Enhanced DPPH Radical Scavenging Activity and Enriched γ-Aminobutyric Acid in Mulberry Juice Fermented by the Probiotic Lactobacillus brevis S3

Round 1

Reviewer 1 Report

The article is interesting; however, authors omitted the characterization of all the bioactive compounds modified during the fermentation process, since only GABA was determined. Even though the strain used is known for GABA production, most components of the mulberry beverage were modified, and thus their bioactive potential. Moreover, the selection of the DPPH radical scavenging assay is not clear in the context of the study.

OK

Author Response

Response to Reviewer 1 Comments

Point 1: The article is interesting; however, authors omitted the characterization of all the bioactive compounds modified during the fermentation process, since only GABA was determined. Even though the strain used is known for GABA production, most components of the mulberry beverage were modified, and thus their bioactive potential.

Response 1: Thank you for your professional comment. The purpose of this study was to explore why Lactobacillus sp. strains cannot produce a significant amount of GABA in high-sugar beverages using mulberry juice as the fermentation substrate. The purpose of this study was to explore why Lactobacillus strains cannot produce a significant amount of GABA in high-sugar beverages using mulberry juice as the fermentation substrate. The study primarily focused on examining the functional changes of mulberry juice after fermentation, with the development of functional beverages as a key focus. During this process, we observed changes in the basic bioactive components of mulberry, including polysaccharides, polyphenols, and flavonoids. Specific alterations in the active constituents of mulberry were not addressed. In future related studies, we will concentrate on the specific changes in functional and active components of fermented mulberry juice to explore the correlation between substances and functionality.

Point 2: Moreover, the selection of the DPPH radical scavenging assay is not clear in the context of the study.

Response 2: Thank you for your suggestion. Aging, inflammation, cancer, and other diseases are directly related to free radicals. Free radicals can interact with components in the body, such as fatty acids and proteins, extracting their hydrogen atoms and causing damage to cellular structure and function. The oxidative byproducts and intermediates of free radicals can also harm biomembranes, vitamins, enzymes, proteins, and the functionality of living cells. Therefore, eliminating free radicals is crucial for antioxidant protection. There are various methods available to measure the capability of free radical scavenging, including superoxide anion, hydroxyl radicals, and DPPH. The DPPH analysis method is a simple approach for screening free radical scavenging capacity and evaluating antioxidant activity. In this study, we aimed to highlight the functional properties and investigate the antioxidant activity of fermented mulberry juice, specifically focusing on the clearance of hydroxyl radicals and DPPH, as well as the total reducing power. These parameters are commonly tested in the development of functional beverages [1, 2]. Through our analysis, we observed that the clearance capacity of hydroxyl radicals and DPPH was enhanced in fermented mulberry juice. In particular, the enhancement in DPPH clearance capacity was significant, making it a noteworthy aspect for evaluating the development of functional mulberry juice beverages. We have already included relevant explanations regarding antioxidant activity in the abstract.

1. Jin Y, Wu J, Hu D, Li J, Zhu W, Yuan L, Chen X, Yao J. Gamma-aminobutyric acid-producing Levilactobacillus brevis strains as probiotics in Litchi juice fermentation. Foods 2023, 12,1-20. doi:10.3390/foods12020302.
2. Wang K, Qi J, Jin Y, Li F, Wang J, Xu H. Influence of fruit maturity and lactic fermentation on physicochemical properties, phenolics, volatiles, and sensory of mulberry juice. Food Bioscience 2022, 48. doi:10.1016/j.fbio.2022.101782.

Reviewer 2 Report

The Ms is very good. Well designed and written. I have few comments to be addressed:

1-At section 2.7.6 please show the details for GABA analysis (used HPLC, column used, mobile phase and flow rate……etc)

2-At fig 1) do the authors confirmed that all isolates are LAB? If no, please replace the x -axis title to GABA- producing isolates.

3-L319 and 345 correct the format of strain

4-Enlarge Fig 7 as the data on x axis are not clear

5-please carefully revise all references as all of them are sticky words. Also confirm the formatting of microorganisms.

Author Response

Response to Reviewer 2 Comments

Point 1: At section 2.7.6 please show the details for GABA analysis (used HPLC, column used, mobile phase and flow rate……etc.)

