Enterococcus faecalis-Induced Biochemical Transformation during Fermentation of Underutilized Solenostemon monostachyus Leaves
, Eze Frank Ahuekwe 2, Precious Amaneshi Garuba 1, Aderinsola Jumai Adigun 1, Oluwatofunmi E. Odutayo 1 and Alaba Oladipupo Adeyemi 1
Round 1
Reviewer 1 Report
Afolabi et al. have reported the assessment of the biochemical impacts of fermenting a probiotic Enterococcus faecalis on promoting the edible properties of the leaves from S. monostachyus.
The α- amylase activity, proteins, carbohydrates, and ash, iron and copper contents significantly increased while fats, crude fibre, cadmium and manganese contents of the fermented S. monostachyus leaves were significantly reduced.
Due to the importance of the research the potential application of this, I could imagine to support the publication of the manuscript. However, in the current form the manuscript has several weak points that need to be addressed prior publication.
In my opinion, the following major and minor changes should be addresses prior publication:
1. Methodology: Section 2.4.1. The preparation of the plant leaves for fermentation is not clear. Authors should modify the details for a complete understanding of the readers.
2. Results; Section 3.1: The plausible reasons for alkalinity following the fermentation should be discussed in detail.
3. Tables: The full names for acronyms such as SM and WL should be mentioned in the footnotes
4. I am a bit confused why the authors used unfermented T. fruticosum as a control rather than the same unfermented plant e.g. S. monostachyus.
5. Figure 1: Why the deviation in the enzyme activities is so high?
6. Manuscript should be thoroughly checked for typos.
Author Response
Dear Reviewer,
We have attended to the comments raised. The comments were indeed extensive, beneficial, constructive, and have immensely improved the quality and understanding of the manuscript. Authors appreciate the efficient and fast review process. We have uploaded the the revised manuscript for your kind consideration. Kindly find below our point-by-point response to each of the comments.
Yours faithfully,
Reviewer 1-Round 1
- Methodology: Section 2.4.1. The preparation of the plant leaves for fermentation is not clear. Authors should modify the details for a complete understanding of the readers.
Response: The description of the preparation was revised to improve understanding as suggested.
- Results; Section 3.1: The plausible reasons for alkalinity following the fermentation should be discussed in detail.
Response: Ammonia and hydroxyl compound production reduces the pH of the fermenting medium to the alkaline region. The requested detail was included in lines 386-389 of the earlier submitted manuscript, and presently in lines 359-360 revised manuscript. A detailed process for biotransformation that generated those compounds has been incorporated as a scheme in the manuscript.
- Tables: The full names for acronyms such as SM and WL should be mentioned in the footnotes
Response: The acronyms had been defined as suggested.
- I am a bit confused why the authors used unfermented T. fruticosum as a control rather than the same unfermented plant e.g. S. monostachyus.
Response: The suggested unfermented leaves of S. monostachyus was also used as the control in the study. We adopted two controls for the study. The use of the unfermented T. fruticosum was to ascertain that the biochemical parameters for the experimental leaves (S. monostachyus) are edible, especially when they are close to those of the already consumed T. fruticosum plant. The relevant statement in lines 16-17 (for abstract), and lines 114-115 was revised for better understanding.
- Figure 1: Why the deviation in the enzyme activities is so high?
Response: A highlighted part of the following reason provide has been incorporated in lines 566-579 of the revised manuscript. The deviation for the almost perfect α- amylase activities in figure 2 went through the same environmental condition, number of replications, spectrophotometer, and isolated from the same homogenate as the lactate dehydrogenase where the slightly high deviation in figure 1 was noticed. Lactate dehydrogenase is a broad-spectrum enzyme that is influenced by numerous factors like pH, and types of phytochemicals (such as 2-hydroxybutanoic acid, 2-hydroxypentanoic acid, 3-phenyllactic acid, cyclopropaneglycodic acid) that are constantly changing during fermentation (McDonald and Keith F. Tipton, 2022; Mahn Joo. Kim and George M. Whitesides, 1988; Rohit Sharma, Bhawna Diwan, Brij Pal Singh & Saurabh Kulshrestha, 2022). The erratic-low preference of the organisms to engage the LDH enzymes compare to the alpha-amylase enzyme under the fermenting conditions used for this experimentation could also generate the indicated in the high deviations in Figure 1 (Peter K. Robinson, 2015). Such a wide spectrum in behaviour influence LDH activity leading to slightly higher erratic behaviour leading to error deviation (Hans Bisswanger, 2014; S Chakraborty et al., 2012; Juan Huang et al., 2021; Robert K Scopes, 2002; Peter K. Robinson, 2015). PLEASE NOTE THAT THE STANDARD DEVIATION WAS BETTER FOR LDH ENZYME AND IT WAS THE PREFERRED ENZYME BY THE SAME ORGANISM WHEN ANOTHER PLANT LEAVES WAS USED AS SOURCE OF PHYTOCHEMICALS DURING ANOTHER OF OUR EXPERIMENTATION (Unpublish report).
