Preservation of Malolactic Starters of Lactiplantibacillus plantarum Strains Obtained by Solid-State Fermentation on Apple Pomace
, Naiquén Elizabeth Flores 1, Manuel Morales 2, Liliana Carmen Semorile 1, Danay Valdes La Hens 1, Adriana Catalina Caballero 2, Barbara Mercedes Bravo-Ferrada 1 and Emma Elizabeth Tymczyszyn 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsI consider this article valuable because it offers an alternative to keeping viable LAB starter cultures used in the winemaking industry for their ability to metabolize malic acid. In addition, a by-product is used. However, some changes need to be made to increase the quality of the manuscript.
L-15: Replace “Organoleptic characteristics” by “sensorial attributes”.
L-24: Remove To this end,
L-54: replace “Thanks” for “due.”
L75-77: Improve writing.
L-106: Correct pH value
L-275: Replace “Given this situation” for “Therefore”,
L-284: Replace ++ for 2+ in Mg and Mn
L-341-359: Reformulate the conclusions according to what was stated in the objective of the work. They must be clearer and more concise.
Comments on the Quality of English Language
Improve English, especially in the conclusion of the manuscript.
Author Response
Dear Editor,
We are now sending you the revised version of the manuscript "Preservation of malolactic starters of Lactiplantibacillus plantarum strains obtained by solid state fermentation on apple pomace". Ref. No.: 3043619, in consonance with the reviewer suggestions.
We are very grateful to the reviewers for their constructive and useful comments. Below are our answers to reviewer’s comments. Changes made to the manuscript are highlighted in yellow in revised manuscript.
Looking forward to hearing from you,
Yours sincerely,
Dr. Elizabeth Tymczyszyn
REVIEWER #1:
I consider this article valuable because it offers an alternative to keeping viable LAB starter cultures used in the winemaking industry for their ability to metabolize malic acid. In addition, a by-product is used. However, some changes need to be made to increase the quality of the manuscript.
L-15: Replace “Organoleptic characteristics” by “sensorial attributes”. DONE
L-24: Remove To this end, DONE
L-54: replace “Thanks” for “due.” DONE
L75-77: Improve writing.:
ANSWER: This sentences were re-written according reviewer suggestions
L-106: Correct pH value. DONE
L-275: Replace “Given this situation” for “Therefore”, DONE
L-284: Replace ++ for 2+ in Mg and Mn DONE
L-341-359: Reformulate the conclusions according to what was stated in the objective of the work. They must be clearer and more concise.
ANSWER: The Conclusion was shortened and re-written for more clarity
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript is comprised of three research items: 1) comparative growth kinetics of two strains of Lactiplantibacillus plantarum on conventional medium (MRS) and on a medium comprised of supplemented (!) apple pomace; 2) comparative survivability of the same strains under different cryo-conservation methods and 3) comparative malate degradation of the previously cryo-conserved cultures.
The results of items 2 and 3 appear sound and are of some interest, in particular item 2 (survivability ~ cryo-conservation methods), however only on a lab scale.
Item 1) on the other hand is flawed in presentation and then in experimental design.
Presentation: The apple pomace medium (AP) is in fact heavily substituted (apple pomace with 1% yeast extract and, additionally, some minerals); this is mentioned in the materials part (and in the discussion) but throughout the manuscript the medium is referred to as “apple pomace”. The experimental design does not allow to make inferences about the suitability of apple pomace the “carry” the growth of LAB, or the suitability of apple pomace to, at least partially, substitute complex (and costly) growth media, nor whether apple pomace had any positive effect on the cryo-preservation.
Consequently, the discussion is in parts suggestive and hypothetical although simple experiments could have been conducted to address precisely these issues (e.g. lines 306-335). For example, freeze drying experiments should have also included LAB grown in MRS (or YE); whether growing LAB on apple pomace (rather in a medium substituted with ~) had any effect on cryo-conservation and the application of re-activated cultures remains unanswered.
The manuscript thus raises more questions than it answers and fails to present conclusive evidence to suggest the growing LAB on apple pomace (in with additional supplements) is in fact a viable alternative, let alone at large (industrial) scale. There was also some evidence one strain struggled in the AP medium.