Response 1: Thank you for your suggestion. We have added the detailed detection method for GABA in the revised manuscript. GABA detection used high-performance liquid chromatography (HPLC, LC-10A, Shimadzu, Japan) followed by the derivative using o-phthaldialdehyde. After derivatization, 10 μL of the sample was injected. Chromatography was conducted using a C18 column (SB-C18, 250 mm×4.6 mm, 5 μm, Agilent, USA). Mobile phase A consisted of 4.52 g/L sodium acetate in the aqueous phase, while mobile phase B consisted of 22.6 g/L sodium acetate in the organic phase, with the addition of 40% acetonitrile and 40% methanol. The analysis temperature was set at 40°C. The detection wavelength was 338 nm. The analysis program was as follows: 0 min, the mobile phase B composition was 8%; from 0 to 20 min, it increased to 46.5% B; from 20 to 22 min, it reached 100% B; and it remained at 100% B from 22 to 25 min. From 26 to 30 min, the mobile phase B composition was decreased back to 8%. The total duration of the analysis was 30 min. The standard curve was established by plotting the concentration of GABA against the corresponding peak area. By measuring the peak area of the sample, the GABA concentration in the sample can be calculated using the standard curve. (Line 244-257)

Point 2: At fig 1) do the authors confirmed that all isolates are LAB? If no, please replace the x -axis title to GABA- producing isolates

Response 2: Thank you for your comments. We have revised the x-axis title to GABA-producing isolates for Figure 1.

 

Point 3: L319 and 345 correct the format of strain

Response 3: Thank you for your suggestion. We have modified the format of the bacterial name for L319 to italics. The bacterial name for L345 has been abbreviated and changed to italics. The format of the bacterial names has been standardized in the revised manuscript. (Line 329, 353)

 

 

Point 4: Enlarge Fig 7 as the data on x axis are not clear

Response 4: Thank you for your suggestion. We have made the requested modifications to the formatting of the x-axis titles for Figure 7 and Figure 9 in the revised manuscript.

 

Point 5: please carefully revise all references as all of them are sticky words. Also confirm the formatting of microorganisms

Response 5: Thank you for your professional comments. We have carefully revised the text and format of all references in the revised manuscript. (Line 643-757)

Author Response File: Author Response.pdf

Reviewer 3 Report

The subject of the study is currently very attractive in science. However, the manuscript must be improved. A major revision is needed. The following comments and suggestions should help the authors improve the manuscript.

Suggestions:

-In the introduction section, the authors list the biological effects of mulberry, its active components, probiotics and GABA. Such enumerated effects without a description of why it is important for their research, or providing a mutual link should be avoided. Therefore, the following parts should be improved (L37 Mulberry exhibits various medicinal effects such as... L44 Anthocyanins in mulberry exert various significant effects, including... L53 mulberry is also rich in polyphenols, which effectively prevent atherosclerosis... L84 γ-aminobutyric acid (GABA)... has various functions, such as antioxidant, anxiolytic, antiaging, hepatoprotective and lowering blood pressure.

(It is recommended that authors link these effects, for example- Mulberries exhibit various medicinal effects such as.... which can be related to their dominant anthocyanin content.)

-The name of strain should be added in the title - L brevis S3

-L51 Furthermore, mulberries contain flavonoid compounds such as anthocyanins, anthocyanin glycosides, rutin, quercetin…

The previous few sentences were about anthocyanins in mulberries, no need to mention them again.

-L72 LAB contribute to a variety of physiological functions, with a few exceptions.

It is necessary to further explain what the authors meant by this "with a few exceptions"

-L90 However, the GABA yield of fermented beverages containing GABA was low.

In what samples was the low yield of GABA? This sentence is out of context, authors should check this part.

-L96 metal ions

Name the metal ions

-L110 Lactic acid bacteria

LAB abbreviation should be used

-L134 LAB abbreviation should be used

Please indicate how many isolates you tested, one or more?

Were anaerobic incubation conditions ensured?

- Subsection 2.3. Preparation of mulberry juice should be transfer before 2.6. Fermentation of mulberry juice by lactic acid bacteria

- L174 antibiotic solution,

Name the used antibiotics

L138 The cell counts were recorded.

How? CFU/mL were determined?

L185 into simulated gastric fluid or simulated intestinal fluid at a 10% inoculum volume.

Provide more details.

