Optimization of the Treatment of Beer Lees for Their Use in Sustainable Biomass Production of Lactic Acid Bacteria

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a concise and well written paper that aims to optimise the treatment of beer lees for the production of lactic acid bacteria. Other than a few very slight grammatical corrections (which can be left to the journal), I have absolutely no major comments to make about this paper which is a very good piece of work and should be accepted for publication.

Author Response

Reviewer #1:

This is a concise and well written paper that aims to optimise the treatment of beer lees for the production of lactic acid bacteria. Other than a few very slight grammatical corrections (which can be left to the journal), I have absolutely no major comments to make about this paper which is a very good piece of work and should be accepted for publication.

 

- Answer: We sincerely thank the reviewer for the positive and encouraging feedback. We are glad to know that our work was considered concise, well written, and of good quality. We truly appreciate the reviewer’s kind comments and support for the publication of our manuscript. Minor grammatical issues will be addressed during the final editing process as suggested.

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer Report for "Optimization of the treatment of beer lees for their use in sustainable biomass production of lactic acid bacteria"

This study aimed to optimize the use of beer lees (BL) as a low-cost alternative to yeast extract (YE) for sustainable biomass production of lactic acid bacteria (LAB). The researchers investigated different lysis treatments of BL and evaluated their suitability to support the growth of two LAB strains, Lactiplantibacillus plantarum UNQLp11 and Lacticaseibacillus paracasei UNQLpc10, in a whey permeate (WP)-based medium. Growth kinetics and cell viability were compared with those obtained in MRS broth. The topic is timely and of interest to the microbiology and food biotechnology community, especially regarding the circular economy and sustainable microbial growth media.

The experimental design is robust, and the results are generally well presented and discussed. However, several aspects need clarification or improvement to strengthen the scientific impact and reproducibility of the study. I recommend minor revision before acceptance.

Major Comments

L13–14: The sentence “evaluating its suitability to support the growth of two LAB strains…” would benefit from directly naming the strains evaluated.

L17-19: The abstract is concise but could benefit from including specific quantitative results (e.g., growth rates, protein content) to highlight the key findings more clearly.

L20–23: The impact of Maillard reaction products on LAB growth is mentioned as a potential explanation but is not experimentally demonstrated. Rephrase as a hypothesis or speculation (e.g., “This may be related to the Maillard reaction products formed during autolysis…”).

L49–50: The statement that “no studies have explored the use of beer lees…” is too strong. Consider softening to “few studies have explored…”.

L56-58: Nutritional benefits of beer lees are mentioned without supporting data. Add a reference or typical values (e.g., protein or amino acid content) for better context.

L59-65: The objectives are well stated, but the hypothesis is implicit. Add a concluding sentence such as: “We hypothesized that sonication would be a more efficient and cost-effective cell disruption method than autolysis for supporting LAB growth.”

L86-87: Please provide the full parameters for the sonication treatment (frequency, power in W or amplitude), and whether direct or indirect sonication was used.

L110-111: Whey permeate composition is briefly described. Provide full nutritional breakdown or cite a technical datasheet or previous study for transparency.

L116-119: Why 28 °C? Since LAB often grow at 30-37 °C, justify this choice

L136-137: The number of replicates for biochemical measurements is not mentioned. Add a statement such as “All measurements were performed in triplicate.”

L138-141: Acid hydrolysis for amino acid quantification may degrade some labile amino acids. Acknowledge this limitation in the discussion, especially for amino acids like tryptophan and cysteine.

L159-160: Statistical Analysis (Missing Details). State the number of replicates and software used.

L181-182: (Figure 1A and B) add error bars (±SD) and note statistical significance

L190-194: The discussion of beer lees’ inhibitory effects is interesting. However, quantifying hop acids or performing a simple extraction and inhibition test would significantly enhance this section.

L202–206: Statistical comparisons are mentioned but not fully detailed in the text. Add a summary table of CFU/mL, OD600, standard deviations, and p-values to reinforce clarity.

L210–213: The differential response between the two LAB strains is mentioned but not discussed. Briefly explain the physiological or genetic differences that may explain this behavior.

L233–234: While sonication appears more efficient, comment on its feasibility at industrial scale, including energy and equipment considerations.

L235-236: Discuss why Kjeldahl (total N) and Lowry (protein) values differ: "Kjeldahl includes non-protein nitrogen (e.g., nucleic acids), while Lowry detects only peptides."