Design: In the current implementation it is not clear whether, the apple pomace has any effect on LAB growth: LAB growth in the AP medium was compared to MRS. The proper design to investigate suitability of apple pomace would have been: a) compare a medium comprised of apple pomace only to b) 1% yeast extract (YE) + minerals medium and then to c) apple pomace + 1% YE + minerals. Omit the MRS, or, alternatively, substitute apple pomace with MRS instead with YE etc. (or vice versa). LAB growth in the AP medium could be simply due to 1% YE. Since the YE is only 1% and typically would be 5% in a full medium; if the intention was to supplement 4/5 of YE with apple pomace, and thus save 4/5 of YE, it has to be shown that the AP actually has an effect. It remains odd the apple pomace would not (also) be supplemented with MRS, a more typical medium to grow LAB.
In addition, there appears to be quite some difference in growth, response freezing and ability to degrade malic acid between the 2 LAB strains, which is a challenge to the generalization of the findings.
As an additional note there appears to be extensive self-citation which in some cases I deem unnecessary (e.g. lines 68-70; lines 140); apparently a lot of what you’re reporting here, you reported previously. 11 of 27 citations are self-citing.
There were some formal flaws like missing whitespaces and decimal points when values reporting some numerical values; figure captions are missing an indication if error bars are standard deviation or standard error of the mean, etc. please check entire manuscript.
some by-line notes
lines 37-44: Consider re-structuring your narrative: Remove the contradiction between “improves taste” (line 37) and “undesirable results” (line 41). All elements may remain. Emphasize spontaneous MLF (undesirable results more likely) vs. MLF using starters (desirable). Consider including that MLF may be a stylistic choice and may not be desired in some beverages.
Line 47: “aromas” change to aroma active compounds; aroma = the sum of sensory impact of all the aroma active compounds (in a given matrix)
lines 48: Please compare to line 37: Here (line 48) you are much more nuanced.
Line 50: Throughout, please abbreviate Lactiplantibacillus using only a single letter, e.g. L.
Line54: consider removing “thanks to 54 the presence of several enzymatic activities”; that’s implied
Line 64: “from industries” -> production of?
Line 65: missing decimal
Line 68-70: (!) Be more specific, esp. since it’s a self-citation: What’s the relevance here: Yeast extract basically is a full medium; I assume LAB grow fine in YE: Where there compounds from the AP required for growth of LAB; and why wouldn’t the LAB still be able to decarboxylate the malate? So please elaborate so readers won’t have to refer to your paper in order to make sense of this paragraph. You cite this work at a later stage of the manuscript at least once more.
Lines 74-77: no, not “On the other hand”; you’re not contrasting the content of this paragraph to what you wrote before; it’s a completely new idea; try Furthermore; or simply start with “The industrial use […] depends [also] on […].” Also: now you’re citing from food industry
Line 77/78: That’s not a new paragraph: You’re expanding on the previous.
Line 93: oenological; but should be “isolated from wine”; you may omit this altogether
Materials & Methods
Line 106: pH 65 - misses a decimal point
Line 109: remove “by-product”
Line 112: replace “characterization” with composition
Line 116: white space in front of mL
Line 118: change “was” to “were”?
Table 1: kg not Kg; change mgN to simply mg/kg
Table 1: what are the values? Mean +/- standard deviation of mean +/- standard error of the mean; how many replicate measurements and were replicate multiple measurements of the same sample or did you take multiple samples; please state IN the table
Table 1: Are these measurements before or after adding yeast extract etc. (lines 113 – 115)?
Line 140: self citation: If your wine-like medium contained only these few components; do not self-cite, please; if other complex components are were included, and you deem them important for outcome and reproducibility, state them in the MS; else remove the citation.
line 149: “0.9% physiological solution”, which when interpreted literally, translates to 0.9% of a 0.9% sodium chloride solution; please replace physiological solution with 0.9 % sodium chloride solution (or whatever was appropriate) throughout.