L200-202

Specifically, 1% (w/v) yeast extract, 0.5% (w/v) peptone, 0.01% (w/v) metal ions (MgSO4, MnSO4 and FeSO4), 1% yeast extract and 0.5% peptone, and 1% yeast extract, 0.5% peptone and 0.01% metal ions were used.

Correct this part, it's repeating itself.

General comments considering Method section: Authors should consider to provide log CFU/mL number of used LAB culture, thus to provide approximal number of used bacteria. The OD number is not informative, unless the authors additionally show a growth curve with a spectrophotometric reading or used the MC Farland standard, in which case I ask the authors to explain how their OD values are so high…

General comments for Results and Discussion section:

-L345 L. brevis writes in italics

-L345 Auto aggregation activity Table 3

Time 2,4,6,8,10,12

In method section it is stated that autoaggregation is measured at every 30 minutes. Please revisit this.

-Please present the results more briefly. Everything can be seen from the figures and from the tables. The authors do not have to describe everything in text. The manuscript will be more readable if the results are shortened.

-Introduce a definition of GABA-producing strains. Considering that authors show in this study their isolate L. brevis S3 belongs to this group, it is important to define GABA-producing strains and to emphasize their benefits.

-Why authors choose to supplemented mulberry juice with peptone, yeast extract and metal ions, detailed explanation should be provided.

L588 It is worth noting that the addition of metal ions led to a decrease in GABA production.

Aren't you aiming for an increase in GABA yield?

Also, if peptone and yeast extract and metal ion combined contribute to increasing the content of GABA, wouldn't it be simpler to choose to make juice from fruits or plants that themselves have a high content of GABA. Or simply add GABA itself. How profitable is it to add all these ingredients?

Author Response

Response to Reviewer 3 Comments

Point 1: In the introduction section, the authors list the biological effects of mulberry, its active components, probiotics and GABA. Such enumerated effects without a description of why it is important for their research, or providing a mutual link should be avoided. Therefore, the following parts should be improved (L37 Mulberry exhibits various medicinal effects such as... L44 Anthocyanins in mulberry exert various significant effects, including... L53 mulberry is also rich in polyphenols, which effectively prevent atherosclerosis... L84 γ-aminobutyric acid (GABA)... has various functions, such as antioxidant, anxiolytic, antiaging, hepatoprotective and lowering blood pressure. (It is recommended that authors link these effects, for example- Mulberries exhibit various medicinal effects such as.... which can be related to their dominant anthocyanin content.)

Response 1: Thank you for your professional comments. We have revised this section as follows: “Mulberry is known to contain more than 150 compounds, including anthocyanins, polyphenols, polysaccharides, and other compounds [2-4]. It prevents cardiovascular diseases, treats chronic obstructive pulmonary diseases, has anti-inflammatory effects, alleviates diarrhea, and improves blood circulation disorders, which can be related to its dominant anthocyanin [5]. Anthocyanins play a crucial role as coloring substances in mulberry, offering characteristics such as high color intensity, water solubility, and safety. Moreover, mulberry anthocyanins can serve as natural antioxidants and natural food colorants, as they offer good stability and are suitable for coloring mildly acidic foods. Preventing atherosclerosis, lowering cholesterol levels, ardiovascular prevention and providing radiation protection are related to polyphenol enrichment [6-12]. Mulberries contain a significant amount of polysaccharides, which serve as important functional factors with high biological activity [3, 13]. Various extraction methods and purification techniques have identified multiple types of polysaccharides in mulberry [3]. Mulberry polysaccharides have been studied for their potential applications in the food and pharmaceutical industries due to their physiological and pathological activities, such as immunoregulation and blood sugar management [14,15]. Mulberries are rich in vitamins A, B, and C, carotenoids, and various amino acids, which are beneficial for anticancer and antiaging activities [9, 16, 17]. These nutritional components make mulberries highly valuable for both nutritional and medicinal benefits, and they hold great potential for further development. γ-Aminobutyric acid (GABA) plays a crucial role as a neurotransmitter in the nervous system and possesses various functions, such as antioxidative, anxiolytic, antiaging, hepatoprotective, and blood pressure-lowering effects [24-27].” (Line 38-57, 77-79)

Point 2: The name of strain should be added in the title - L brevis S3

Response 2: Thank you for your suggestion. We have added strain S3 to the title. The title has been rewritten as “Enhanced DPPH radical scavenging activity and enriched γ-aminobutyric acid in mulberry juice fermented by the probiotic Lactobacillus brevi S3”. (Line 2-4)

Point 3: L51 Furthermore, mulberries contain flavonoid compounds such as anthocyanins, anthocyanin glycosides, rutin, quercetin…The previous few sentences were about anthocyanins in mulberries, no need to mention them again.