- Manuscript should be thoroughly checked for typos.
Response: The grammar quality has been critically improved as suggested.
Reviewer 2 Report
This article is about describing changes in leaves of a local African plant during fermentation by E. faecalis. The authors cultivated the bacteria on leaves of the plant for 5 days and the analysis was carried out to determine various chemicals in extract and in fermented leaves.
1. Although analysis is extensive, yet the experimental design lacks the rigor. The selection of the plant under investigation and control is not stated. Also, why this particular organism was used? Any previous study, or a link with the constituent of this plant should work. Since the authors have suggested the fermented food for human consumption so it would be better if the authors could have mentioned about taste and other sensory properties of the fermented leaves. The probable ‘way of consumption’ of the fermented leaves in the context of the local practices would find interest of the readers.
2. Abstract needs improvement to make it easy to follow for a wise readership
3. Lines 51-57 do not differ to a greater extent than the lines 58-65
4. What was the rationale to use water leaf as control?
5. L114: What is meant by ‘prepared fermenter’?
6. Section 2.4.2: State the size of inoculum added. Specify the cultivation conditions. Also mention the approx. size of the chopped leaves
7. Section 2.5.1 can be merged with the previous or subsequent section
8. Section 3.1 This can be attributed to the medium constituent utilized by the organism, such as carbohydrate utilization leads to acid formation and proteinaceous compounds usually end up with alkaline products. Also, link it with the ability of organism as already reported.
9. Table 2: Why some values are given for the day 0 but mentioned as ‘not applicable’ for day 3 or 5?
10. Table 3: State full forms of abbreviations used in this table.
11. Improve the presentation of table 4. The values given in the last two columns are not easy to track
12. Fig. (1 and 2): Include full form of abbreviations. Present units of enzyme in standard form
13. L472: State other examples of ‘food’ where similar situation is encountered and how this compound is removed?
14. L476: Avoid writing incomplete sentences
Author Response
Dear Reviewer,
We have attended to the comments raised. The comments were indeed extensive, beneficial, constructive, and have immensely improved the quality and understanding of the manuscript. Authors appreciate the efficient and fast review process. We have uploaded the the revised manuscript for your kind consideration. Kindly find below our point-by-point response to each of the comments.
Yours faithfully,
Reviewer 2
Comments and Suggestions for Authors
General statement: This article is about describing changes in leaves of a local African plant during fermentation by E. faecalis. The authors cultivated the bacteria on leaves of the plant for 5 days and the analysis was carried out to determine various chemicals in extract and in fermented leaves.
Response: Line 52-54 was revised to reflect the following highlighted statement in the revised manuscript (Lines 63-65) to indicate the benefits of the plant is not limited to Africans alone. The plant has the potential to benefit humans globally since it is also geographically located in Costa Rica, and some other parts of the world (Brenes-Prendas et al., 2013; Afolabi et al., 2012).
- Although analysis is extensive, yet the experimental design lacks the rigor.
a.) The selection of the plant under investigation and control is not stated.
Response: The intention of the reviewer was not very clear, but we suspect the reviewer wants an explanation of why we selected the plant. Therefore, we have included the following statement to improve understanding (Lines 78-83). The leaves of S. monostachyus plant were selected for this study due to the numerous medicinal benefits like antisickling potential, helping to facilitate child-birth and other associated with it. The leaves present low toxicity and may be a useful source of health-beneficial phytochemicals and nutrients when administered and consumed nutritionally after improvements with necessary processing techniques (Olusa and Oyemitan, 2021).
b.) Also, why this particular organism was used? Any previous study, or a link with the constituent of this plant should work.
Response: The E. faecalis was selected for this study since it provides the best probiotic characteristics of all probiotic organisms identified during spontaneous fermentation experimentation on T. catappa seeds (Odutayo et al., 2021). This reason was provided in section 2.3.1, lines 102-105 of the previous manuscript, and can be found in the revised manuscript (Lines 117-120).
c.) Since the authors have suggested the fermented food for human consumption so it would be better if the authors could have mentioned about taste and other sensory properties of the fermented leaves. The probable ‘way of consumption’ of the fermented leaves in the context of the local practices would find interest of the readers.
Response: we have included the following statement in the manuscript (SEE Lines 52-56 of the revised manuscript) as a reason for not determining the taste and sensory parameters in the study. ‘The nutritional quality is the primary reason for consuming leafy vegetables, which are usually bitter and sour in their natural state (Yiming Feng et al., 2018). The taste and sensory properties of vegetables are of secondary importance since they are not often consumed directly. These properties during consumption of leafy vegetables are most usually influenced by those of the spices, and oil added to improve their edible qualities (Juliana R. Fritts et al., 2018; Annie Thomas et al., 2021’.
