Microbial Production and Enzymatic Biosynthesis of γ-Aminobutyric Acid (GABA) Using Lactobacillus plantarum FNCC 260 Isolated from Indonesian Fermented Foods

Round 1

Reviewer 1 Report

Manuscript ID: processes-1039480

Title: Microbial production and enzymatic biosynthesis of γ-aminobutyric acid (GABA) using Lactobacillus plantarum FNCC 260 isolated from Indonesian fermented foods

This study presents a newly isolated strain of Lactobacillu plantarum FNCC260 with a high efficiency of γ-aminobutyric acid (GABA) production. It is an interesting study with potential applications, however, a major revision is required before acceptance.

Critical remarks.

1. Present a short state of the art on biological sources of GABA. What bacteria produce GABA;  what plants, maybe animals?

2. Materials and methods.

Section 2.2. Give references to the primers bak 4 and bak11. What size of the PCR product is obtained? There is no information about sequencing.

3. Results and Discussion

Screening of GABA-producing LAB selected two strains of Lactobacillus plantarum FNCC260 and FNC344. For further analysis the former was used. In the Abstract we can read: “The highest yield of GABA production obtained from the fermentation of L. plantarum FNCC 260 was 809.2 mg/l of culture medium after 60 h of cultivation”. What was the GABA production from the fermentation of FNC344 strain? If FNC344 strain is not subjected for further analysis, give a reason, and remove it from the abstract. The abstract suggests that a lower production of GABA was obtained for FNC344 strain but there were no data in the Results section.

Legends to Figures 2, 3, 4 and 5 inform that “Data shown as mean ± SD with SD less than 5%. The experiments were conducted at least as duplicates”. In the case of duplicates it is impossible to calculate SD.

You use expression “at various concentrations”. In fact there were two or three concentration, give values instead of “various concentration”. The interval 0-0.6mM for PLP is also incorrect. Just 0.2 and 0.6 mM. 0 mM is an obvious control.

Section 3.3 lines 294-295 “addition of 0.2 mM and 0.6 mM PLP led to the increase in GABA production, reaching 903.0 mg/L and 945.3 mg/L after 108 h of cultivation, respectively” – the increase in comparison to what values?  

There is a common characteristic observation for Figures 3 and 4 – till the ~70^th hour of cultivation the GABA concentration is comparable independently on the PLP or pyridoxine addition. The differences are observed later, in the control samples the concentration of GABA decreases whereas it shows a stable or increasing tendency if the FNC344 strain is grown in the medium containing  PLP or pyridoxine. This observation is completely ignored by the authors. Why? Discuss this observation/tendency. Statistical analysis should be done to show the differences. Avoid the words significant and significantly without p values.

Figures 3 and 4, give the names: PLP and pyridoxine in the legend to curves in the graph showing concentrations as it was done in Figure 5.

Lines 317 – 320. “It is shown that the addition of 0.1 mM pyridoxine had a better enhancement on GABA production compared to higher concentrations of pyridoxine tested. The addition of 0.1 mM pyridoxine enhanced GABA production reaching 727.5 mg/L after 60 h of cultivation (Figure 4a), which are significantly higher compared to the cultivation without pyridoxine.” Really? I can see that after 60 h the highest concentration of GABA is observed for the control sample.

Section 3.4. Figure 5. No control curve is added. How to compare? How to analyse the effect of MSG? Coming back to Figures 3 and 4, if we assume that the control curve is the same, the same effect (as in Figures 3 and 4) was observed, i.e. stable production of GABA after 60 hours. But the reader or reviewer can not guess.

Line 331, give the full name of MSG and then the abbreviation as for PLP.

There is a rule to give a full name of compounds at the beginning of the manuscript and then to use abbreviations. Improve the manuscript.

Figure 6. “M” and “1” are incorrectly placed in the figure.

Figure 8. There should be control curves, without the enzyme and/or without the substrate.

Author Response

Critical remarks.

1. Present a short state of the art on biological sources of GABA. What bacteria produce GABA;  what plants, maybe animals?

Respond: we already mentioned the biological sources of GABA from LAB in the introduction. Almost plants produce GABA as a self defends to extreme conditions and in animals, GABA is located in the brain.

2. Materials and methods.

Section 2.2. Give references to the primers bak 4 and bak11. What size of the PCR product is obtained? There is no information about sequencing.

Respond:Done, we already add the references for primer bak 4 and 11, the references from our previous publication and the size of the PCR product is already added in the text.