Response 3: Thank you for your suggestion. We have deleted this sentence in the revised manuscript.

Point 4: L72 LAB contribute to a variety of physiological functions, with a few exceptions. It is necessary to further explain what the authors meant by this "with a few exceptions"

Response 4: Thank you for your professional comments. Most LAB are probiotic and play an important role in the human gastrointestinal system. However, a few LAB, such as Listeria monocytogenes and Streptococcus pneumoniae, can be harmful to the human body. This study focuses on LAB that have significant probiotic effects. We have rewritten this sentence as “Most LAB contribute to a variety of physiological functions” in the revised manuscript. (Line 65-66)

Point 5: L90 However, the GABA yield of fermented beverages containing GABA was low.In what samples was the low yield of GABA? This sentence is out of context, authors should check this part.

Response 5: Thank you for your suggestion. Compared to using commercially complex culture media to produce GABA, directly fermenting using pure fruit juices such as apple juice, lychee juice, grape juice, and mulberry juice as the culture medium does not yield high levels of GABA in the fruit juice. The GABA yield typically ranges from a few hundred milligrams per liter (mg/L) to a few grams per liter (g/L). We revised this sentence as follows: “Compared to using commercially complex culture media to produce GABA, using pure fruit juices as the culture medium did not yield high levels of GABA in the fruit juice. It is important to note that using pure fruit juice as the sole substrate for lactic acid bacteria cultivation may not promote bacterial growth and synthesis of GABA, resulting in lower GABA production.” (Line 83-87)

Point 6: L96 metal ions:Name the metal ions

Response 6: Thank you for your comments. We have added the detailed names of the metal ion mixture and nitrogen source in the revised manuscript. By adding various nutrient components, including nitrogen sources (yeast extract or peptone) and metal ion mixtures (Mn2+, Fe2+, Mg2+, Na+) in mulberry juice, the cell viability, nutritional composition, antioxidative properties, and GABA production were analyzed. (Line 89-91)

Point 7: L110 Lactic acid bacteria, LAB abbreviation should be used

Response 7: Thank you for your suggestion. We have abbreviated lactic acid bacteria as LAB and standardized the term “lactic acid bacteria” in the revised manuscript. (Line 62, 63, 81, 85, 105, 128, 129, 283, 287, 291, 297, 385, 447, 458, 482, 497, 593)

Point 8: L134 LAB abbreviation should be used. Please indicate how many isolates you tested, one or more? Were anaerobic incubation conditions ensured?

Response 8: Thank you for your suggestion. We have abbreviated these lactic acid bacteria and indicated the number of strains used in this study. LAB (25 strains) stored at -80°C were streaked onto GYP agar plates and incubated at 37°C for 48 h. These strains were cultured on a standard shaking incubator at 200 rpm. These cultures were conducted in aerobic conditions. This primary seed culture was then inoculated at 10% (v/v) into GYP medium and incubated at 37°C and 200 rpm under aerobic conditions for 15 h to obtain the secondary seed culture. The fermentation seed culture was inoculated at 10% into sterilized mulberry juice and incubated at 37°C and 200 rpm under aerobic conditions for 48 h. (Line 129-130, 131-133, 137-138)

Point 9: Subsection 2.3. Preparation of mulberry juice should be transfer before 2.6. Fermentation of mulberry juice by lactic acid bacteria

Response 9: Thank you for your suggestion. To isolate suitable LAB for mulberry fermentation, the selection medium was prepared with pure mulberry juice. The section of 2.3 preparation of mulberry juice should be before the section of 2.4 screening and identification of LAB. To enhance the readability, we transferred the section of 2.6 fermentation of mulberry juice by L. brevis S3 after the section of 2.4. We have rewritten the title of 2.5 Influence of nutritional components on fermentation of L. brevis S3 in mulberry juice. (Line 150-164)

Point 10: L174 antibiotic solution, Name the used antibiotics

Response 10: Thank you for your suggestion. We have added the names and concentrations of the antibiotic solutions in the revised manuscript. The antibiotic solution included erythromycin (40 μg/μL), penicillin (50 μg/μL), rifampicin (4 μg/μL), cefalexin (30 μg/μL), tetracycline (30 μg/μL), chloromycetin (30 μg/μL), vancomycin (30 μg/μL), and kanamycin (30 μg/μL). (Line 183-185)

Point 11: L138 The cell counts were recorded. How? CFU/mL were determined?