L248-249: Table 2: The "difference in sonicated samples" column is confusing. Replace with "% change vs. autolyzed BL" for clarity.

L251–259: The discussion of amino acids could be expanded to explain why higher concentrations of certain amino acids did not correlate with better growth.

L295–296: The association between Maillard reaction product color change and metal ions is speculative; please support with data or cite more specific evidence.

L303–307: Maillard reaction products (MRPs) are suggested to reduce growth, but this is not experimentally tested here. Suggest future experiments with isolated MRPs or antioxidant assays to confirm this mechanism.

L317–320: The conclusion that autolysis reduces growth-promoting potential due to nutrient degradation is reasonable but not directly proven. Rephrase to clarify it as a hypothesis.

Discuss the environmental impact of BL valorization (e.g., carbon footprint reduction) in the conclusion.

The manuscript is suitable for publication after addressing the above points. The study is well-designed, and the findings are significant for sustainable biomass production. With minor revisions, it will make a valuable contribution to the field.

Author Response

Reviewer #2:

Specific comments:

L13–14: The sentence “evaluating its suitability to support the growth of two LAB strains…” would benefit from directly naming the strains evaluated.

- Answer: As recommended, we have modified the sentence in the abstract to include the names of the evaluated strains: Lactiplantibacillus plantarum UNQLp11 and Lacticaseibacillus paracasei UNQLpc10 (line 15).

L17-19: The abstract is concise but could benefit from including specific quantitative results (e.g., growth rates, protein content) to highlight the key findings more clearly.

-Answer: We have revised the abstract to include specific quantitative data that highlight our main findings. We believe these additions improve the clarity and impact of the abstract (lines 17-18 and lines 20-21).

L20–23: The impact of Maillard reaction products on LAB growth is mentioned as a potential explanation but is not experimentally demonstrated. Rephrase as a hypothesis or speculation (e.g., “This may be related to the Maillard reaction products formed during autolysis…”).

-Answer: We appreciate the reviewer’s insightful comment. In response, we have rephrased the sentence to indicate that the impact of Maillard reaction products on LAB growth is a hypothesis rather than a demonstrated fact. The revised sentence now reads: “These changes, which may be related to the formation of Maillard reaction products during the autolysis process, could have negatively affected the nutritional quality of the extract and thus reduced its effectiveness as a bacterial growth promoter” (lines 23-25).

L49–50: The statement that “no studies have explored the use of beer lees…” is too strong. Consider softening to “few studies have explored…”.

-Answer: We have modified the sentence accordingly, replacing “no studies have explored” with “few studies have explored” to reflect a more accurate and balanced statement (line 51).

L56-58: Nutritional benefits of beer lees are mentioned without supporting data. Add a reference or typical values (e.g., protein or amino acid content) for better context.

-Answer: We appreciate the reviewer’s suggestion to provide more context regarding the nutritional composition of beer lees. In response, we have added appropriate references, to strengthen the statement and provide better context for their potential as a nutrient source (lines 60-61).

L59-65: The objectives are well stated, but the hypothesis is implicit. Add a concluding sentence such as: “We hypothesized that sonication would be a more efficient and cost-effective cell disruption method than autolysis for supporting LAB growth.”

-Answer: We thank the reviewer for this valuable suggestion. Following the recommendation, we have added a concluding sentence to explicitly state our working hypothesis: “It was hypothesized that sonication would represent a superior method of cell disruption, both in terms of efficiency and cost-effectiveness, compared to autolysis, for enhancing the growth of lactic acid bacteria (LAB)”. We believe this addition helps clarify the rationale behind our experimental design (lines 64- 66).

L86-87: Please provide the full parameters for the sonication treatment (frequency, power in W or amplitude), and whether direct or indirect sonication was used.

-Answer: We have revised the Materials and Methods section to include the full sonication parameters. Specifically, we clarified that an ultrasonic cleaner (BioBase, Shandong, China) operating at a frequency of 40 kHz and power of 180 W was used. This information has been added to provide better reproducibility of the method (lines 92-93).

L110-111: Whey permeate composition is briefly described. Provide full nutritional breakdown or cite a technical datasheet or previous study for transparency.

-Answer: We thank the reviewer for this observation. The whey permeate used in this study is a commercial product provided by Arla Foods Ingredients S.A. We have included the nutritional information supplied by the manufacturer as supplementary material. This same whey permeate was used in previous studies conducted in our laboratory (Cerdeira et al., 2019; Brizuela et al., 2021).