3. Results and Discussion

Screening of GABA-producing LAB selected two strains of Lactobacillus plantarum FNCC260 and FNC344. For further analysis the former was used. In the Abstract we can read: “The highest yield of GABA production obtained from the fermentation of L. plantarum FNCC 260 was 809.2 mg/l of culture medium after 60 h of cultivation”. What was the GABA production from the fermentation of FNC344 strain? If FNC344 strain is not subjected for further analysis, give a reason, and remove it from the abstract. The abstract suggests that a lower production of GABA was obtained for FNC344 strain but there were no data in the Results section.

Respond: Done, we already removed strain FNCC 343 since GABA production from this strain was lower than FNCC 260.

You use expression “at various concentrations”. In fact there were two or three concentration, give values instead of “various concentration”. The interval 0-0.6mM for PLP is also incorrect. Just 0.2 and 0.6 mM. 0 mM is an obvious control.

Respond: Done,we already change the word “various concentration”.

Section 3.3 lines 294-295 “addition of 0.2 mM and 0.6 mM PLP led to the increase in GABA production, reaching 903.0 mg/L and 945.3 mg/L after 108 h of cultivation, respectively” – the increase in comparison to what values?  

Respond: the values were compared with the control (0 mM), since not all species/strains of LAB able to synthesize PLP in their cells. We already mentioned in the text that after 72 h the GABA production without PLP were rapidly decreased

There is a common characteristic observation for Figures 3 and 4 – till the ~70^th hour of cultivation the GABA concentration is comparable independently on the PLP or pyridoxine addition. The differences are observed later, in the control samples the concentration of GABA decreases whereas it shows a stable or increasing tendency if the FNC344 strain is grown in the medium containing PLP or pyridoxine. This observation is completely ignored by the authors. Why? Discuss this observation/tendency. Statistical analysis should be done to show the differences. Avoid the words significant and significantly without p values.

Figures 3 and 4, give the names: PLP and pyridoxine in the legend to curves in the graph showing concentrations as it was done in Figure 5.

Respond: we would like to clarify that we already described in the discussion that PLP/pyridoxine increased GABA production.

We already correct the name of PLP and Pyridoxine in the legend.

Lines 317 – 320. “It is shown that the addition of 0.1 mM pyridoxine had a better enhancement on GABA production compared to higher concentrations of pyridoxine tested. The addition of 0.1 mM pyridoxine enhanced GABA production reaching 727.5 mg/L after 60 h of cultivation (Figure 4a), which are significantly higher compared to the cultivation without pyridoxine.” Really? I can see that after 60 h the highest concentration of GABA is observed for the control sample.

Respond: We are sorry for the incorrect typing of the values, after 60 h (72 h) the GABA production the medium with 0.1 mM pyridoxine was 839 mg/L whereas the GABA production after 60 h was 754 mg/L. we already fixed the values in the text.

Section 3.4. Figure 5. No control curve is added. How to compare? How to analyse the effect of MSG? Coming back to Figures 3 and 4, if we assume that the control curve is the same, the same effect (as in Figures 3 and 4) was observed, i.e. stable production of GABA after 60 hours. But the reader or reviewer can not guess.

Respond: from our previous observation, the strain cannot produce GABA in the absence of MSG in the medium, GABA production can only be triggered by MSG and for that reason we did not add the control curve (0 mM MSG). below the figures 3 and 4 we already mentioned the initial MSG concentration in the medium was 118 mM (equivalent to 2%) and that concentration was not meant to be a control samples (0 mM MSG) as you’re expected.

Line 331, give the full name of MSG and then the abbreviation as for PLP.

Respond: Done, we already give the full name of MSG and the abbreviation of PLP is already mentioned in the text.

There is a rule to give a full name of compounds at the beginning of the manuscript and then to use abbreviations. Improve the manuscript.

Respond: Done, we already check and correct the abbreviations.

Figure 6. “M” and “1” are incorrectly placed in the figure.

Respond: Done, we already fixed the incorrect place

Figure 8. There should be control curves, without the enzyme and/or without the substrate.

Respond: thank you for your suggestion, however our goal is to synthesize GABA enzymatically, it is clear that without enzyme or substrate the reaction will not occur.

