Silkworm Pupae Coupled with Glucose Control pH Mediates GABA Hyperproduction by Lactobacillus hilgardii
Round 1
Reviewer 1 Report
The Manuscript [fermentation-2513251] entitled (Silkworm pupae coupled with glucose control pH mediates GABA hyperproduction by Lactobacillus hilgardii) needs to be revised with these comments
1- In abstract, lines 21 and 23, Abbreviate the Genus name of Lactobacillus
2- In Keywords: delete Lactobacillus hilgardii and others should be in alphabetical.
3- Line 70, add Family and Order of Bombyx mori
4- Line 79: it is the first mention of Lactobacillus hilgardii in the text, thus, abbreviate the genus after that as in lines; 83, 98, 109. ……..
5- Line 88: abbreviate the genus Bombyx and in the remaining text.
6- In Methods: Authors should add a section of: Statistical analysis to indicate how it was done.
7- In results: lines; 187-188, 244, 270, 337. Authors should add the values of F, df, and P
8- In All figure: letters of significance (a,b,c, ….), it should be added
Author Response
Response to Reviewer 1 Comments
Point 1: In abstract, lines 21 and 23, Abbreviated the Genus name of Lactobacillus
Response 1: Thank you for your suggestion. We have abbreviated the genus name of Lactobacillus in the abstract. (Line 21 and 23)
Point 2: Keywords: delete Lactobacillus hilgardii and others should be in alphabetical.
Response 2: Thank you for your comment. We have deleted the keyword Lactobacillus hilgardii and rearranged other keywords in alphabetical. The keywords are γ-aminobutyric acid (GABA), automatic control, fermentation, glucose, and silkworm pupae. (Line 25)
Point 3: Line 70, add Family and Order of Bombyx mori.
Response 3: Thank you for your suggestion. We have added the family (Bombycidae) and order (Lepidoptera) of Bombyx mori L. The sentence had been rewritten as “Silkworm pupae (Lepidopyera, Bombycidae, Bombyx mori L) are a byproduct of the silk reeling industry” in the revised manuscript. (Line 78)
Point 4: Line 79: it is the first mention of Lactobacillus hilgardii in the text, thus, abbreviate the genus after that as in lines; 83, 98, 109. ……..
Response 4: Thank you for your suggestion. We have abbreviated the genus Lactobacillus in our revised manuscript. (Line 55, 56, 59, 60, 62, 95, 99, 113, 124, 129, 139, 160, 224, 225, 226, 227, 242, 247, 252, 253, 257, 280, 345, 357, 361, 371, 381, 389, 400, 401, 406)
Point 5: Line 88: abbreviate the genus Bombyx and in the remaining text.
Response 5: Thank you for your suggestion. We have abbreviated the genus Bombyx in the revised manuscript. (Line 103)
Point 6: In Methods: Authors should add a section of: Statistical analysis to indicate how it was done.
Response 6: Thank you for your professional comments and valuable suggestion. We have added this information to the Materials and Methods section in the revised manuscript. Each experiment was carried out with three biological replicates. Statistical analysis of the sample was performed at intervals of 12 h. At the same time, analysis of variance (ANOVA) was conducted with culture condition as the main factor. This analysis was performed to determine whether the silkworm pupae replaced the nitrogen source, and the concentration of silkworm pupae, glucose, glucose and monosodium glutamate had significant effects on cell growth, fermentation pH and GABA production in L. hilgardii at each time point. The significant difference between two samples was analyzed by an independent sample T test. The data were analyzed using SPSS software, version 26. ANOVA and T test analysis were conducted with a confidence level of 95%. P<0.05 was considered a significant difference. Figures were exported by GraphPad Prism 9 and expressed as the mean and standard deviation of the three biological repetitions. Error bars represent the standard deviation of the mean. The significant differences are marked on the point. The figure captions contained explanations. (Line 188-199)
Point 7: In results: lines; 187-188, 244, 270, 337. Authors should add the values of F, df, and P.