Table 1S.- Nutritional information of whey permeate

Parameter	Unit
Dry matter (%)	16-20
Lactose (%)	80
Crude protein (%)	3
Fat (%)	0.15
Ash (%)	10.25
Calcium (%)	0.98
Phosphorous (%)	0.82
Sodium (%)	1.2
Magnesium (%)	0.15
Potassium (%)	2.1
ME (Mcal/kg of DM)	3.6

ME = Metabolizable Energy
DM = Dry Matter

L116-119: Why 28 °C? Since LAB often grow at 30-37 °C, justify this choice

-Answer: Thank you for this important question. The optimal growth temperature for lactic acid bacteria (LAB) generally ranges from 20 to 45°C, although it varies depending on the species. Certain LAB strains exhibit optimal growth at 40–45°C, while others prefer temperatures between 30 and 37°C. Our laboratory’s extensive experience in the isolation, identification, and selection of oenological strains has demonstrated that native Argentine LAB strains typically show optimal growth at approximately 28°C (Bravo Ferrada et al., 2011, 2013; Brizuela et al., 2017; Valdes la Hens et al., 2015). Consistent with our findings, at 28°C these oenological strains reach the stationary growth phase in under 48 hours.

L136-137: The number of replicates for biochemical measurements is not mentioned. Add a statement such as “All measurements were performed in triplicate.”

-Answer:  Thank you for your valuable suggestion. We have addressed this by adding a dedicated section in the Methods where we detail the statistical analyses and specify the number of replicates for all biochemical measurements. Specifically, we clarify that all measurements were performed in triplicate. This information can now be found in the revised manuscript under the section titled 2.8 Reproducibility of results and statistical analysis (lines 174-179).

L138-141: Acid hydrolysis for amino acid quantification may degrade some labile amino acids. Acknowledge this limitation in the discussion, especially for amino acids like tryptophan and cysteine.

-Answer: Thank you for your comment. As indicated in lines 296–301, acid hydrolysis used for amino acid analysis can lead to the degradation of labile amino acids. We have also included additional references to support this statement.

L159-160: Statistical Analysis (Missing Details). State the number of replicates and software used.

-Answer: As mentioned above, a proper section discussing the statistical analysis performed was added in the Material and methods section for more clarity (lines 174 - 179).

L181-182: (Figure 1A and B) add error bars (±SD) and note statistical significance

-Answer:  Figure 1 has been revised, and details of the statistical analysis have been added to the Materials and Methods section (lines 126–133). Regarding the kinetics analysis, the Boltzmann model was used, as it provides an appropriate fit for the data.

L190-194: The discussion of beer lees’ inhibitory effects is interesting. However, quantifying hop acids or performing a simple extraction and inhibition test would significantly enhance this section.

-Answer: We appreciate the reviewer’s insightful suggestion. We agree that quantifying hop acids or performing an inhibition assay with extracted compounds would provide a more robust confirmation of their inhibitory role. However, due to current limitations in sample availability and resources, it is not feasible for us to conduct additional experiments at this stage. Nonetheless, we have revised the discussion to more clearly acknowledge the potential involvement of hop acids in the observed inhibitory effect, and to highlight this as an important aspect for future research (lines 222-229). We hope the reviewer finds this contextual clarification acceptable.

L202–206: Statistical comparisons are mentioned but not fully detailed in the text. Add a summary table of CFU/mL, OD600, standard deviations, and p-values to reinforce clarity.

-Answer: Thank you for your suggestion. We have added Table 2, which summarizes the kinetic growth parameters along with the corresponding statistical analyses (lines 213-215). This addition enhances the clarity and completeness of the data presentation.

L210–213: The differential response between the two LAB strains is mentioned but not discussed. Briefly explain the physiological or genetic differences that may explain this behavior.

-Answer: We thank the reviewer for this valuable observation. We have now added a brief discussion to address this point in the revised manuscript. Specifically, Lpb. plantarum UNQLp11 and Lcb. paracasei UNQLpc10 differ in their origin, genomic characteristics, and metabolic profiles. For instance, Lpb. plantarum and Lcb. paracasei are known for its high metabolic versatility and tolerance to different environmental stresses. However, tolerance to various stressors or potential inhibitory compounds—possibly present in the different yeast extract preparations—may be strain-dependent. This point has been clarified in the manuscript (lines 245-249).

