitrogen in fungal cultures. It is a complex mixture mainly composed of amino acids, small peptides, water-soluble vitamins (especially B-complex), carbohydrates, and minerals. Because of this balanced composition, yeast extract serves as an excellent organic nitrogen and growth factor source for microbial cultures.

 

Comments 6: When applied to the hydrolysis of healthy and defective coffee beans, the enzyme preparation can recover approximately 25% mannose, demonstrating its potential as a mannose source. However, is the waste product (coffee cake) economically feasible for this purpose?

Response: As coffee cake is a waste product (a by-product of the pressing process), it has high potential for application in mannan and mannose recovery processes. Currently, with only bench-scale cultivation data, we cannot perform a detailed and accurate economic evaluation. The enzymatic hydrolysis stage is at Technology Readiness Levels (TRL) 1-3, corresponding to the laboratory research and proof of concept phase. Subsequent stages, if there is business interest, will include studying process scaling and assessing economic feasibility.

 

Comments 7: Is mannanase production from coffee cake more suitable than the plant or its byproduct, which is the best mannanase source? A comparative discussion is needed.

Response: Ground coffee beans and coffee cake, both rich in mannan, were used as carbon sources to induce the production of mannanases by the filamentous fungus Aspergillus niger (CFAM 1234). The comparative analysis between the coffee beans and coffee press cake, which is already a byproduct, is presented in Figure 1. The presence of mannan is essential for the induction of mannanases. Other parts or residues from the coffee production chain that do not contain mannan are not suitable for mannanases production. As discussed for the first comment, one of the most important factors is the chemical composition of the residue, which can induce or inhibit fungal growth and/or enzyme production. For example, as indicated in the text following the Figure 1: “The presence of lipids in green coffee beans negatively affects enzymatic activity compared to coffee cake. The highest activities were obtained in media containing coffee cake of different particle sizes, with no significant differences among them (p ≤ 0.05).”

 

Comments 8: To what extent did the oil remaining after the oil in ground coffee is removed by pressing contribute to the production of mannanase? This is because peroxidation and lipase activity caused the material to freeze.

Response: Pressing is known for not completely removing lipids from oily materials; however, it is a sustainable, scalable, and economically viable technique that is widely implemented on an industrial scale. The pressing process applied to coffee generates a solid residue (coffee cake), which currently has no specific destination (sometimes being used irregularly in the roasting of beans). Given this scenario, the present study investigated the potential of this residue as a substrate to produce mannanases.

Table 1 shows that lipid content negatively affects mannanases production. Ground coffee beans showed enzymatic activity close to 16 U/mL, while coffee grounds resulted in values above 20 U/mL, after 7 days of cultivation in both cases. To improve the discussion, the text below Figure 1 was supplemented with: “A hypothesis for this is that the fungus may be using the lipids present in the substrate as its main source of carbon, redirecting its metabolism toward the production of enzymes related to lipid degradation, such as lipases, to the detriment of mannanases. Lin et al. (2004) [48] indicate that the presence of oil in copra (a fat-rich matrix) may depress mannanase production.”. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 The article focuses on the biotechnological potential of the by-products of green Arabica coffee processing in terms of its application in microbial fermentation processes for both β-mannanase production and subsequent enzymatic conversion into value-added products. The research is interesting combining approach to microbial production of mannanase based on novel promising waste substrate and its following application for hydrolysis of polymers in untreated coffee beans. Generally, the manuscript is quite well and clearly written, and qualifiedly designed. Nevertheless, there are problems in some concepts, methodology and data presentation.

 

Abstract:

L21: "enzymes obtained were evaluated” – It is indefinite phrase (for Abstract content) in terms of enzyme preparation composition and activity (…ties).

L22: “Statistical optimization” - The essence and effectiveness of the optimization here remains unclear, given that enzyme production increases only slightly, except that the cultivation time is reduced and some salts are eliminated. In particular, the initial production of the mannanase in presence of several coffee cake substrates illustrated in Fig. 1 indicates the close production levels (more than 20 U/mL). And this does not take into account the influence of cultivation time, when the enzyme synthesis reaches 25 U/ml on the 17^th day (Fig. 2). Thus, it is necessary to specify the initial cultivation parameters and the main carbon source for non-optimized conditions.

L25-26: “within only 3 days” - Was cultivation time used as one of the independent factors for optimization? The presented value (22.4 U/mL) does not agree with the any data presented in Results for 3^rd day (Figs. 1-3, Tables 5-6).

L26: “42.9% improvement” – Specify non-optimized conditions.

L27: “β-mannosidase activity…1.39 U/mL” – The level of β-mannosidase activity is relatively low compared to β-mannanase that does not allow the preparation to be considered as highly suitable for effective hydrolysis of coffee beans to mannose (monomer). In this view, a general determination of soluble sugars and mannooligosaccharides in particular would be more informative for assessment of the substrate conversion.

L29: “25% mannose recovery” – This value is misleading indicating really the percent ratio to acid hydrolysis (Table 7).The conversion degree should be based on real mannose production (~4.7-4.8%).

Overall: The main analytical methods and enzymatic conversion parameters should be mentioned in Abstract.

Keywords:

L34: “mannanases and β-mannosidases’ should be written in the singular.

Introduction:

L63: “a mannan-degrading enzymes” – remove the repeating fragment. The paragraph should be supplemented with information about presence of other hemicellulases than mannans in coffee press cake.

L64-67: Please, add the biochemical nomenclature numbers for several mentioned mannan-degrading enzymes.

Materials and methods

2.1.2. Elementary organic analysis in the best carbon source

L134-138: Add more detail description of sample prep of the substrate for element analysis.

2.1.3. Microorganism

L146-148: It is unclear whether such procedure was carried out to preparation of inoculate, but then why store and freeze it? Commonly, the freshly prepared spore suspension is used as inoculate.

 

2.2.1. Preliminary growth medium composition and cultivation

Table 1: If the buffer is indicated as one of constituents of the media, then its content must be indicated in relative volumetric ratio. In the presented view it is necessary to indicate the content of individual salts. The similar remark regards the presentation of concentration for coffee cake extract.

Justify and explain within the main text the necessity of Al₂O₃ supplement in nutritional medium.

2.2.3. Statistical experimental design for mannanases production

Add more detail description of used PBD and CCRD with refs.

2.2.4. Determination of β-mannosidase activity

In logical terms, this section should be combined in a single section Enzymatic assays as sub-section.

 

2.3.1. Enzymatic hydrolysis

L200: Crude enzyme preparation should be characterized by enzymatic activities levels and composition and protein concentration. Specify the process (conditions) where the preparation was prepared (or ref to previous sections).

L202: The pre-treatment of coffee beans for hydrolysis process should be described.

L205: “after 6, 24 and 48 h” – Why the withdrawal was carried out in these time points?

L207-210: The analysis of oligosaccharides would be informative supplement for assessment of hydrolysis efficiency. The indicated column allows to fractionating di-, tri- and tetra-saccharides.

L214: Why fructose was used as additional standard in HPLC-RI analysis? Or it is one of constituents of coffee beans’ glucans? In this case the estimation of efficiency of enzymatic hydrolysis is questionable.

3.1.1. Influence of carbon source arrangement

L229: “were tested as carbon to” – carbon sources.

3.1.2. Influence of cultivation time

L275: “mediums” – media.

 

3.1.3. Preliminary test

The principle difference of this section with data above and Fig. 3 with Figs. 1-2 is unclear.

3.1.4. Statistical experimental design

The comments / explanations for the factors VI_1-5 (Table 5) should be added.

3.1.5. Determination of β-mannosidase enzyme activity

L406: “Using coffee cake as a carbon source for β-mannosidase production…” – This comment requires the presentation of data of β-mannosidase production in presence of several substrates as in Fig. 1.

3.2. Hydrolysis experiments of coffee beans

L427: “arabinogalactan proteins” – Check the terminology correctness.

Fig. 6: the caption “Figure 61” – Correct. “using the optimized enzyme productized in conditions” – Revise.