Response 7: Thank you for your suggestion. We have added the values of F, df, and P in lines 187-188. The sentence has been rewritten as follows: “When silkworm pupae meal replaced peptone, the cell growth of the GABA-producing strain L. hilgardii GZ2 was significantly higher than that in GYP complex medium (Fig. 1a). In particular, the difference between these two samples was extremely significant at 72 h (P=0.000002<0.001, F=607.439, df=16)”. (Line212-215)
The significance analysis of Figure 3b has been added to the revised manuscript. Figure 3b showed that the trend of fermentation pH was not changed by different concentrations of silkworm pupa meal to replace tryptone, and the trend of fermentation pH increased in the same way. Finally, all pH values reached approximately 8.6 at 72 h, which did not indicate a difference in pH value. Therefore, the sentence has been rewritten as “The trend of pH was basically consistent at the different concentrations of silkworm pupae meal replacing tryptone (Fig. 3b). The pH ultimately increased to approximately 8.6, indicating that hydrogen ions dissociated from organic acids were utilized in the fermentation broth”. (Line 273-276)
The significant difference in Figure 4b has been added. We have added the values of F, df and P at 72 h with 10 g/L and 20 g/L glucose used as carbon sources in the revised manuscript. The sentence has been rewritten as “However, the pH of the fermented supernatant significantly changed (Fig. 4b). For example, the p value of significant difference was 1.37×10-10 between 10 g/L and 20 g/L glucose used as carbon source at 72 h (F=128, df=16)”. (Line 302-304)
The sentence in line 337 indicate that the GABA yield is high in L.brevis, but the fermentation cost is high too, by using glutamic acid adjusting pH. Compared to flask fermentation, GABA was slightly increased in the fermenter. Therefore, we have rewritten the sentence as follows: “Researchers used glutamic acid ($2.76 per kg) to adjust the fermentation pH in L. brevis. Although the GABA yield was 321.9 g/L in the 3 L fermenter, there was little increase compared to flask fermentation (approximately 320 g/L). The cost of glutamic acid was high, which was not conducive to industrial implementation.” (Line 375-378)
Point 8: All figure: letters of significance (a,b,c, …. ), it should be added
Response 8: Thank you for your professional comments. We have analyzed the significant differences in every figure and added the letters of significance in the revised manuscript. (Line 235-237, 262-264, 289-292, 322-325, 350-353, 395-397)
Reviewer 2 Report
The authors reported the use of low-cost and safe fermentation strategies to improve the efficiency of GABA production by Lactobacillus hilgadii. The manuscript is well-structured and contains interesting results. The most expressive limitation of the manuscript is the total absence of statistical analysis. The collected data needs of ANOVA + post-ANOVA test. A robust investigation performed in similar articles involves the use of response surface methodology. Another point to be considered before the manuscript publication is the absence of chemical characterization of the silkworm pupae used as a nitrogen source. Due to the missing analysis, I believe the manuscript will be suitable for publication in Fermentation after considering these points. Other minor issues to be considered by the authors are listed below:
(1) Explain better in what context GABA may be used as a food additive
(2) Gamma-aminobutyric acid and γ-Aminobutyric acid are used in different places, please use the same term in all the places
(3) references [1-5] are not appropriate for the sentence about the roles of GABA
(4) all the graphs are without asterisks highlighting the statistical differences between the experimental groups at each analysis time. Statistical analysis was not performed. ANOVA followed by a means test comparison is a minimum expected in an article and must be performed aiming to evaluate how intense are the differences shown by the authors
(5) the numerical values used in the description of the results are without standard deviation
(6) a critical limitation of the manuscript is the absence of a factorial design and the validation of response surfaces. This statistical tool is widely used in investigations involving the optimization of fermentation conditions.
(7) the chemical characterization of the material used as an alternative nitrogen source is absent. The centesimal composition and more refined analysis could be improving the quality of results and yield more impressive results.
Minor editing of English language required
Author Response
Response to Reviewer 2 Comments
Point 1: Explain better in what context GABA may be used as a food additive
Response 1: Thank you for your suggestion. We have added the national requirements for GABA as a food additive in the revised manuscript. The European Food Safety (EFSA) and the U.S. Food and Drug Administration (FDA) recognizes GABA produced through lactic acid bacteria fermentation as a natural food additive. EFSA allows the addition of GABA to food, with a maximum recommended dietary intake of 550 mg/day for GABA. The FDA states that the addition of GABA to food is safe, and its usage includes beverages, coffee, tea, and chewing gum, but it is not permitted in baby food, meat products, or products containing meat. In 2001, Japan included GABA in its range of food products. In 2009, the Chinese Ministry of Health approved GABA produced through lactic acid fermentation as a new resource food. It stipulates that GABA intake should not exceed 500 mg/day, and its usage includes beverages, cocoa products, chocolate and its products, candy, baked goods, and puffed snacks, but it cannot be added to baby food. (Line 35-45)
Point 2: Gamma-aminobutyric acid and γ-Aminobutyric acid are used in different places, please use the same term in all the places
Response 2: Thank you for your suggestion. We have unified the term γ-aminobutyric acid in the revised manuscript. (Line 28)
Point 3: references [1-5] are not appropriate for the sentence about the roles of GABA
Response 3: Thank you for your professional comments. We agree with your suggestion. The appropriate references have been added and changed in the revised manuscript. We have checked the other references and confirmed the correct references. (Line 30-32)
- Hou, D., Tang, J., Feng, Q., Niu, Z., Shen, Q., Wang, L., Zhou, S. Gamma-aminobutyric acid (GABA): a comprehensive review of dietary sources, enrichment technologies, processing effects, health benefits, and its applications. Crit. Rev. Food Sci. Nutr.2023, 1-23. doi:10.1080/10408398.2023.2204373.