L233–234: While sonication appears more efficient, comment on its feasibility at industrial scale, including energy and equipment considerations.

-Answer: We agree that industrial scalability is a key factor when considering sonication as a lysis method. We have now included a brief discussion addressing the feasibility of scaling up sonication, noting current challenges such as energy demands, equipment design, and batch versus continuous operation. This new addition can be found in lines 327-334 of the revised manuscript.

L235-236: Discuss why Kjeldahl (total N) and Lowry (protein) values differ: "Kjeldahl includes non-protein nitrogen (e.g., nucleic acids), while Lowry detects only peptides."

-Answer: We have clarified this point in the revised manuscript. The differences observed between the nitrogen content determined by the Kjeldahl method and protein concentration measured by the Lowry assay can be attributed to the analytical principles of each method. The Kjeldahl method quantifies total nitrogen, which includes nitrogen from proteins as well as non-protein sources such as nucleic acids, amino sugars, and free amino acids. In contrast, the Lowry assay specifically detects peptides and proteins based on their aromatic amino acid content. This clarification has been added to the relevant paragraph (lines 272–279).

L248-249: Table 2: The "difference in sonicated samples" column is confusing. Replace with "% change vs. autolyzed BL" for clarity.

-Answer: The column name was changed as suggested and highlighted in yellow in the manuscript.

L251–259: The discussion of amino acids could be expanded to explain why higher concentrations of certain amino acids did not correlate with better growth.

-Answer: We have expanded the discussion to address this point (lines 302–315). While autolyzed yeast extract contained higher concentrations of amino acids essential for LAB growth, this did not result in improved cell biomass. We now discuss possible explanations, including the reduced bioavailability of amino acids due to Maillard reaction product formation, the degradation of heat-sensitive nutrients during autolysis, and the potential presence of growth-inhibitory compounds. This addition provides a more comprehensive understanding of the complex relationship between nutrient content and microbial growth performance.

L295–296: The association between Maillard reaction product color change and metal ions is speculative; please support with data or cite more specific evidence.

-Answer: We have revised the manuscript to include more specific references that support the role of metal ions in enhancing Maillard reaction progression and influencing pigment formation. Several studies have demonstrated that transition metal ions (e.g., Fe²⁺, Cu²⁺) can catalyze Maillard reactions, accelerating browning and melanoidin formation by promoting oxidative steps. We have modified the sentence accordingly and included appropriate citations (lines 359-363).

L303–307: Maillard reaction products (MRPs) are suggested to reduce growth, but this is not experimentally tested here. Suggest future experiments with isolated MRPs or antioxidant assays to confirm this mechanism.

-Answer: We appreciate the reviewer’s thoughtful suggestion. While we did not directly isolate or quantify Maillard reaction products (MRPs) in this study, we agree that their potential inhibitory effect on LAB growth should be confirmed in future work. Accordingly, we have included a new sentence in the Discussion section (lines 374–379), suggesting future experiments involving the isolation of MRPs and their direct assessment on LAB growth and antioxidant capacity.

L317–320: The conclusion that autolysis reduces growth-promoting potential due to nutrient degradation is reasonable but not directly proven. Rephrase to clarify it as a hypothesis.

-Answer: We thank the reviewer for this valuable observation. We agree that the statement regarding the reduction in growth-promoting potential due to nutrient degradation during autolysis should be presented as a hypothesis rather than a definitive conclusion. We have revised the sentence accordingly to reflect this (lines 389-394).

Discuss the environmental impact of BL valorization (e.g., carbon footprint reduction) in the conclusion.

-Answer: We appreciate the reviewer’s suggestion to include a discussion of the environmental impact of beer lees (BL) valorization. We have added a sentence to the conclusion emphasizing the potential environmental benefits of reusing this brewing by-product, such as reducing organic waste and contributing to a lower carbon footprint (lines 396-398).

The manuscript is suitable for publication after addressing the above points. The study is well-designed, and the findings are significant for sustainable biomass production. With minor revisions, it will make a valuable contribution to the field.

-Answer: We sincerely thank the reviewer for their thoughtful evaluation and encouraging comments. We are pleased to know that the study was considered well-designed and that its findings are viewed as significant for sustainable biomass production. We have carefully addressed all the points raised and implemented the suggested revisions to improve the clarity and scientific rigor of the manuscript. We truly appreciate your feedback, which has helped us strengthen our work and improve its overall quality.