Response 11: Thank you for your suggestion. The cell counts were recorded as log₁₀ CFU/mL for the safety evaluation. The cell counts were recorded by CFU/mL in mulberry fermentation juice. We have rewritten these sentences in the revised manuscript. (Line 196, 201-202, 208-209, 221-222)

Point 12: L185 into simulated gastric fluid or simulated intestinal fluid at a 10% inoculum volume.

Provide more details.

Response 12: Thank you for your suggestion. We have added details in sections 2.6.7 and 2.6.8. The cells cultured for 24 h were resuspended in physiological saline to achieve an OD₆₀₀ of 10, resulting in a cell count of approximately 2.0×1010 CFU/mL. A 10% volume ratio of the cell suspension was inoculated into simulated gastric fluid or simulated intestinal fluid. The culture was incubated at 37°C and 200 rpm, and then the cell count was recorded as log10 CFU/mL. Using the same cell culture (OD600=10), a 10% (v/v) inoculation volume was inoculated into MRS medium containing 0%, 0.3%, 0.6%, and 1% (w/v) sodium cholate or 0%, 1%, 2%, and 4% (w/v) salt. (Line 198-202, 204-206)

Point 13: L200-202: Specifically, 1% (w/v) yeast extract, 0.5% (w/v) peptone, 0.01% (w/v) metal ions (MgSO4, MnSO4 and FeSO4), 1% yeast extract and 0.5% peptone, and 1% yeast extract, 0.5% peptone and 0.01% metal ions were used. Correct this part, it's repeating itself.

Response 13: Thank you for your professional suggestion. We have deleted this sentence and revised the part. The strain that was cultured for 24 h was collected by centrifugation at 4°C and 5000 rpm for 5 min. The cells were then washed twice with physiological saline solution and resuspended to achieve an optical density (OD₆₀₀) of 100. The cell counts were 2.1±0.2×10¹¹ CFU/mL. This resulting cell suspension was used as a fermentation starter for mulberry juice fermentation with a 1% (v/v) volume ratio. In the mulberry juice fermentation process, different supplements were added to the mulberry juice. The fermentation starter was inoculated into each of these mulberry juice samples and incubated at 37°C with agitation at 200 rpm for 72 h. Each resulting fermented sample was designated as follows: Y group: mulberry juice supplemented with 1% (w/v) yeast extract, P group: mulberry juice supplemented with 0.5% (w/v) peptone, M group: mulberry juice supplemented with 0.01% (w/v) metal ions (MgSO4, MnSO4 and FeSO4), Y+P group: mulberry juice supplemented with 1% yeast extract and 0.5% peptone, and Y+P+M group: mulberry juice supplemented with 1% yeast extract, 0.5% peptone, and 0.01% metal ions (Line 151-164).

Point 14: General comments considering Method section: Authors should consider to provide log CFU/mL number of used LAB culture, thus to provide approximal number of used bacteria. The OD number is not informative, unless the authors additionally show a growth curve with a spectrophotometric reading or used the MC Farland standard, in which case I ask the authors to explain how their OD values are so high…

Response 14: Thank you for your professional comments. We have provided the cell count in the revised manuscript. The cell counts were 2.5±0.5×10¹⁰ CFU/mL with an OD₆₀₀ of 10 for the safety evaluation. The cell counts were 2.1±0.2×10¹¹ CFU/mL for mulberry fermentation. with an OD₆₀₀ of 100. (Line 153-154, 193, 199)

Point 15: L345 L. brevis writes in italics.

Response 15: Thank you for your suggestion. We have converted “L. brevis” to italics and standardized the formatting of all bacterial names in the revised manuscript (Line 329, 353)

Point 16: L345 Auto aggregation activity Table 3. Time 2,4,6,8,10,12. In method section it is stated that autoaggregation is measured at every 30 minutes. Please revisit this.

Response 16: Thank you for your suggestion. We have revised this sentence in section 2.6.5 Autoaggregation ability. The bacterial cell density in the supernatant was measured every 2 h for 12 h. (Line 188-189)

Point 17: Please present the results more briefly. Everything can be seen from the figures and from the tables. The authors do not have to describe everything in text. The manuscript will be more readable if the results are shortened.