 

Reviewer 2 Report

The work presented in this manuscript about the microbial and enzymatic production of GABA using a Lactobacillus plantarum spp isolated by the authors from fermented food, showed a very complete work on the discovery of new GABA-producing spp as well as a good characterization of the implicated enzyme and the activity of this one. The structure of the paper is very logic to follow and the description of the method used very clearly explained. 

In the introduction of the paper, the author mentioned the inconvenience of adding synthetic GABA to food (line 57) but that could be more explained in the text. Moreover, the use of biosynthetic GABA formed by LAB was highlighted by the authors as a promising strategy (line 75 and 76) for the probiotic addition of the bacteria and that it is something that could be more deeply study in future work. Mostly because it seems more advantageous the enzymatic production of GABA in term of yield then the microbial fermentation.

In the results and discussion section, line 231, 232, 233, the authors pointed the production of GABA by two of the isolated strains using 118mM of MSG. Why to use this initial concentration of substrate?

Later when they presented the results of the production by L. plantarum they mentioned a lower production then in the initial screening cause of the use of a different MSG substrate (261 line). In the material and method we can see two different brand for the same compound, but could the authors give an hypothesis of what could affect the difference in the production (purity, type of source, ...)

In the graph where the author presents the production of GABA over time ( as Figure 2) it could be very interesting to see as well the consumption of the MSG to correlate the total intake of the initial substrate with the maximum product formation. 

In the line 297 the author commented the non inhibition of the cell growth by the addition of PLP, there is any paper that already presented impact of the PLP in cell culture growth?

While comparing the use of PLP and Pyridoxine as cofactor, did the author tried the highest concentration used for PLP 0.6mM for the other compound, because they used showed a lower addition of pyridoxine (half of the maximum compared with PLP).

Did they author perform any statistical analysis in the results from the use of different pyridoxine concentration to support an statistical better results with the lowest concentration of the cofactor (0.1mM)? (line 318, 319)

In the line 324 about the possible degradation of the pyridoxine in the culture, could be that characterize somehow during the kinetic of the experiment?

In the line 335, could the maximum yield on GABA production be related somehow with the total consumption of MSG?

At the end of the 3.4 point, the author discussed about the lower yield obtained with 118mM of MSG compared with 100mM for the negative effect of glutamate concentration on LAB bacterial growth. But since they did not report any difference on the bacterial growth using the 2 MSG concentration, could they find another explanation for this results? or could it be try a higher concentration on MSG, maybe 150 mM to confirm that higher MSG affect the bacteria and further the GABA production on the culture?

In the section 3.6 about the production of GABA from recombinant expression of the gene in E. coli. The authors showed a very good increase on the production yield by using the enzyme instead than the fermentation with the LAB. But I would like to have an explanation of this fact. Before in the text while describing the results with the bacterial fermentation, they discussed the possible interaction of other enzymes that degradate GABA over the time. Could be the specific use of ecombinant GAD with no other enzymes the explanation of the increase in GABA production? If yes, that should be more clearly explained in the text as final discussion. 

In the figure 9, the figure description is not clear enough, since the author mentioned a media culture of 200mM of MSG (line 447) when this condition was never presented before in the text. Could that be an error and the concentration is 100mM? 

Author Response

In the introduction of the paper, the author mentioned the inconvenience of adding synthetic GABA to food (line 57) but that could be more explained in the text. Moreover, the use of biosynthetic GABA formed by LAB was highlighted by the authors as a promising strategy (line 75 and 76) for the probiotic addition of the bacteria and that it is something that could be more deeply study in future work. Mostly because it seems more advantageous the enzymatic production of GABA in term of yield then the microbial fermentation.

In the results and discussion section, line 231, 232, 233, the authors pointed the production of GABA by two of the isolated strains using 118mM of MSG. Why to use this initial concentration of substrate?

Respond: the reason is that concentration of 118 mM (equivalent to 2%) of MSG is quite high for some species/strain and also showed toxicity effects to certain species and for that, we want to know the ability of our strain to produce GABA at high MSG concentration and it is shown in Figure 2 that apparently the strain can not produce GABA at higher MSG concentration.

Later when they presented the results of the production by L. plantarum they mentioned a lower production then in the initial screening cause of the use of a different MSG substrate (261 line). In the material and method we can see two different brand for the same compound, but could the authors give an hypothesis of what could affect the difference in the production (purity, type of source, ...)

Respond: the reason is that for screening purposes, utilization of MSG from Ajinomoto is lot cheaper since we screened many isolates from our previous publication, and also different purity might affect GABA production.