The mannose / fructose concentration increased slightly between 6 and 24 h. In this view, additional points (1-3 h, 12 h) would be informative. The control (o h) is also desirable. Decrease of mannose / fructose recovery after 48 h should be commented / explained.

 

L439-440: “enzymatic hydrolysis was 24% for healthy beans and 20% for defective beans” – It contradicts to data in Fig. 6 and Table 7.

L441: “untreated biomass’ – Again, it is worth note in what form was this raw material used (grinding degree, etc.).

L458-459: “good potential for recovery of glucose (76% in healthy beans and 72% in defective beans)” – According to Table 7, glucose recovery was 90% for defective beans;

L460-462: “integrated strategies should be adopted, including appropriate pretreatments, optimization of hydrolysis parameters (enzyme loading, pH, and temperature)” – Such moderate optimization could be implemented within this study.

Conclusions:

L475: “increased enzyme productivity by 42.9%” – specify initial and final substrate and parameters.

L479: “reached only~25%” - Either emphasize that this is a relative value, or present the absolute recovery for a clear understanding.

L484-485: “capable of capable” – Correct.

Author Response

We sincerely appreciate the thorough review and constructive contributions provided. All comments and suggestions were carefully considered and have led to substantial improvements in the writing style, clarity of discussion, and consistency of the results presented. We acknowledge that the comments made contributed substantially to strengthening the scientific rigor of the work, and we are grateful for the attention and dedication during the review process.

 

Abstract:

Comments 1: L21: "enzymes obtained were evaluated” – It is indefinite phrase (for Abstract content) in terms of enzyme preparation composition and activity (…ties).

Response: The sentence has been changed to: “Furthermore, the enzymes obtained were tested for mannose recovery in the enzymatic hydrolysis of healthy and defective coffee beans to investigate their hydrolytic potential.”

 

Comments 2: L22: “Statistical optimization” - The essence and effectiveness of the optimization here remains unclear, given that enzyme production increases only slightly, except that the cultivation time is reduced and some salts are eliminated. In particular, the initial production of the mannanase in presence of several coffee cake substrates illustrated in Fig. 1 indicates the close production levels (more than 20 U/mL). And this does not take into account the influence of cultivation time, when the enzyme synthesis reaches 25 U/ml on the 17^th day (Fig. 2). Thus, it is necessary to specify the initial cultivation parameters and the main carbon source for non-optimized conditions.

Response: Thank you for this valuable observation. The initial production of mannanases in the presence of various coffee substrates (illustrated in Fig. 1) showed enzyme production levels close to 20 U/mL after 7 days of cultivation. After optimization, this same activity level was achieved in only 3 days, representing a substantial advantage in terms of industrial process time. Additionally, the optimization allowed to eliminate several medium components, thereby reducing production costs (as discussed in the last paragraph of Section 3.1.4). To clarify this point, the following excerpt in the Abstract was changed to: "Statistical optimization (Plackett–Burman and Central Composite Rotatable Design) simplified the culture medium composition to coffee press cake (48.78 g.L⁻¹), yeast extract (4 g.L⁻¹), and potassium phosphate (0.25 g.L⁻¹, pH 5.5), and increased mannanases productivity to 22.4 ± 0.6 U.mL⁻¹ within only 3 days (a 42.9% improvement compared to non-optimized conditions, which were: 30 g.L⁻¹, carbon source, 4 g.L⁻¹ yeast extract, 1 g.L⁻¹ Al₂O₃, 0.5 g.L⁻¹ potassium phosphate buffer (pH 5.5), 0.5 g.L⁻¹ of MgSO₄.7H₂O, and 0.05 g.L⁻¹ of CaCl₂.2H₂O, which reached a maximum of ~20 U.mL⁻¹ in 7 days)”.

 

Comments 3: L25-26: “within only 3 days” - Was cultivation time used as one of the independent factors for optimization? The presented value (22.4 U/mL) does not agree with the any data presented in Results for 3^rd day (Figs. 1-3, Tables 5-6).

Response: Culture time is not usually included as an independent variable in bioprocess optimization because its effect tends to be more pronounced than that of other factors, which could mask the influence of critical variables. Therefore, optimization is generally conducted at fixed culture times, while enzyme production is evaluated independently at different cultivations periods.

In this study, samples were collected after 3, 5, and 7 days, and statistical analyses were performed separately for each period. Although cultivation time was not included as one of the independent factors for optimization, it was systematically investigated throughout the experiments. The value used for the CCRD was 5 days of cultivation, but the value of 22.4 U/mL (for 3 days) is reported in line 367: “Additional assays under the optimized condition showed activities of 22.43 ± 0.59 U.mL⁻¹ at three days and 17.26 ± 0.56 U.mL⁻¹ at seven days of cultivation. The activities on days three and five were not significantly different (p ≤ 0.05), indicating that the peak of enzymatic activity shifted to the third day of cultivation.”. Thus, the value of 22.4 U mL⁻¹ refers to the enzymatic activity obtained at the third day under optimized conditions, confirming that the highest productivity was achieved earlier in the process.

 

Comments 4: L26: “42.9% improvement” – Specify non-optimized conditions.

Response: The non-optimized conditions have now been explicitly specified in the revised Abstract. The following excerpt was added: “(… a 42.9% improvement compared to non-optimized conditions, which were: 30 g.L⁻¹, carbon source, 4 g.L⁻¹ yeast extract, 1 g.L⁻¹ Al₂O₃, 0.5 g.L⁻¹ potassium phosphate buffer (pH 5.5),            0.5 g.L⁻¹ of MgSO₄.7H₂O, and 0.05 g.L⁻¹ of CaCl₂.2H₂O, with a peak close to 20 U.mL⁻¹ in 7 days)”.

 

Comments 4: L27: “β-mannosidase activity…1.39 U/mL” – The level of β-mannosidase activity is relatively low compared to β-mannanase that does not allow the preparation to be considered as highly suitable for effective hydrolysis of coffee beans to mannose (monomer). In this view, a general determination of soluble sugars and mannooligosaccharides in particular would be more informative for assessment of the substrate conversion.

Response: Thank you for the comment. We agree that a more balanced ratio between β-mannosidase and β-mannanase activities would be ideal to accelerate the release of mannose monomers. Enzymatic formulations are often optimized to achieve more suitable activity ratios, include complementary enzymes, and consequently reduce hydrolysis time. In the present study, however, the objective was not to optimize the enzymatic formulation but to demonstrate the potential of the enzymes produced by Aspergillus niger (CFAM 1234) using coffee press cake as substrate. For this reason, an extended hydrolysis time (up to 48 h) was applied. Nevertheless, even without enzymatic formulation optimization, the results obtained after 24 h were already significant.

Regarding the analyses, the hydrolysates were evaluated by HPLC for monosaccharides detection and quantification. No peaks corresponding to mannobiose were observed. The major sugar is mannose. However, the possible presence of mannanoligosaccharides (β-MOS) is acknowledged (at very low concentration) and is currently being investigated in a complementary study conducted in our laboratory. The identification and quantification of β-MOS is not trivial. Therefore, it is being carried out within the scope of another project. It requires time, a study of kinetics and enzymatic formulations, as well as resources for chromatographic columns and specific standards. For these reasons, oligosaccharide analysis was not included in the current assessment of hydrolysis efficiency. The focus of this work was to demonstrate the feasibility and hydrolytic potential of the enzyme produced from coffee residue, corresponding to early stages of technological maturity (TRL 1–4).

To clarify these points, the following paragraphs were added to the revised manuscript:

Section 3.1.5 (β-mannosidases activities): “Although this is a very positive result, the addition of an external β-mannosidase (either commercial or laboratory-concentrated) to balance the β-mannosidase/β-mannanase ratio could enhance the enzymatic hydrolysis of this material, enabling the complete conversion of polysaccharides into monosaccharides.”

Section 3.2: (Enzymatic hydrolysis): “Although only monosaccharides were quantified, presented in Table 7, mannanoligosaccharides (MOS) may also have been formed and remained in the reaction medium without being converted to mannose, due to the imbalance between β-mannosidase and β-mannanase activities.”.