Line 180: you’re omitting again the AP medium was supplemented apple pomace
Line 184/184: “Also, UNQLp11 showed a fast adaptation to the AP medium and no significant difference with the control in MRS.“ how do you figure “adaptation”; p11 grows faster than p155 in either medium; if anything p155 struggles in the AP medium; but note that growth rate of p155 appears to be lower in general (compare CFU increase after 24h in MRS)
lines 196/197 that’s materials and methods; remove here; all figures are in log
btw – state if log is natural or log10
figure 1State if bars are standard deviation, standard error of mean and or min/max; with 3 replaces you could (should in my opinion) opt to show all 3 lines per condition.
figure 2: On a log scale, error bars are not symmetric: Since lower error bars are not visible: Please plot the lower half. Also state if bars are standard deviation, standard error of mean and or min/max.
Author Response
Dear Editor,
We are now sending you the revised version of the manuscript "Preservation of malolactic starters of Lactiplantibacillus plantarum strains obtained by solid state fermentation on apple pomace". Ref. No.: 3043619, in consonance with the reviewer suggestions.
We are very grateful to the reviewers for their constructive and useful comments. Below are our answers to reviewer’s comments. Changes made to the manuscript are highlighted in yellow in revised manuscript.
Looking forward to hearing from you,
Yours sincerely,
Dr. Elizabeth Tymczyszyn
REVIEWER #2:
The manuscript is comprised of three research items: 1) comparative growth kinetics of two strains of Lactiplantibacillus plantarum on conventional medium (MRS) and on a medium comprised of supplemented (!) apple pomace; 2) comparative survivability of the same strains under different cryo-conservation methods and 3) comparative malate degradation of the previously cryo-conserved cultures.
The results of items 2 and 3 appear sound and are of some interest, in particular item 2 (survivability ~ cryo-conservation methods), however only on a lab scale.
Item 1) on the other hand is flawed in presentation and then in experimental design.
Presentation: The apple pomace medium (AP) is in fact heavily substituted (apple pomace with 1% yeast extract and, additionally, some minerals); this is mentioned in the materials part (and in the discussion) but throughout the manuscript the medium is referred to as “apple pomace”.
ANSWER: The reviewer is right and all mention of apple pomace was replaced by supplemented apple pomace (sAP).
The experimental design does not allow to make inferences about the suitability of apple pomace the “carry” the growth of LAB, or the suitability of apple pomace to, at least partially, substitute complex (and costly) growth media, nor whether apple pomace had any positive effect on the cryo-preservation.
Consequently, the discussion is in parts suggestive and hypothetical although simple experiments could have been conducted to address precisely these issues (e.g. lines 306-335). For example, freeze drying experiments should have also included LAB grown in MRS (or YE); whether growing LAB on apple pomace (rather in a medium substituted with ~) had any effect on cryo-conservation and the application of re-activated cultures remains unanswered
ANSWER: Is important to consider that Lactic acid bacteria are considered fastidious microorganism and they need a rich medium for growth. An appropriate culture medium for lactobacilli should have sugars (i.e., lactose or glucose) as carbon source, peptides as a nitrogen source, yeast extract as growth factor, magnesium and manganese in optimal concentrations.Yeast extract provides vitamins, amino acids, purines and pyrimidines, Mg2+ and Mn2+ are essential ions for nucleic acids, phospholipids, and ATP synthesis. Tween provides essential fatty acids for LAB lipid membrane synthesis, mainly oleic acid and C19cyc11, which have been related to increase stress tolerance during preservation.(Djeghri-Hocine et al. 2010, Guerrini et al 2022,Remize et al 2005, Chen et al 2022). In previous works, we studied the same strains in the whey permeate and apple pomace based medium, and found that supplementation with both, yeast extract and salts, were essential to obtain high levels of biomass, nearly to the obtained with MRS (86 and 94% for Lp155 and Lp11 respectively). Whereas, the biomass in AP without supplementation was lower than 30 %, AP with YE was 54-56 %. (Cerdeira et al 2019 and 2021). Taking this into account, more information about nutritional requirement of BAL was added in the introduction.
Regarding the cost of supplementation, it is important to note that apple pomace is an abundant by-product, usually AP is driedto produce material suitable for combustion or composted. Also, yeast extract could be obtained at low cost by autolysis of beer lees (by product of brewery’s). Under these conditions, the sAP results an economic way to produce biomass, replacing the commercial medium MRS and adding value to a waste produced from juice industry.
Regarding freeze-drying in MRS, more results in this medium was added as positive control in the revised manuscript.