Response 17: Thank you for your professional suggestion. We have shortened and deleted the repeat part from the figures and tables in the results and discussion in the revised manuscript.

Point 18: Introduce a definition of GABA-producing strains. Considering that authors show in this study their isolate L. brevis S3 belongs to this group, it is important to define GABA-producing strains and to emphasize their benefits.

Response 18: Thank you for your suggestion. We have added the definition of the GABA-producing strain. Microorganisms capable of producing GABA are known as GABA-producing strains. GABA-producing strains include LAB, yeast, Escherichia coli, and mold. Among them, LAB are commonly used in the food industry for the fermentation and production of GABA. (Line 284-287)

1. brevis is a common plant-based lactic acid bacterium found in fermented foods such as kimchi and pickles. It has resistance to acid and salt, allowing it to survive in harsh environments. Although it is not naturally present in the human body, L. brevis can reach the intestinal tract when consumed live and produce significant amounts of lactic acid to inhibit the growth of harmful bacteria and regulate the gut environment. L. brevis has been associated with several beneficial effects, including enhancing immune function, promoting healthy skin, reducing cholesterol levels, and suppressing allergic reactions. Therefore, based on the above safety and potential value, L. brevis S3 is an effective probiotic strain. (Line 413-421)

Point 19: Why authors choose to supplemented mulberry juice with peptone, yeast extract and metal ions, detailed explanation should be provided.

Response 19: Thank you for your suggestion. Lactic acid bacteria require a rich nutrient environment for their growth, including carbon sources, organic nitrogen sources, and mineral salts. However, mulberries mainly contain high concentrations of sugars, which cannot provide a rich nutrient environment for the growth and production of GABA by lactic acid bacteria. Yeast extract and peptone are among the most commonly used organic nitrogen sources for cultivating LAB. Additionally, we determined whether the lack of mineral salts affects GABA synthesis in the strain. Commonly found metal ions in LAB culture media include magnesium, iron, manganese, and others. These metal ions play important roles in the growth and metabolism of lactic acid bacteria. The presence of these metal ions provides a favorable growth environment for LAB and plays a significant role in their normal metabolic functions. Therefore, based on the composition of commercial LAB culture medium, including organic nitrogen sources and a mixture of metal ions, this study aims to explore the factors that influence the high production of GABA by LAB in high-sugar fruit juice. This study provides a reference for the future development of GABA-enriched functional fruit juice beverages. We have revised the text as follows: “LAB, as demanding microorganisms, rely on several essential nutrients, including carbon sources, organic nitrogen sources and mineral salts, for their growth and metabolic activities. While mulberry juice naturally contains sugars, amino acids, and minerals that can support the growth of LAB, it lacks a sufficient organic nitrogen source [43]. Additionally, we determined whether the lack of mineral salts affects GABA synthesis in the strain. Therefore, based on the composition of commercial LAB culture medium, including organic nitrogen sources and a mixture of metal ions, we explored the factors that influence the high production of GABA by LAB in high-sugar fruit juice. Yeast extract and peptone are among the most commonly used organic nitrogen sources for cultivating LAB. These metal ions (Mn²⁺, Mg²⁺, and Fe²⁺) are commonly found in LAB culture media. This targeted approach of supplementation is expected to significantly improve the overall performance of LAB in mulberry juice fermentation, leading to enhanced survival ability and ultimately higher GABA production levels.” (Line 422-435)

 

Point 20: L588 It is worth noting that the addition of metal ions led to a decrease in GABA production. Aren't you aiming for an increase in GABA yield?

Response 20: Thank you for your suggestion. Our research aimed to enhance the growth and production of GABA by LAB in mulberry juice through the addition of various nutrient components. We hypothesized that the addition of metal ions would enrich the nutrient composition of the juice. However, our results showed that compared to the control group without metal ion addition, the presence of metal ions did not lead to higher GABA production in mulberry juice. It is possible that the mineral salts naturally present in the mulberry juice already provided sufficient nutrients for the strain’s growth, and the additional metal ions had a negative effect on bacterial growth. This indicates that the addition of metal ions does not increase GABA production in mulberry juice. Therefore, we have revised the sentence to “However, the M group did not increase the GABA content or cell survival in mulberry juice. It was likely that the mineral salts present in mulberry juice already fulfill the growth requirements of the bacterial strains, and extra addition of metal ions might hinder their growth”. (Line 588-591)

Point 21: Also, if peptone and yeast extract and metal ion combined contribute to increasing the content of GABA, wouldn't it be simpler to choose to make juice from fruits or plants that themselves have a high content of GABA. Or simply add GABA itself. How profitable is it to add all these ingredients?