 

Comments 5: L29: “25% mannose recovery” – This value is misleading indicating really the percent ratio to acid hydrolysis (Table 7).The conversion degree should be based on real mannose production (~4.7-4.8%).

Response: We acknowledge that the 25% value refers to the ratio between mannose released by enzymatic hydrolysis and that obtained by acid hydrolysis, which was used as the 100% reference value, as described in Section 2.3.2. To avoid any possible misinterpretation, the following clarification was added to the title of Figure 6 and to the corresponding text: “The mannose value obtained by acid hydrolysis was considered to be 100%.”.

 

Comments 6: Overall: The main analytical methods and enzymatic conversion parameters should be mentioned in Abstract.

Response: The following content has been added to the abstract: “Mannanases production was investigated using various carbon sources, including ground coffee beans, coffee press cake, different particle sizes of coffee press cake, aqueous coffee cake extract (prepared at 30 g.L^-1 under constant stirring (300 rpm) at 80°C for 2h, followed by filtration.), and a commercial galactomannan, locust bean gum (LBG). CNHSO analysis was performed in the best carbon source (coffee press cake) and LBG.”

Regarding mannose recovery, the sentence was modified to include an explanation of the calculation, thus becoming: “When applied to the hydrolysis of untreated healthy and defective coffee beans, the enzyme preparation enabled ~25% mannose recovery (considering the value obtained through acid hydrolysis as 100%), highlighting its potential as a mannose resource.”

 

Comments 7: Keywords: L34: “mannanases and β-mannosidases’ should be written in the singular.

Response: The terms were corrected to the singular forms “mannanase” and “β-mannosidase” in the list of keywords.

 

Introduction:

Comments 8: L63: “a mannan-degrading enzymes” – remove the repeating fragment. The paragraph should be supplemented with information about presence of other hemicellulases than mannans in coffee press cake.

Response: The redundant phrase was removed. Additionally, the paragraph was supplemented with the following sentence: "This by-product is rich in the polysaccharide fraction of green coffee, mainly composed of cellulose, type II arabinogalactans, and galactomannans [22]."

 

Comments 9: L64-67: Please, add the biochemical nomenclature numbers for several mentioned mannan-degrading enzymes.

Response: The corresponding Enzyme Commission (EC) numbers were added to the text: " β-mannanase (EC 3.2.1.78)" and the " β-mannosidase (EC 3.2.1.25)".

 

Materials and methods

Comments 10: 2.1.2. Elementary organic analysis in the best carbon source. L134-138: Add more detail description of sample prep of the substrate for element analysis.

Response: No specific sample preparation was required for CHNSO analysis using the Unicube elemental analyzer. Approximately 2 g of the ground and homogenized sample was directly introduced into the instrument. The elemental composition was determined by combustion analysis with thermal conductivity detection.

 

Comments 11: 2.1.3. Microorganism. L146-148: It is unclear whether such procedure was carried out to preparation of inoculate, but then why store and freeze it? Commonly, the freshly prepared spore suspension is used as inoculate.

Response: We appreciate the reviewer’s observation. To clarify this point, the expression “(for later use in inoculation)” was added at the end of the sentence. This indicates that the spore suspension was prepared and stored under appropriate conditions specifically for subsequent inoculation procedures, ensuring culture reproducibility across experiments.

 

Comments 12: 2.2.1. Preliminary growth medium composition and cultivation. Table 1: If the buffer is indicated as one of constituents of the media, then its content must be indicated in relative volumetric ratio. In the presented view it is necessary to indicate the content of individual salts. The similar remark regards the presentation of concentration for coffee cake extract.

Response: The following paragraph was added to the section 2.2.1: “Potassium phosphate buffer was prepared by mixing 9.55 g of KH2PO4 with 0.24 g of K2HPO4, dissolving it in 800 mL of distilled water, and adjusting the pH to 5.5 with KOH or HCl. The final solution was brought to a volume of 1 L using a volumetric flask.”.

 

Comments 13: Justify and explain within the main text the necessity of Al₂O₃ supplement in nutritional medium.

Response: The following sentence was added: “Aluminum oxide (Al₂O₃) was used to prevent excessive growth of filamentous fungi, acting to reduce the size of fungal pellets and, consequently, helping to control hyphal development and minimize cell leakage [39].”

 

Comments 14: 2.2.3. Statistical experimental design for mannanases production. Add more detail description of used PBD and CCRD with refs.

Response: The following paragraph was added to section 3.1.4, expanding the methodological description and including references: “A Plackett–Burman (PB) design was employed to evaluate the influence of six independent variables and identify those with significant effects on mannanase production. Subsequently, a Central Composite Rotatable Design (CCRD) was applied to optimize the significant variables, enabling a more detailed evaluation of their individual and interactive effects through a full factorial approach. The statistical analyses were performed using response surface methodology (RSM) and analysis of variance (ANOVA), following established procedures for bioprocess optimization [56,57].”

 

Comments 15: 2.2.4. Determination of β-mannosidase activity. In logical terms, this section should be combined in a single section Enzymatic assays as sub-section.

Response: We appreciate the reviewer’s suggestion. However, the determination of β-mannosidase activity was performed only after establishing the optimal conditions for mannanase production, as this enzyme was not the primary target throughout the study. We believe that combining the two methodologies into one section might lead the reader to believe that both will be determined from the initial experiments. To clarify this point, the following sentence was added before the description of the β-mannosidase assay: “The specific production of β-mannosidases was evaluated in the culture medium optimized for mannanase production after 3, 5, and 7 days of cultivation.”.

 

Comments 16: 2.3.1. Enzymatic hydrolysis. L200: Crude enzyme preparation should be characterized by enzymatic activities levels and composition and protein concentration. Specify the process (conditions) where the preparation was prepared (or ref to previous sections).

Response: The enzymatic activity of mannanases in the crude enzyme preparation was used to calculate the volume of enzyme to be used in hydrolysis, to obtain 15 U per gram of dry biomass. The following sentence is found in the text: “Enzymatic hydrolysis was performed with 12% (w/v) dry biomass, enzymatic activity of 15 U.g^-1 of dry biomass, and 100 mM sodium citrate buffer (pH 4.8) to complete 10 mL medium.”

 

Comments 17: L202: The pre-treatment of coffee beans for hydrolysis process should be described.

Response: No pre-treatment was applied to the coffee beans prior to enzymatic hydrolysis. The following text was added to topic 2.1.1: “For enzymatic hydrolysis, the coffee beans were ground using a Pulverisette 19 knife mill (Fritsch, USA) equipped with a 1mm sieve.”.

 

Comments 18: L205: “after 6, 24 and 48 h” – Why the withdrawal was carried out in these time points?

Response: Aliquots were collected during enzymatic hydrolysis at 6, 24, and 48h, following the time intervals adopted in previous studies by our research group (Moro et al., 2017; Fasheun et al., 2022; Guimarães et al., 2025). The 6h starting point was selected because it represents sufficient time for the effective onset of polysaccharide hydrolysis, ensuring reproducible and consistent results. The 48h endpoint was established to represent a long yet practical reaction duration, since extending hydrolysis beyond this period may not be economically feasible in industrial applications.

Moro et al. (2017) - https://doi.org/10.1016/j.indcrop.2016.12.051; Fasheun et al. (2022) - https://doi.org/10.1016/j.carbpol.2022.119256; Guimarães et al. (2025) - https://doi.org/10.1007/s12155-025-10856-6.

 

Comments 19: L207-210: The analysis of oligosaccharides would be informative supplement for assessment of hydrolysis efficiency. The indicated column allows to fractionating di-, tri- and tetra-saccharides.

Response: We agree that the analysis of oligosaccharides could provide additional insight into hydrolysis efficiency. However, in the present study, mannose was considered the primary indicator of hydrolysis efficiency, as it represents the major monomeric product of mannan degradation. Nevertheless, preliminary analyses using thin-layer chromatography (TLC) (data not shown) indicate that the enzymatic hydrolysis produces a very high proportion of mannose, while the formation of mannanoligosaccharides (β-MOS) is comparatively very low. Regarding the HPLC analyses, no peaks corresponding to mannobiose were observed. The major and significant sugar is mannose. Additionally, the focus of the present research was to demonstrate the feasibility and hydrolytic potential of the enzyme produced using a coffee residue, corresponding to the early stages of technological maturity (TRL 1–4).