Abstract needs improvement to make it easy to follow for a wise readership
Response: The grammar quality has been critically improved as suggested, and to improve understanding of the content of the abstract.
Lines 51-57 do not differ to a greater extent than the lines 58-65.
Response: Lines 51-57 (now lines 56-62 in the revised manuscript) described the plant, origin and emphasised it possesses the ability to serve the global community. However, lines 58-65 (now lines 66-73 in the revised manuscript) described the need to address the increasing global food security. The grammar quality has been critically revised to improve coherence and understanding.
- What was the rationale to use water leaf as control?
Response: We adopted two controls for the study. The use of the unfermented T. fruticosum was to ascertain that the biochemical parameters for the experimental leaves (S. monostachyus) are edible, especially when they are close to those of the already consumed T. fruticosum plant. The relevant statement in lines 16-17 (for abstract), and lines 114-115 was revised for better understanding.
- L114: What is meant by ‘prepared fermenter’?
Response: the section consisting of the statements had been revised to improve understanding of the manuscript.
- Section 2.4.2: State the size of inoculum added. Specify the cultivation conditions. Also mention the approx. size of the chopped leaves
Response: The size of the inoculum (2 x 103 CFU/ml) is now included as suggested. The size of chopped leaves was previously stated as 1.0 Kg (Line 121 of the previous manuscript, and now line 139 of the revised manuscript) and the cultivation conditions as 37 â—¦C (Line 123 of the previous manuscript, and now line 141 of the revised manuscript), and the pH in Table 1 as suggested. The grammar quality was also critically improved to improve understanding.
- Section 2.5.1 can be merged with the previous or subsequent section
Response: Section 2.5.1 has been merged with the previous section as stated.
- Section 3.1 This can be attributed to the medium constituent utilized by the organism, such as carbohydrate utilization leads to acid formation and proteinaceous compounds usually end up with alkaline products. Also, link it with the ability of organism as already reported.
Response: A detailed scheme had been provided to illustrate the biotransformation of the phytochemical constituents (Scheme 1). The scheme depicts some of the alkaline products formed during fermentation.
- Table 2: Why some values are given for the day 0 but mentioned as ‘not applicable’ for day 3 or 5?
Response: the highlighted part of the following response was included in lines 273-277 of the revised manuscript for improved quality. The unfermented plant (for water leaf) only served as the control for the experimentation, and the objective of the study does not include studying the effect of the fermentation on the control leaves. The values of the control leaves for day 0 are sufficient for the purpose it serves in the experimentation. Also, the need to extensively appreciate the bioremediation role of the E. faecalis encouraged the extension of the relevant control to day 3 and day 5. The ash content obtained was generally less the 50 % of the corresponding values for the S. monostachyus leaves (Table 1). Hence, the ash samples generated from water leaves were extremely small after fermentation due to the nature of the control leaves.
- Table 3: State full forms of abbreviations used in this table.
Response: The full details of the abbreviations have been included as suggested.
- Improve the presentation of table 4. The values given in the last two columns are not easy to track
Response: The values in the last two columns were now revised for clarity as suggested.
- (1 and 2): Include full form of abbreviations. Present units of enzyme in standard form
Response: the full abbreviations had been incorporated into Fig. 1 and Fig. 2 as suggested. Also, the units of enzymes activities for the two graphs were updated accordingly.
- L472: State other examples of ‘food’ where similar situation is encountered and how this compound is removed?
Response: we have added the following statement to boost the quality of the previous content, which previously exist in lines 324-329 as suggested (lines 495-502): The compound (1,2-dimethylhydrazine) was previously identified in grilled foods, and heat-treated foods-containing preservatives (Mihail Silviu Tudosie et al., 2022). Consumption of gallic and other extracts from plants has proven to alleviate or eliminate the toxic effects since they provide relevant therapeutic phytochemicals (Takefumi Katsuki et al., 2006; Fatemeh Torabi et al., 2015). We suggest processing the leaves of the S. monostachyus with palm oil since it is usually used to reduce poisons and harmful compounds similar to 1,2-dimethylhydrazine. Palm oil is incorporated into the processing of cassava, and the products of cassava (garri) to counter the effects of cyanide toxicity (Paul Chidoka Chikezie and Okey A. Ojiako, 2013; Ihedioha, 2002- 10.1023/a:1021824031799; Uvere, 1999- https://doi.org/10.1023/A:1008053813164).
- L476: Avoid writing incomplete sentences
Response: The incomplete sentence had been deleted as suggested.
Round 2
Reviewer 1 Report
The authors have addressed all the concerns raised by this reviewer. The manuscript can be accepted for publication.