Response 21: Thank you for your suggestion. I agree with your point. It is indeed ideal to produce GABA-rich juice beverages using fruits or plants that have a high GABA content. Daily intake of 30-100 mg of GABA has positive effects on the human body. To meet the consumption requirements with a low volume of beverage, researchers have focused on increasing GABA production in fermented foods. Our research achieved a GABA production of 7.29 g/L, while consumers only needed to consume less than 15 mL of the beverage daily. The lower the GABA content in the fermented beverage, the higher the volume of the beverage that can be consumed. The GABA content in plants or fruits is typically only a few tens of micrograms per gram. Therefore, it would be highly desirable to significantly increase the GABA content in plants or fruits to use them as raw materials for producing GABA-rich beverages.

GABA can be directly added to beverages, but the price of pure GABA is $200,000 per ton. Based on this, to produce a beverage with a GABA content of 7.29 g/L, only 1.4 kg of yeast extract, 0.7 kg of peptone, and 14 g of a metal ion complex would be needed, which is much lower in cost compared to pure GABA. Therefore, using GABA-producing lactic acid bacteria to ferment and produce GABA-rich beverages is the most economical approach.

 

Round 2

Reviewer 1 Report

Authors addressed all the recommendations suggested by the reviewers.

Minor editing

Author Response

Response to Reviewer 1 Comments

Point 1: Authors addressed all the recommendations suggested by the reviewers.

Response: Thank you very much for your comments and review. Our manuscript has been greatly improved.

Comments on the Quality of English Language: Minor editing

Thank you for your comments. We have checked and edited the language in the revised manuscript.

Reviewer 3 Report

The manuscript has been improved.

L221 The cell count on the plate was recorded by CFU/mL or log10 CFU/mL.

After the plates were counted, the results were presented as the log of the mean number of the colony-forming units (log CFU/mL).

L284 Microorganisms capable of producing GABA are known as GABA-producing strains. GABA-producing strains include LAB, yeast, Escherichia coli, and mold. Among them, LAB are commonly used in the food industry for the fermentation and production of GABA.

Provide reference

Author Response

Response to Reviewer 3 Comments

Point 1: The manuscript has been improved.

Response: Thank you very much for your comments and review. Your comments have provided us with great help to revise and improve our manuscript.

Point 2: L221 The cell count on the plate was recorded by CFU/mL or log10 CFU/mL. After the plates were counted, the results were presented as the log of the mean number of the colony-forming units (log CFU/mL).

Response: Thank you for your suggestion. We recorded CFU/mL and log10 CFU/mLlog10 to assess the cell survival of L. brevis S3 under various conditions. For cell survival in gastric acid, simulated gastric juice, simulated intestinal juice and tolerance of bile salt, salt and phenol, L. brevis S3 exhibited strong tolerance. The cell count remained relatively stable after treatment under these conditions. Using log10 CFU/mL to record the cell count, the figure looked simpler and clearer. However, for the cell count in mulberry juice, we used CFU/mL to record. This counting method is more intuitive and reflects the impact of different nutrients on cell survival in mulberry juice, which is particularly relevant for beverages and food products where consumers are interested in the number of viable cells. We have revised the sentence as follows: “The cell count on the plate was recorded as CFU/mL (cell survival in mulberry juice) or log10 CFU/mL (safety evaluation).” (Line 221-222)

Point 3: L284 Microorganisms capable of producing GABA are known as GABA-producing strains. GABA-producing strains include LAB, yeast, Escherichia coli, and mold. Among them, LAB are commonly used in the food industry for the fermentation and production of GABA. Provide reference.

Response: Thank you for your professional suggestion. We have added the references in the revised manuscript. Microorganisms capable of producing GABA are known as GABA-producing strains. GABA-producing strains include LAB, yeast, Escherichia coli, and mold [1-4]. Among them, LAB are commonly used in the food industry for the fermentation and production of GABA [1, 5].

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