As mentioned before, the possible presence of β-MOS (very low concentration) is acknowledged and is currently being investigated in a complementary study conducted in our laboratory. The objective of this other project is to find the best conditions to increase B-MOS production and reduce the amount of mannose. The identification and quantification of β-MOS is not trivial. It requires time, a study of kinetics and enzymatic formulations, as well as resources for chromatographic columns and specific standards. For these reasons, oligosaccharide analysis was not included in the current assessment of hydrolysis efficiency.

 

 

Comments 20: L214: Why fructose was used as additional standard in HPLC-RI analysis? Or it is one of constituents of coffee beans’ glucans? In this case the estimation of efficiency of enzymatic hydrolysis is questionable.

Response: Fructose was used as an additional standard in the HPLC-RI analysis, since it coelutes with mannose in the column used. Coffee beans contain sucrose at very low concentration, so the hydrolysates may contain low concentration of fructose, as A. niger  also produce an enzyme that catalysis the hydrolysis of sucrose into glucose and fructose. However, the mannose from mannan hydrolysis is substantially higher than that of fructose; therefore, the quantification obtained can be considered correct.

 

Comments 21: 3.1.1. Influence of carbon source arrangement. L229: “were tested as carbon to” – carbon sources.

Response: The sentence has been changed to “carbon sources.”

 

Comments 22: 3.1.2. Influence of cultivation time. L275: “mediums” – media.

Response: The sentence has been changed.

 

Comments 21: 3.1.3. Preliminary test. The principle difference of this section with data above and Fig. 3 with Figs. 1-2 is unclear.

Response: The results presented in section 3.1.3 was designed to validate and assess the baseline production of mannanases under unoptimized conditions, after evaluation of different coffee substrates and particle sizes, as well as cultivation time. The preliminary test thus serves as an initial screening step, providing context and justification for selecting the conditions evaluated and validated in the experiments shown in Figure 3. We have changed the Section name to “Validation of preliminary tests”.

 

Comments 22: 3.1.4. Statistical experimental design. The comments / explanations for the factors VI_1-5 (Table 5) should be added.

Response: The following explanation has been added to Table 5: “… IV_1-5 - dummy variables, included to complete the design matrix, estimate the experimental error, and allow identification of the real factors that have a significant effect on mannanases production.”.

 

Comments 23: 3.1.5. Determination of β-mannosidase enzyme activity. L406: “Using coffee cake as a carbon source for β-mannosidase production…” – This comment requires the presentation of data of β-mannosidase production in presence of several substrates as in Fig. 1.

Response: The determination of β-mannosidase activity was performed only under the optimal conditions for mannanase production, using coffee press cake as the carbon source. Production of this enzyme was not evaluated for all substrates tested for β-mannanase activity. Nevertheless, the β-mannosidase levels obtained with coffee press cake are comparable to values reported in the literature for specific β-mannosidase studies, supporting the potential of coffee press cake as a viable substrate for producing this enzyme.

 

Comments 24: 3.2. Hydrolysis experiments of coffee beans. L427: “arabinogalactan proteins” – Check the terminology correctness.

Response: The text has been changed to “arabinogalactans”.

 

Comments 25: Fig. 6: the caption “Figure 61” – Correct. “using the optimized enzyme productized in conditions” – Revise.

Response: The sentence has been changed to: “Figure 6. Mannose plus fructose recovery (%) obtained from the enzymatic hydrolysis of healthy and defective coffee beans in 50ºC and 250 rpm; using the optimized enzyme productized.”.

 

Comments 26: The mannose / fructose concentration increased slightly between 6 and 24 h. In this view, additional points (1-3 h, 12 h) would be informative. The control (o h) is also desirable. Decrease of mannose / fructose recovery after 48 h should be commented / explained.

Response: We acknowledge the reviewer’s suggestion. In the present study, the primary objective was to demonstrate the feasibility and hydrolytic potential of the enzyme produced from coffee residue, corresponding to early stages of technological maturity (TRL 1–4) and laboratory-scale optimization. Therefore, only the 6, 24, and 48 h time points were evaluated, as performed for other works from our group (Moro et al., 2017; Fasheun et al., 2022; Guimarães et al., 2025). Looking ahead to this work, we intend to scale up production using bioreactors and optimize other parameters by monitoring mannanase production at shorter time intervals. Thus, more detailed kinetic studies including additional time points will be addressed in future work to refine hydrolysis efficiency assessment.

Moro et al. (2017) - https://doi.org/10.1016/j.indcrop.2016.12.051; Fasheun et al. (2022) - https://doi.org/10.1016/j.carbpol.2022.119256; Guimarães et al. (2025) - https://doi.org/10.1007/s12155-025-10856-6.

Regarding the slight decrease in mannose/fructose recovery observed at 48 h, this may be attributed to minor sugar degradation or adsorption phenomena in the reaction medium during prolonged incubation, which has been reported in similar enzymatic hydrolysis studies, but for lignocellulosic biomass.

 

Comments 27: L439-440: “enzymatic hydrolysis was 24% for healthy beans and 20% for defective beans” – It contradicts to data in Fig. 6 and Table 7.

Response: We acknowledge the error in the original text. There was indeed an error in the text; 20% was changed to 26%. The correct values for enzymatic hydrolysis are 23.68 ± 0.03% for healthy beans and 25.98 ± 0.10% for defective beans after 24 h. The text has been updated accordingly: “… by enzymatic hydrolysis was 23.68 ± 0.03% for healthy beans and 25.98 ± 0.10% for defective beans, in 24h.”.

 

Comments 28: L441: “untreated biomass’ – Again, it is worth note in what form was this raw material used (grinding degree, etc.).

Response: “For enzymatic hydrolysis, the coffee beans were ground in a knife mill with an internal 1.0 mm sieve”. These data were added in section 2.1.1. Sample preparation.

 

Comments 29: L458-459: “good potential for recovery of glucose (76% in healthy beans and 72% in defective beans)” – According to Table 7, glucose recovery was 90% for defective beans;

Response: We thank the reviewer for pointing out this discrepancy. There was an error in the reported glucose content in the table for the enzymatic hydrolysis of defective beans. The value has now been corrected to 3.96 ± 0.39 g/100g, corresponding to a recovery of 72%, consistent with the data in Table 7.

 

Comments 30: L460-462: “integrated strategies should be adopted, including appropriate pretreatments, optimization of hydrolysis parameters (enzyme loading, pH, and temperature)” – Such moderate optimization could be implemented within this study.

Response: We appreciate the reviewer’s suggestion. The focus of the present study was to demonstrate the feasibility of mannanase production using coffee waste as a carbon source. Further studies are currently being conducted by our research group to evaluate the most effective pretreatments for this biomass, as well as to optimize hydrolysis parameters such as enzyme loading, pH, and temperature. These investigations will allow a more comprehensive assessment of hydrolysis efficiency in future work.

 

Conclusions:

Comments 31: L475: “increased enzyme productivity by 42.9%” – specify initial and final substrate and parameters.

Response: The following paragraph was added: “The comparison of enzyme productivity was performed using the same carbon source (coffee press cake). In the non-optimized medium, the composition was: 30 g·L⁻¹ coffee cake, 4 g·L⁻¹ yeast extract, 1 g·L⁻¹ Al₂O₃, 0.5 g·L⁻¹ potassium phosphate buffer (pH 5.5), 0.5 g·L⁻¹ MgSO₄·7H₂O, and 0.05 g·L⁻¹ CaCl₂·2H₂O, resulting in a peak mannanase activity of ~20 U·mL⁻¹ after 7 days. After statistical optimization, the medium composition was sim-plified to 48.78 g·L⁻¹ coffee cake, 4 g·L⁻¹ yeast extract, and 0.25 g·L⁻¹ potassium phosphate buffer (pH 5.5), which increased mannanase productivity to 22.4 ± 0.6 U·mL⁻¹ within 3 days, representing a 42.9% improvement.”