References
* Guerrini S, Bastianini A, Granchi L, Vincenzini M. Effect of oleic acidon Oenococcus oeni strains and malolactic fermentation in wine. Curr Microbiol. 2002;44(1):5-9.
* Remize F, Augagneur Y, Guilloux-BenatierM, Guzzo J. Effect of nitrogenlimitation and nature of the feed upon Oenococcus oeni metabolism andextracellular protein production. J ApplMicrobiol. 2005;98(3):652-661.
* Yoo, H., Rheem, I., Rheem, S., & Oh, S. (2018). Optimizing medium components for the maximum growth of Lactobacillus plantarum JNU 2116 using response surface methodology. Koreanjournalforfoodscience of animal resources, 38(2), 240.
* Cheng, Z., Yan, X., Wu, J., Weng, P., & Wu, Z. (2022). Effects of freeze drying in complex lyoprotectants on the survival, and membrane fatty acid composition of Lactobacillus plantarum L1 and Lactobacillus fermentum L2. Cryobiology, 105, 1-9.
The manuscript thus raises more questions than it answers and fails to present conclusive evidence to suggest the growing LAB on apple pomace (in with additional supplements) is in fact a viable alternative, let alone at large (industrial) scale. There was also some evidence one strain struggled in the AP medium.
ANSWER: The use of AP supplemented is suitable at industrial scale and it has been scaled up for wine yeast starter cultures (Bravo et al 2019). In this work, the application of alternative production is investigated for enological LAB
Design: In the current implementation it is not clear whether, the apple pomace has any effect on LAB growth: LAB growth in the AP medium was compared to MRS. The proper design to investigate suitability of apple pomace would have been: a) compare a medium comprised of apple pomace only to b) 1% yeast extract (YE) + minerals medium and then to c) apple pomace + 1% YE + minerals. Omit the MRS, or, alternatively, substitute apple pomace with MRS instead with YE etc. (or vice versa). LAB growth in the AP medium could be simply due to 1% YE. Since the YE is only 1% and typically would be 5% in a full medium; if the intention was to supplement 4/5 of YE with apple pomace, and thus save 4/5 of YE, it has to be shown that the AP actually has an effect. It remains odd the apple pomace would not (also) be supplemented with MRS, a more typical medium to grow LAB.
ANSWER: The selection of supplementation is based on the composition of MRS, and different carbon sources (in this case AP) are enriched with other factors, such as yeast extract. As mention above, the supplementation with both, yeast extract and salts, is essential. The supplementation with MRS was not considered because of the high cost of this medium. To clarify these queries, more information about previous work was added in the introduction. Our aim was to add value to apple pomace and optimize the production of dried malolactic starters. The characteristic of semi-solid state of AP difficults the correlation of these results with strains growth in MRS. However, more information about preservation of culture growth in MRS was added in the revised version.
When bacteria are grown in MRS, cells must be acclimated previous to inoculation in wine (Bravo Ferrada et al 2014). The acclimation medium consists in MRS modified by addition of fructose, ethanol and low pH. The acclimation process duplicates the cost and time to obtain malolactic starter cultures. Previous to freeze-drying, cultures have to be centrifugated, washed and then resuspended in acryoprotective agent, such as trehalose. Also,previous to inoculation in wine, dried cultures must be rehydrated in a rich medium (such as MRS) to be more tolerant to the harsh wine condition (Brizuela et al 2021).
For all thesereasons, we considered that the results in the present work represent and advance to produce dried cultures in a sustainable form and low cost, facilitating its use on a large scale. Mainly avoiding centrifugation of AP, acclimation, separation of cells from growing medium and also the addition of buffer to neutralize the pH for improve cell survival after freeze-drying.
References
* Bravo‐Ferrada, B. M., Tymczyszyn, E. E., Gómez‐Zavaglia, A., & Semorile, L. (2014). Effect of acclimation medium on cell viability, membrane integrity and ability to consume malic acid in synthetic wine by oenological Lactobacillus plantarum strains. Journal of applied microbiology, 116(2), 360-367.
* Brizuela, N. S., Arnez-Arancibia, M., Semorile, L., Bravo-Ferrada, B. M., & Tymczyszyn, E. E. (2021). Whey permeate as a substrate for the production of freeze-dried Lactiplantibacillus plantarum to be used as a malolactic starter culture. World Journal of Microbiology and Biotechnology, 37(7), 115.