In the graph where the author presents the production of GABA over time ( as Figure 2) it could be very interesting to see as well the consumption of the MSG to correlate the total intake of the initial substrate with the maximum product formation. 

Respond: thank you for your suggestion, we did not measure the MSG consumption/MSG residues in the medium. However, we can calculate GABA conversion efficiently using formula:

GABA%= (GABA)-(GABA)0/(MSG)X103.12/169.13X100. based on that formula, the GABA conversion of our strain was only 6.2%, indicating that our strain can not convert high concentration of MSG (118 mM) to GABA efficiently. Possible reason is that GABA production is strain/species dependent and also source of isolation might affect the production, since the source of this strain is from fermented cassava which not contain high glutamate.

In the line 297 the author commented the non inhibition of the cell growth by the addition of PLP, there is any paper that already presented impact of the PLP in cell culture growth?

Respond: yes there are several papers mentioned the effect of PLP on cell growth, doi:10.1016/j.fm.2005.01.002, doi:10.1186/1475-2859-9-85

While comparing the use of PLP and Pyridoxine as cofactor, did the author tried the highest concentration used for PLP 0.6mM for the other compound, because they used showed a lower addition of pyridoxine (half of the maximum compared with PLP).

Respond: We did not try the highest concentration of PLP. However, we did characterize the enzyme and see the effect of PLP on its activity. Apparently, the higher PLP concentration did not increase the enzyme activity. So for that reason, we hypothesize that the higher PLP concentration did not increase GABA production in the cells.

Did they author perform any statistical analysis in the results from the use of different pyridoxine concentration to support an statistical better results with the lowest concentration of the cofactor (0.1mM)? (line 318, 319)

Respond: unfortunately, we did not perform any statistical analysis, because as shown in the figure, there’s a clear differences between concentrations and hence, statistical analysis can be ignored to draw a conclusion.

In the line 324 about the possible degradation of the pyridoxine in the culture, could be that characterize somehow during the kinetic of the experiment?

Respond: unfortunately, we did not measure the degradation of pyridoxine in the culture because our main focus is to determine the effect of pyridoxine on GABA production. However, we did characterized the enzyme and the characterized enzyme will be published in later publication.

In the line 335, could the maximum yield on GABA production be related somehow with the total consumption of MSG?

Respond: yes is related with the consumption of MSG, as already described above, our strain cannot efficiently convert all MSG to GABA.

At the end of the 3.4 point, the author discussed about the lower yield obtained with 118mM of MSG compared with 100mM for the negative effect of glutamate concentration on LAB bacterial growth. But since they did not report any difference on the bacterial growth using the 2 MSG concentration, could they find another explanation for this results? or could it be try a higher concentration on MSG, maybe 150 mM to confirm that higher MSG affect the bacteria and further the GABA production on the culture?

Respond: from the figure it’s shown that the strain cannot produce GABA after 60^th of cultivation at 118 mM MSG. as mentioned above, GABA production is strain/species dependent and also the source of isolation. L. brevis is considered as high GABA-producing LAB and there are already many publications about this species and yet, not many publications reveal the ability of L. plantarum as GABA-producing ability. The sources of isolation also affect GABA production, some study suggest that foodstuffs enriched in glutamate are important isolation sources for high GABA-producing LAB (DOI 10.1007/s00726-010-0582-7). Another explanation is that L. plantarum only carry one gene i.e gadB while L. brevis carry two genes i.e gadR and gadB, this also affect GABA production (doi:10.3390/microorganisms8121923)

In the section 3.6 about the production of GABA from recombinant expression of the gene in E. coli. The authors showed a very good increase on the production yield by using the enzyme instead than the fermentation with the LAB. But I would like to have an explanation of this fact. Before in the text while describing the results with the bacterial fermentation, they discussed the possible interaction of other enzymes that degradate GABA over the time. Could be the specific use of recombinant GAD with no other enzymes the explanation of the increase in GABA production? If yes, that should be more clearly explained in the text as final discussion. 

Respond: during enzymatic conversion there are no other enzymes involved and that explain the increase in GABA production. And those explanation we already described in section 3.4 and we describe it in section 3.6

In the figure 9, the figure description is not clear enough, since the author mentioned a media culture of 200mM of MSG (line 447) when this condition was never presented before in the text. Could that be an error and the concentration is 100mM? 

Respond: we are sorry, we already correct the sentence and we mistyped the sentence.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you for the answers.