 

Comments 32: L479: “reached only~25%” - Either emphasize that this is a relative value, or present the absolute recovery for a clear understanding.

Response: We have clarified in the text that the reported recovery is relative to the mannose content determined by acid hydrolysis, which was considered 100%. The sentence in the conclusion now reads: “… (using the acid hydrolysis value at 100%)”.

 

Comments 33: L484-485: “capable of capable” – Correct.

Response: The sentence has been corrected in the text.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In the current study, the authors have investigated the potential of coffee press cake as a mannan-rich carbon source for the production of mannanases by submerged fermentation. The study explores, the hydrolytic potential of coffee waste promoting the concepts of integrated waste valorization and circular bioeconomy opportunities. Despite the study being interesting and relevant, the authors fail to highlight how their work is different from the previously published studies. Coffee byproducts have their own applications well documented in literature, using them just as another source of carbon for enzyme production might not bring in novelty to the readership of the journal Processes. Unfortunately, the current version of the manuscript cannot be accepted for publication.

The manuscript that I reviewed significantly lacks scientific novelty in its present version. It is important for the authors to revise their manuscript highlighting how green arabica coffee waste as a source for mananases production is different from any other carbon source. The experimental design and results are in good shape, but these are just repetition of previously published studies where a different carbon source is used for the production of mananases or any other enzyme. Any carbon source could be used for production of enzymes via fermentation, so it is difficult for me to identify if this work in its present form adds any knowledge to the journal.

Further, coffee byproducts have many applications due to the presence of high value bioactives in them so how those bioactives affect the production of enzymes (if they do so) is an important question here. 

I would appreciate if the authors could add to their paper the research gaps that they identified and what they did to fill those research gaps. That will highlight the novelty of their study.

Author Response

We sincerely thank the reviewer for the valuable feedback. We fully understand the concern regarding the novelty of the study and have revised the manuscript to better emphasize the original aspects and contributions.

 

Comments: In the current study, the authors have investigated the potential of coffee press cake as a mannan-rich carbon source for the production of mannanases by submerged fermentation. The study explores, the hydrolytic potential of coffee waste promoting the concepts of integrated waste valorization and circular bioeconomy opportunities. Despite the study being interesting and relevant, the authors fail to highlight how their work is different from the previously published studies. Coffee byproducts have their own applications well documented in literature, using them just as another source of carbon for enzyme production might not bring in novelty to the readership of the journal Processes. Unfortunately, the current version of the manuscript cannot be accepted for publication.

The manuscript that I reviewed significantly lacks scientific novelty in its present version. It is important for the authors to revise their manuscript highlighting how green arabica coffee waste as a source for mananases production is different from any other carbon source. The experimental design and results are in good shape, but these are just repetition of previously published studies where a different carbon source is used for the production of mananases or any other enzyme. Any carbon source could be used for production of enzymes via fermentation, so it is difficult for me to identify if this work in its present form adds any knowledge to the journal.

Further, coffee byproducts have many applications due to the presence of high value bioactives in them so how those bioactives affect the production of enzymes (if they do so) is an important question here.

I would appreciate if the authors could add to their paper the research gaps that they identified and what they did to fill those research gaps. That will highlight the novelty of their study.

Response: The main innovation of this work lies in the first-time use of green Arabica coffee press cake, a mannan-rich coproduct from oil extraction, as a carbon source for the submerged fermentation of Aspergillus niger aimed at mannanase and β-mannosidase production. Unlike other coffee by-products (grounds, husks, or spent extracts) that have been investigated for enzyme production, the press cake remains largely unexplored despite its high mannan content and unique chemical profile, which includes lipids, phenolics, caffeine, and serotonin amides. These compounds can influence fungal metabolism and enzyme induction, and our results demonstrate that A. niger efficiently adapted to this complex substrate, achieving high enzyme yields without inhibition.

We have highlighted this novelty in the introduction, "Thus, this is the first study to report the use of Arabica coffee press cake as a carbon source for the submerged fermentation of Aspergillus niger aimed at mannanase production.". In addition, we have emphasized the amount of defective beans and coffee press cake, which remains unexplored for mannanase, although rich in mannans. We have added: “Among agro-industrial residues, those generated by the coffee industry are particularly noteworthy, given that, according to International Coffee Organization (2023) [7] only 5% of the coffee pulp remains in the final beverage for consumers. Waste from the coffee sector accounts for approximately 40% of the total production volume, consisting of coffee husks, pulp, mucilage, parchment, silverskin, and coffee grounds [8–11]. In addition, other residues can be considered, such as coffee press cake (which represents approximately 90% of the residual biomass from green coffee oil extraction [12]) and defective beans, which represent up to 20% of green coffee production [13].”   and “This by-product is rich in the polysaccharide fraction of green coffee, mainly composed of cellulose, type II arabinogalactans, and galactomannans [22].”

Additionally, the manuscript now clearly highlights that the optimized medium, composed solely of coffee press cake, yeast extract, and phosphate buffer, led to a 42.9% increase in mannanase productivity while reducing cultivation time from seven to three days. This represents a significant advancement toward simplified and cost-effective enzyme production, eliminating the need for inorganic salts or synthetic inducers. Since few commercial sources of mannans are available and those that exist are highly expensive (like LBG - approximately 100g costs R$1,308.00 - Sigma-Aldrich, 2025), it is of great relevance to investigating industrial residues that can serve as alternative substrates to produce these enzymes. This discussion can be found below Figure 1: “The medium containing only LBG, a commercially available galactomannan, resulted in lower mannanases activity compared to media containing coffee cake or green coffee beans. These results indicate that coffee beans are an excellent substrate for mannanases production. Furthermore, since coffee cake is an industrial waste product, its use in the enzyme production process could be highly cost-effective.”.

To further reinforce the originality and applicability of this approach, we explored the use of the produced enzymes to hydrolyze untreated healthy and defective coffee beans, achieving approximately 25% mannose recovery (relative to acid hydrolysis). This integration between enzyme production and biomass valorization demonstrates a circular bioeconomy strategy, in which one coffee residue (press cake) is used to generate enzymes capable of valorizing another (defective beans).

In summary, we have expanded the introduction and discussion sections to explicitly state the research gap and how our study addresses it: (i) Coffee press cake has not been previously reported as a substrate for mannanase production; (ii) The co-production of β-mannanase and β-mannosidase using this residue adds functional value to the enzymatic extract; (iii) The study demonstrates the feasibility of replacing expensive commercial mannans (e.g., locust bean gum) with a low-cost industrial coproduct, significantly reducing process costs; (iv) The approach aligns with sustainability principles, converting low-value residues into high-value bioproducts for industrial and nutritional applications.

Finally, we note that the number of studies on mannanases remains significantly lower than those on other hydrolases, as confirmed by a recent Scopus search (November 2025): (i) Mannanase: 2,226 documents; (ii) Xylanase: 14,308 documents; (iii) Cellulase: 36,928 documents; and (iv) Amylase: 93,070 documents.

This disparity further underscores the relevance of exploring new, sustainable, and low-cost sources for mannanase production. Thus, the lack of information on mannanases can already be identified as a technological bottleneck for the use of mannan-rich biomass in a bioeconomy context.

Considering these aspects, we believe the manuscript now clearly highlights its originality and fits well within the scope of the Special Issue “Enzyme Production Using Industrial and Agricultural By-Products.”

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The work entitled “Valorization of green Arabica coffee coproducts for mannanases production and carbohydrate recovery” is focused on an interesting research topic. It evaluates coffee industrial by-products to yield a highly nutritious material to be used by A. niger to produce mannases, which could be further utilized to hydrolyze other discarded materials (residues, byproducts) for added-value components recovery. The study employs an appropriate methodology, however, some issues have been detected in the presentation of the results and the discussion, thus the following revisions should be addressed prior to publication:

INTRODUCTION:

• Line 50: Specify why these residues are particularly noteworthy.
• Line 55: Please provide any information about the amount of byproducts generated from coffee.
• Line 60: After oil extraction, what is the proportion of residue (coffee press cake) related to the initial material submitted to defatting?