In addition, there appears to be quite some difference in growth, response freezing and ability to degrade malic acid between the 2 LAB strains, which is a challenge to the generalization of the findings.
ANSWER: The results of both strains growth in MRS and then freeze-dried were added in the supplementary material (also, a figure could be added in the manuscript if reviewer considered it). As can we see, UNQLp11 and UNQL155 showed difference in the tolerance to dehydration and inoculation in the harsh wine conditions, which could be due to the fact that tolerance to stress factors is strain dependent, making it difficult to generalize bacteria response (Brizuela et al 2018).
References
* Brizuela, N. S., Bravo-Ferrada, B. M., Curilén, Y., Delfederico, L., Caballero, A., Semorile, L., ... & Tymczyszyn, E. E. (2018). Advantages of using blend cultures of native L. plantarum and O. oeni strains to induce malolactic fermentation of Patagonian Malbec wine. Frontiers in Microbiology, 9, 2109.
As an additional note there appears to be extensive self-citation which in some cases I deem unnecessary (e.g. lines 68-70; lines 140); apparently a lot of what you’re reporting here, you reported previously. 11 of 27 citations are self-citing.
ANSWER: The reviewer is right, the self citation was reduced. References number 5, 14, 15 and 21 were replaced by others, and ref 17 was deleted.
There were some formal flaws like missing whitespaces and decimal points when values reporting some numerical values; figure captions are missing an indication if error bars are standard deviation or standard error of the mean, etc. please check entire manuscript.
ANSWER: The errors were corrected, and the error bars were indicated in the legend of figures.
some by-line notes
.lines 37-44: Consider re-structuring your narrative: Remove the contradiction between “improves taste” (line 37) and “undesirable results” (line 41). All elements may remain. Emphasize spontaneous MLF (undesirable results more likely) vs. MLF using starters (desirable). Consider including that MLF may be a stylistic choice and may not be desired in some beverages.
ANSWER:The paragraph was modified
Line 47: “aromas” change to aroma active compounds; aroma = the sum of sensory impact of all the aroma active compounds (in a given matrix) DONE
lines 48: Please compare to line 37: Here (line 48) you are much more nuanced. DONE
Line 50: Throughout, please abbreviate Lactiplantibacillus using only a single letter, e.g. L. DONE
Line54: consider removing “thanks to 54 the presence of several enzymatic activities”; that’s implied DONE
Line 64: “from industries” -> production of? DONE
Line 65: missing decimal DONE
Line 68-70: (!) Be more specific, esp. since it’s a self-citation: What’s the relevance here: Yeast extract basically is a full medium; I assume LAB grow fine in YE: Where there compounds from the AP required for growth of LAB; and why wouldn’t the LAB still be able to decarboxylate the malate? So please elaborate so readers won’t have to refer to your paper in order to make sense of this paragraph. You cite this work at a later stage of the manuscript at least once more. DONE
Lines 74-77: no, not “On the other hand”; you’re not contrasting the content of this paragraph to what you wrote before; it’s a completely new idea; try Furthermore; or simply start with “The industrial use […] depends [also] on […].” Also: now you’re citing from food industry DONE
Line 77/78: That’s not a new paragraph: You’re expanding on the previous. DONE
Line 93: oenological; but should be “isolated from wine”; you may omit this altogether
ANSWER: OENOLOGICAL WAS OMITED
Materials & Methods
Line 106: pH 65 - misses a decimal point DONE
Line 109: remove “by-product” DONE
Line 112: replace “characterization” with composition DONE
Line 116: white space in front of mL DONE
Line 118: change “was” to “were”? DONE
Table 1: kg not Kg; change mgN to simply mg/kg DONE
Table 1: what are the values? Mean +/- standard deviation of mean +/- standard error of the mean; how many replicate measurements and were replicate multiple measurements of the same sample or did you take multiple samples; please state IN the table.
ANSWER: Data are presented as mean ±SDand sampling information was added in the table 1
Table 1: Are these measurements before or after adding yeast extract etc. (lines 113 – 115)?.
ANSWER: Table 1 correspond to AP previous supplementation, this was aclared in the revised manuscript.