MATERIALS AND METHODS:

• Table 1: Cite the references used for collecting the cultivation media formulation information in the table title.

RESULTS:

• Line 250-251: “The presence of lipids in green coffee beans negatively affects enzymatic activity compared to coffee cake”. And what about the mechanical pressing process itself to extract lipids from the coffee bean as responsible for modifying the structure, and releasing nutritional substances? Only the lack of lipids was the responsible for improving the enzymatic activity? Anything in literature?
• Line 265-266: check the value of the enzymatic activity (15.6 U/mL), does not agree with that reported in the figure (<15).
• Table 4: Please include the statistical analysis to see significant differences.
• Figure 3: These results, specifically for day 3 and 5, seem very different from those represented in figure 2. In the first evaluation, enzyme activity in day 3 and 5 is close to 10 U/mL while in the second evaluation is around 17-21 U/mL, reaching the peak at day 7 in the first evaluation, while in this case it was reached at 5^th
• Line 320-322: “Only assays 13 and 14 presented values below 15 U.mL-1.” Check. Values below 15 U/mL are found for run 8 and 12.
• Table 5: Specify the units for each variable (footnotes). What do VI1 to VI5 mean? Specify units also in Table 6.
• Line 341-342: Check. Highest activity was found also in run 8.
• Figure 6: Check fig. numeration. Include statistical analysis. Check title spell.
• Line 438: “The best mannose yield was obtained after 24 h, as shown in Table 7.” Check reference, this is depicted in figure 6.
• Line 438-440: “The efficiency of mannose recovery by enzymatic hydrolysis was 24% for healthy beans and 20% for defective beans.” At which time? According to figure 6, recovery was aprox. 22-21% (6h), 26-24% (24h) and 23-21% (48h). In all cases, recovery was higher in defective beans compared to healthy ones, also in the results included in table 7 (4.66% for healthy and 4.81% for defective beans).
• Table 7. Specify the conditions of hydrolysis process (time, temperature).
• Line 453: “enzyme cocktail”, specify the enzyme content and temperature.
• Line 459: According to data shown in table 7 (4.97% for enzymatic and 5.52% for acid hydrolysis in defective beans), glucose extraction yield is 90%. Check.
• Line 484-485: Check the spell.

OTHERS:

• Homogenize the format to define the degrees’ values when talking about temperature (ºC).
• Eliminate numbers in the keywords.
• Ensure variety names appear in italics throughout all the manuscript.

 

Author Response

We sincerely appreciate the thorough review and constructive contributions provided. All comments and suggestions were carefully considered and have led to substantial improvements in the writing style, clarity of discussion, and consistency of the results presented. We acknowledge that the comments made contributed substantially to strengthening the scientific rigor of the work, and we are grateful for the attention and dedication during the review process.

 

 

INTRODUCTION:

Comment 1: Line 50: Specify why these residues are particularly noteworthy.

Response: The sentence was completed: “Among agro-industrial residues, those generated by the coffee industry are particularly noteworthy, given that, according to International Coffee Organization (2023) [7] only 5% of the coffee pulp remains in the final beverage for consumers.”. In this way, thousands of tons of waste from the coffee production chain are generated annually, as pointed out in the introduction.

 

Comments 2: Line 55: Please provide any information about the amount of byproducts generated from coffee.

Response: The following text was added to the introduction: "Waste from the coffee sector accounts for approximately 40% of the total production vol-ume, consisting of coffee husks, pulp, mucilage, parchment, silverskin, and coffee grounds [8–11]. In addition, other residues can be considered, such as coffee press cake (which represents approximately 90% of the residual biomass from green coffee oil extraction [12]) and defective beans, which represent up to 20% of green coffee production [13].".

 

Comments 3: Line 60: After oil extraction, what is the proportion of residue (coffee press cake) related to the initial material submitted to defatting?

Response: This point was addressed together with the previous comment, through the addition of the following paragraph to the Introduction: " Waste from the coffee sector accounts for approximately 40% of the total production vol-ume, consisting of coffee husks, pulp, mucilage, parchment, silverskin, and coffee grounds [8–11]. In addition, other residues can be considered, such as coffee press cake (which represents approximately 90% of the residual biomass from green coffee oil extraction [12]) and defective beans, which represent up to 20% of green coffee production [13].”

 

MATERIALS AND METHODS:

Comments 4: Table 1: Cite the references used for collecting the cultivation media formulation information in the table title.

Response: The references for the prospecting were cited in the table title: “Table 1. Initial cultivation media formulated based on scientific prospecting [41–45].”

 

RESULTS:

Comments 5: Line 250-251: “The presence of lipids in green coffee beans negatively affects enzymatic activity compared to coffee cake”. And what about the mechanical pressing process itself to extract lipids from the coffee bean as responsible for modifying the structure, and releasing nutritional substances? Only the lack of lipids was the responsible for improving the enzymatic activity? Anything in literature?

Response: The mechanical pressing process used for lipid extraction from coffee beans may promote slight modifications in the recalcitrant structural of the material, which may facilitate accessibility. However, since mannanases production between the 3^rd and 5^th day of cultivation was very similar, the hypothesis is that the presence of residual lipids and their hydrophobic interactions with the cell wall exert a greater influence on the expression and release of enzymes than any changes in the crystallinity of the substrate. To improve the discussion, the text below Figure 1 was supplemented with: “A hypothesis for this is that the fungus may be using the lipids present in the substrate as a source of carbon, redirecting its metabolism toward the production of enzymes related to lipid degradation, such as lipases, to the detriment of mannanases. Lin et al. (2004) [48] indicate that the presence of oil in copra (a fat-rich matrix) may depress mannanase production.”.

 

Comments 6: Line 265-266: check the value of the enzymatic activity (15.6 U/mL), does not agree with that reported in the figure (<15).

Response: We appreciate the reviewer’s observation. The value was replaced in the text with the correct one (12.44 U/mL), as shown in Figure 2.

 

Comments 7: Table 4: Please include the statistical analysis to see significant differences.

Response: The statistical analysis (Student's t test) to verify significant differences in the elements between the carbon sources is included in Table 4. A table note was added: “The comparison of elementary organic analysis between LBG and coffee cake was performed using  one-way ANOVA. Identical letters, in the same element, indicate no significant difference (p ≤ 0.05) between the carbon sources, according to the Student’s t test.

 

Comments 8: Figure 3: These results, specifically for day 3 and 5, seem very different from those represented in figure 2. In the first evaluation, enzyme activity in day 3 and 5 is close to 10 U/mL while in the second evaluation is around 17-21 U/mL, reaching the peak at day 7 in the first evaluation, while in this case it was reached at 5^th

Response: The results presented in section 3.1.3 was designed to validate and assess the baseline production of mannanases under unoptimized conditions, after evaluation of different coffee substrates and particle sizes, as well as cultivation time. The preliminary test thus serves as an initial screening step, providing context and justification for selecting the conditions evaluated and validated in the experiments shown in Figure 3. We have changed the Section name to “Validation of preliminary tests”.

 

Comments 9: Line 320-322: “Only assays 13 and 14 presented values below 15 U.mL-1.” Check. Values below 15 U/mL are found for run 8 and 12.

Response: Thank you. The number of assays has been corrected in the text: “Only assays 8 and 12 presented values below 15 U.mL^-1.”

 

Comments 10: Table 5: Specify the units for each variable (footnotes). What do VI1 to VI5 mean? Specify units also in Table 6.

Response: We thank the reviewer for observation. The matrices presented in Tables 5 and 6 were shown in their coded level format (-1, 0 and +1) and therefore do not include units. The corresponding concentrations and units are provided in Table 2 and 3. The following explanation has been added to Table 5: “… IV_1-5 - - dummy variables, included to complete the design matrix, estimate the experimental error, and allow identification of the real factors that have a significant effect on mannanases production.”.

 

Comments 11: Line 341-342: Check. Highest activity was found also in run 8.

Response: We appreciate the reviewer’s observation. Run 8 has been included in the text, and the sentence has been revised to read as follows: “The highest mannanases activities were obtained in assays 8, 9 and 10, of which two tests correspond to the central points of the design.”.

 

Comments 12: Figure 6: Check fig. numeration. Include statistical analysis. Check title spell.

Response: The figure numbering and title have been corrected as follows:

“Figure 6. Mannose plus fructose recovery (%) obtained from the enzymatic hydrolysis of healthy and defective coffee beans at 50 °C and 250 rpm, using the optimized enzyme product.”. The standard deviation is already shown in the figure, and the statistical tests have been added to the footnotes in Figure 6: “The comparison of mannose+fructose recovery between cultivation days and between biomass was performed using a one-way ANOVA. Identical uppercase letters indicate no significant difference (p ≤ 0.05) between hydrolysis time, in the same biomass, for mannose recovery, according to the Tukey’s test. Identical lowercase letters indicate no significant difference (p ≤ 0.05) between biomass, in the same hydrolysis time, for mannose recovery, according to the Student’s t test.”.

 

Comments 13: Line 438: “The best mannose yield was obtained after 24 h, as shown in Table 7.” Check reference, this is depicted in figure 6.

Response: The reference has been corrected, and Table 7 has been replaced by Figure 6 in the sentence.

 

Comments 14: Line 438-440: “The efficiency of mannose recovery by enzymatic hydrolysis was 24% for healthy beans and 20% for defective beans.” At which time? According to figure 6, recovery was aprox. 22-21% (6h), 26-24% (24h) and 23-21% (48h). In all cases, recovery was higher in defective beans compared to healthy ones, also in the results included in table 7 (4.66% for healthy and 4.81% for defective beans).

Response: There was indeed an error in the text; 20% was changed to 26%. The values and corresponding time have been corrected in the text. The revised sentence now reads: “… by enzymatic hydrolysis was 23.68 ± 0.03% for healthy beans and 25.98 ± 0.10% for defective beans, in 24h of reaction.”.

 

Comments 15: Table 7. Specify the conditions of hydrolysis process (time, temperature).

Response: The following sentence was added to Table 7: “Enzymatic hydrolysis was performed at 50°C and 200 rpm.”

 

Comments 16: Line 453: “enzyme cocktail”, specify the enzyme content and temperature.

Response: The following sentence was added: “… and an enzyme cocktail (cellulase - 17.74 mg.g^-1 of dry biomass; pectinase – 16 mg.g^-1 of dry biomass) for 24 h …”.

 

Comments 17: Line 459: According to data shown in table 7 (4.97% for enzymatic and 5.52% for acid hydrolysis in defective beans), glucose extraction yield is 90%. Check.

Response: Thank you for your great attention to the numbers. There was indeed an error in the glucose value for the enzymatic hydrolysis of defective beans in Table 7. The correct value is 3.96 ± 0.39%, resulting in a recovery yield of 72%.

 

Comments 18: Line 484-485: Check the spell.

Response: The term has been corrected.

 

OTHERS:

Comments 19: Homogenize the format to define the degrees’ values when talking about temperature (ºC).

Response: The degree symbol (°) has been standardized throughout the manuscript to ensure consistency in all temperature values (°C).

 

Comments 20: Eliminate numbers in the keywords.

Response: The numbers have been deleted in the keywords.

 

Comments 21: Ensure variety names appear in italics throughout all the manuscript.

Response: The scientific names have been carefully reviewed, and all fungal species are now presented in italics throughout the manuscript. In addition, Coffea arabica L. has been formatted in italics as required.

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

The manuscript presented by Raquel Coldibelli Ribeiro et al., entitled Valorization of green Arabica coffee coproducts for mannanases production and carbohydrate recovery, describes the optimization process for utilizing an industrial residue from the coffee industry to produce the hydrolytic enzyme mannanase, and ultimately to fully valorize this biomass through the production of mannose.

For obvious reasons the specific interest is limited to the place where the coffee is produced in large amounts, however the methodology applied is of general interest.

The scientific study is sound and generally well-executed, but there are several aspects that require further attention and improvement, then requiring a major revision.

Points to be addressed.

Line 56 Use italics for the plant species.
Line 159 Table 1 correct the text using proper chemical nomenclature and graphical conventions.

Line 196 Remove U ml-1

Line 202 The expression unit per gram of dry biomass, as written is quite puzzling, better use biomass, or dry biomass, as subscript.

Line 241 Figure 1. The comparison is performed within the same day, one-way ANOVA, this point must be clarified and use the same color code for bars and Tukey’s test letters. The grayscale figure doesn’t help. Similar suggestion for Figure 2.

Line 261 Figure 2, add the productivity curve, will help the reader to better follow the text below. Same as figure 3.

Line 270. If the peak activity occurred on day 17 at 24.4 U/ml, when was the value of 25.15 U/ml obtained?

Line 345 In Table 6 add the calculated value obtained with equation 2, with the standard error.

Line 357 Equation 2 is written based on the Pareto chart, which shows it as a function of Z1 in both linear and quadratic form. Given this equation, how can Figure 5b be drawn?
Doing the experiments at yeast extract at different concentration see table 6 line 7 and 8 gives different values. According to the graph, the best yield in mannanase is when the yeast extract is between a concentration from 6.7 to 10.7 g/l. This fact must be deeply discussed in the text.
In addition, the results presented in Table 6, Figure 5b, indicate that the surface is relatively flat, with minimal variation. Therefore, accepting an error margin of 10% raises concerns about the usefulness of Equation 2, potentially rendering it useless.

I also suggest that after fitting the full model, term selection should be guided by ANOVA results, p-values, and residual analysis to support robust decisions.

Lines 366 to 369. The authors reported three values: 22.43 ± 0.59 on day 3, 25.63 ± 0.48 on day 5 (Table 6, ID9), and 17.26 ± 0.56 on day 7. I did the t Student test by myself, and I found that between day 3 and day 5  p <0.05, so the null hypothesis must be rejected, which usually states that there is no difference, in simple words the results at day 3 and 5 are different, but in the text is stated the contrary. I calculated using 3 runs, the combined standard error, the t value, the degree of freedom, and finally the p value. This point requires an explanation.

Line 435 Fix the figure number.

Line 438. Add some comments on the reason for decreasing the amount of mannose/fructose recovery after 48 hours.

Author Response

We appreciate the detailed review and valuable suggestions provided. All observations were carefully analyzed, and the recommended changes were incorporated into the manuscript, resulting in significant improvements in the clarity and quality of the text.

 

Points to be addressed.

Comment 1: Line 56: Use italics for the plant species.

Response: The scientific names have been carefully reviewed, and all fungal species are now presented in italics throughout the manuscript. In addition, Coffea arabica L. has been formatted in italics as required.

 

Comments 2: Line 159: Table 1 correct the text using proper chemical nomenclature and graphical conventions.

Response: There was a layout error in the submission. The subscript numbers have been corrected.

 

Comments 3: Line 196: Remove U ml-1

Response: The “mL^-1” has been removed.

 

Comments 4: Line 202: The expression unit per gram of dry biomass, as written is quite puzzling, better use biomass, or dry biomass, as subscript.

Response: For better understanding, the text was modified to: "... enzymatic activity of 15 U.g^-1 of dry biomass".

 

Comments 5: Line 241: Figure 1. The comparison is performed within the same day, one-way ANOVA, this point must be clarified and use the same color code for bars and Tukey’s test letters. The grayscale figure doesn’t help. Similar suggestion for Figure 2.

Response: The figures have been enhanced with color, and the letters indicating statistical significance have been updated accordingly. The title of Figure 1 has been supplemented with: “The comparison of mannanases activity between cultivation days was performed using a one-way ANOVA. Identical letters, in the same day, indicate no significant difference (p ≤ 0.05) between carbon sources for mannanases production, according to the Tukey’s test.”. The title of Figure 2 has been supplemented with: “The comparison of mannanase activity between cultivation days was performed using a one-way ANOVA. Identical letters, in the same carbon source, indicate no significant difference (p ≤ 0.05) between cultivation days for mannanases production, according to the Tukey’s test.”.

 

Comments 6: Line 261: Figure 2, add the productivity curve, will help the reader to better follow the text below. Same as figure 3.