Line 140: self citation: If your wine-like medium contained only these few components; do not self-cite, please; if other complex components are were included, and you deem them important for outcome and reproducibility, state them in the MS; else remove the citation. DONE
line 149: “0.9% physiological solution”, which when interpreted literally, translates to 0.9% of a 0.9% sodium chloride solution; please replace physiological solution with 0.9 % sodium chloride solution (or whatever was appropriate) throughout. DONE
Line 180: you’re omitting again the AP medium was supplemented apple pomace DONE
Line 184/184: “Also, UNQLp11 showed a fast adaptation to the AP medium and no significant difference with the control in MRS.“ how do you figure “adaptation”; p11 grows faster than p155 in either medium; if anything p155 struggles in the AP medium; but note that growth rate of p155 appears to be lower in general (compare CFU increase after 24h in MRS).
ANSWER: The sentences was corrected according reviewer suggestion
lines 196/197 that’s materials and methods; remove here; all figures are in log DONE
btw – state if log is natural or log10
ANSWER: Log correspond to Log in base 10, this was aclared in M&M of revised version
figure 1State if bars are standard deviation, standard error of mean and or min/max; with 3 replaces you could (should in my opinion) opt to show all 3 lines per condition.
ANSWER: Data are presented as mean ± SD and sampling information was added in legend of figure
figure 2: On a log scale, error bars are not symmetric: Since lower error bars are not visible: Please plot the lower half. Also state if bars are standard deviation, standard error of mean and or min/max.
ANSWER: The results of number of viable cells were expressed as Log10 and then was calculated the mean and SD of this values, for this reason the error bars are simetric.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn this paper the authors the preservation of malolactic starters obtained by solid state fermentation on apple pomace. This is a short and simple study providing however interesting data regarding Lactiplantibacillus plantarum strains viability and survaval to different freeze-drying conditions (with or without osmoprotectant)
Overall the manuscript is well written and easy to read. In the Materials and Methods section, the author should specify how long the strains are kept freezed-dried before being tested for the viability/survival.
The authors should also explain why did the choose to freeze the samples at -20°C rather than at -80°C which could provide more stability of the strains. Cell viabilty and survival are not the only parameters to consider when dealing with production and conservation of starters. The authors should at least mention this point in their conclusion if they have not integrated it into their study.
Author Response
Dear Editor,
We are now sending you the revised version of the manuscript "Preservation of malolactic starters of Lactiplantibacillus plantarum strains obtained by solid state fermentation on apple pomace". Ref. No.: 3043619, in consonance with the reviewer suggestions.
We are very grateful to the reviewers for their constructive and useful comments.Below are our answers to reviewer’s comments. Changes made to the manuscript are highlighted in yellowin the revised manuscript.
Looking forward to hearing from you,
Yours sincerely,
Dr. Elizabeth Tymczyszyn
REVIEWER #3:
In this paper the authors the preservation of malolactic starters obtained by solid state fermentation on apple pomace. This is a short and simple study providing however interesting data regarding Lactiplantibacillus plantarum strains viability and survaval to different freeze-drying conditions (with or without osmoprotectant)
Overall the manuscript is well written and easy to read. In the Materials and Methods section, the author should specify how long the strains are kept freezed-dried before being tested for the viability/survival.
ANSWER: the reviewer is right, more specifications about freeze-dried samples were added in the Material and Methods section.
The authors should also explain why did the choose to freeze the samples at -20°C rather than at -80°C which could provide more stability of the strains.
ANSWER: Preliminary results showed no significant differences between freezing at -80ºC or -20ºC, and considering that freezing at -20 ºC is more accessible at an industrial level, only this temperature was tested.
Cell viabilty and survival are not the only parameters to consider when dealing with production and conservation of starters. The authors should at least mention this point in their conclusion if they have not integrated it into their study.
ANSWER: According to reviewer suggestion, information about other studies necessaries to complement the results of the present manuscript was added in the conclusion.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe revised manuscript improves upon key aspects of the initial version. Throughout, greater detail or a more nuanced account was provided. Formal issues in material and methods section, figure and table captions were remedied.
In its revised form, the manuscript may be accepted for publication as any interested reader may now judge its merits on their own based on the presented content, which is now in sufficiently good shape.