Response: Including productivity in the graph resulted in excessive information, making it difficult to visualize small variations over the growing days. This occurred because productivity values ​​remained close to 1, while enzymatic activity values ​​were significantly higher, reaching up to 25 U.mL⁻¹. If it is necessary to include this data, we can submit it as supplementary material. An example of Figure 2 with the inclusion of productivity is shown below:

 

Comments 7: Line 270: If the peak activity occurred on day 17 at 24.4 U/ml, when was the value of 25.15 U/ml obtained?

Response: The peak value has been corrected in the text, being 25.15 U/mL as shown in Figure 2.

 

Comments 8: Line 345 In Table 6 add the calculated value obtained with equation 2, with the standard error.

Response: Table 6 presents the coded concentration values of coffee press cake and yeast extract, along with their corresponding mannanase activity results. Based on these data, Pareto charts, ANOVA, and response surface analyses were performed. The results obtained from these analyses enabled the development of a mathematical model capable of predicting the mannanase activity corresponding to each combination of coffee press cake and yeast extract concentrations. The resulting equation is presented below Figure 5. The value predicted by the mathematical model under the optimized conditions is described in the paragraph above the equation: “The model predicted a mannanase production (Manprod) of 24.32 U.mL⁻¹.”.

 

Comments 9: Line 357 Equation 2 is written based on the Pareto chart, which shows it as a function of Z1 in both linear and quadratic form. Given this equation, how can Figure 5b be drawn?

Response: For a better understanding of the response surface used, the text has been changed to: “The mathematical model (Equation 2), obtained for Pareto chart, was used even though the variables corresponding to yeast extract (L and Q) were not statistically significant, due to the necessity of a nitrogen source to support fungal growth. Although yeast extract did not have a significant effect on the evaluated response, it is well known to be an essential nutrient source. Therefore, since the central composite rotatable design (CCRD) did not include experiments with zero concentration of this component, the minimum studied value was adopted.”.

 

Comments 10: Doing the experiments at yeast extract at different concentration see table 6 line 7 and 8 gives different values. According to the graph, the best yield in mannanase is when the yeast extract is between a concentration from 6.7 to 10.7 g/l. This fact must be deeply discussed in the text.

In addition, the results presented in Table 6, Figure 5b, indicate that the surface is relatively flat, with minimal variation. Therefore, accepting an error margin of 10% raises concerns about the usefulness of Equation 2, potentially rendering it useless.

I also suggest that after fitting the full model, term selection should be guided by ANOVA results, p-values, and residual analysis to support robust decisions.

Response:  The Central Composite Rotational Design (CCRD) is a statistical tool widely used in process optimization experiments, especially in biotechnology. It allows for the simultaneous evaluation of linear, quadratic, and interaction effects between independent variables on a response, with the goal of determining optimal operating conditions. This type of design consists of three main parts: a full factorial (with coded levels of -1 and +1), axial points (levels 1.41 and +1.41), and repetitions of the central point (level 0), used to estimate the pure error and verify reproducibility, and which allow for model curvature and increase the predictive capacity of the resulting equation. In the present study, 4 repetitions of the central point were used for greater reliability of the results. Thus, the CCRD provided a second-order mathematical model that describes the response surface of the studied system.

The yeast extract did not show a significant effect on mannanase activity within the tested ranges. However, since yeast extract is an essential component for microbial growth and enzyme production (being the main source of nitrogen), it was retained in the mathematical model equation. For experimental validation of the model, the lowest coded concentration value (-1.41), corresponding to 4 g/L of yeast extract, was used, ensuring that the medium formulation continued to support fungal metabolism, even at reduced concentrations. These explanations can be found below Figure 5 and also below Equation 2.

It is important to highlight that, in bioprocesses, an experimental error of up to 10% is widely accepted due to the biological nature of the systems involved. Factors such as cellular variability, small fluctuations in environmental conditions, and differences in microorganism physiology can affect the reproducibility of the results. This variation is even more relevant when dealing with enzymes produced by filamentous fungi, such as mannanases, since these microorganisms have complex morphologies and are sensitive to changes in culture conditions, which can directly impact enzyme production and secretion.

 

Comments 11: Lines 366 to 369. The authors reported three values: 22.43 ± 0.59 on day 3, 25.63 ± 0.48 on day 5 (Table 6, ID9), and 17.26 ± 0.56 on day 7. I did the t Student test by myself, and I found that between day 3 and day 5  p <0.05, so the null hypothesis must be rejected, which usually states that there is no difference, in simple words the results at day 3 and 5 are different, but in the text is stated the contrary. I calculated using 3 runs, the combined standard error, the t value, the degree of freedom, and finally the p value. This point requires an explanation.

Response: The analysis used was Tukey's test because it involved the comparison between 3 different data sets (3, 5, and 7 days). The accepted significance of the test was 95%. The determination of the enzymatic activity of the samples was carried out on the same day and, therefore, with the same associated experimental error. The values ​​used were 22.43 ± 0.59 U/mL for 3 days, 21.85 ± 0.73 U/mL for 5 days and 17.26 ± 0.56 U/mL for 7 days, as described in the text (paragraph below Equation 2). There was no significant difference between the samples from days 3 and 5, while the samples from days 3 and 7 showed a difference, as did those from days 5 and 7.

Adjustment P value: 0.5364 (3-5); 0.0001 (3-7); 0.0003 (5-7).

 

Comments 12: Line 435 Fix the figure number.

Response: The numbers in the figures and tables have been fixed so that there are no changes.

 

Comments 13: Line 438. Add some comments on the reason for decreasing the amount of mannose/fructose recovery after 48 hours.

Response: Regarding the slight decrease in mannose/fructose recovery observed at 48 h, this may be attributed to minor sugar degradation or adsorption phenomena in the reaction medium during prolonged incubation, which has been reported in similar enzymatic hydrolysis studies, but for lignocellulosic biomass.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 In general, the revised article is improved and does not raise any serious concerns. Just one small remark regarding the section Conclusions, as it's possible one of the reviewer’s comments on the previous manuscript was misunderstood. It would be better to remove the detailed list of media compositions from the Conclusions, leaving the most important parameters regarding simplifying the salt composition and reducing the total fermentation time while increasing the mannanase yield by a certain amount of units / percentage.

Author Response

Comment: In general, the revised article is improved and does not raise any serious concerns. Just one small remark regarding the section Conclusions, as it's possible one of the reviewer’s comments on the previous manuscript was misunderstood. It would be better to remove the detailed list of media compositions from the Conclusions, leaving the most important parameters regarding simplifying the salt composition and reducing the total fermentation time while increasing the mannanase yield by a certain amount of units / percentage. 

Response: The conclusion was modified to remove the detailed list of the composition of the culture media, and the sentence was modified to: “The simplified medium composition—based solely on coffee press cake, yeast extract, and phosphate buffer—further enhances economic feasibility by eliminating the need for additional supplements (Al₂O₃, MgSO₄·7H₂O, and CaCl₂·2H₂O).”. 

We thank the reviewer for the final evaluation and for approving the manuscript for publication. We express our gratitude for the time dedicated to the evaluation process and for the contributions offered in the previous stages, which certainly improved the quality of the work. 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have successfully revised the manuscript by incorporating the changes suggested by the reviewers and have also done a good job in highlighting the novelty of their study. The manuscript can now be accepted for publication.

Author Response

Comment: The authors have successfully revised the manuscript by incorporating the changes suggested by the reviewers and have also done a good job in highlighting the novelty of their study. The manuscript can now be accepted for publication 

Response: We would like to express our gratitude to the reviewer for approving the manuscript at this final stage of the editorial process. The considerations and recommendations presented earlier were duly addressed and were essential for improving the work. We therefore thank you for your attention and rigor in the evaluation. 

Reviewer 5 Report

Comments and Suggestions for Authors

The revised manuscript is now suitable to be considered for publication.

Author Response

Comment: The revised manuscript is now suitable to be considered for publication. 

Response: We express our sincere gratitude to the reviewer for the final analysis and for accepting the article for publication. We acknowledge the relevance of the observations presented in the previous stages of the review, which were fully incorporated and contributed significantly to strengthening the study.