- Ngo, D.H., Vo, T.S. An updated review on pharmaceutical properties of gamma-aminobutyric acid. Molecules 2019, 24. doi:10.3390/molecules24152678.
- Cao, Q., Wang, J., Hao, Y., Zhao, F., Fu, R., Yu, Y., Wang, J., Niu, R., Bian, S., Sun, Z. Exercise ameliorates fluoride-induced anxiety- and depression-like behavior in mice: role of GABA. Biol. Trace Elem. Res.2022, 200,678-688. doi:10.1007/s12011-021-02678-2.
- Chen, H.H., Cheng, P.W., Ho, W.Y., Lu, P.J., Lai, C.C., Tseng, Y.M., Fang, H.C., Sun, G.C., Hsiao, M., Liu, C.P., Tseng, C.J. Renal denervation improves the baroreflex and GABA system in chronic kidney disease-induced hypertension. Sci. Rep.2016, 6,38447. doi:10.1038/srep38447.
- Sears, S.M., Hewett, S.J. Influence of glutamate and GABA transport on brain excitatory/inhibitory balance. Exp. Biol. Med. (Maywood)2021, 246,1069-1083. doi:10.1177/1535370221989263.
Point 4: all the graphs are without asterisks highlighting the statistical differences between the experimental groups at each analysis time. Statistical analysis was not performed. ANOVA followed by a means test comparison is a minimum expected in an article and must be performed aiming to evaluate how intense are the differences shown by the authors
Response 4: Thank you for your professional comments and valuable suggestion. We have conducted a significance analysis on all the figures. Significance analysis was performed using ANOVA on multiple samples at the same time point. The significant difference between two samples was analyzed by an independent sample T test. These data were analyzed using SPSS software, version 26. ANOVA and T test analysis are conducted with a confidence level of 95%. Different small letters indicate significant differences at p<0.05 in every figure. The detailed method of statistical analysis has been added to the Materials and Methods and figure captions in the revised manuscript. (Line 188-199, 235-237, 262-264, 289-292, 322-325, 350-353, 395-397)
Point 5: the numerical values used in the description of the results are without standard deviation
Response 5: Thank you for your suggestion. We have added the standard deviation for the value in the results. The points of figures were expressed as the mean and standard deviation of the three biological repetitions. Error bars represent the standard deviation of the mean. (Line 223, 272, 281, 301, 308, 309, 310, 338, 339, 342, 359, 363)
Point 6: a critical limitation of the manuscript is the absence of a factorial design and the validation of response surfaces. This statistical tool is widely used in investigations involving the optimization of fermentation conditions.
Response 6: Thank you for your professional comments and valuable suggestion. Factorial design and response surface validation are highly important in determining the optimal process parameters and serve as vital approaches in optimizing fermentation conditions. The objective of this study was to reduce the cost of GABA synthesis in lactic acid bacteria culture medium by replacing expensive nitrogen sources with cost-effective silkworm pupae in GYP complex medium. During the study, an interesting phenomenon was observed. A pH of approximately 5 could be achieved by adjusting the ratio of glucose to monosodium glutamate. The concentrations of glucose and monosodium glutamate are not the only ones. To improve production efficiency, we chose 50 g/L glucose and 50 g/L monosodium glutamate. Regarding the response of pH, it is not necessary and impossible to precisely control it to a specific value. The objective is to maintain the pH relatively stably at approximately 5, while the GABA yield was higher than that of nonfed batch fermentation. This objective can be achieved by implementing online cascade control in the fermentation tank. If the pH increases or decreases during the fermentation process, glucose or sodium glutamate can be supplied directly to adjust the pH. However, the control is not immediately executed. Fluctuations in pH are caused by multiple factors, such as substrate, product and cell growth. As long as the strain is not at a high pH for a long time, GABA will be produced. For industrial fermentation, yield and productivity are important, and production optimization of fermentation tanks is necessary. Therefore, to achieve efficient GABA production and automated pH control in industrial production, this study directly utilized fermentation tanks for process optimization. Ultimately, through fermentation optimization, strategies were implemented to automatically control pH by adjusting the concentrations of monosodium glutamate and glucose, resulting in high GABA production by Lactobacillus hilgardii. The factorial design or response surface analysis methods were not employed for parameter optimization. In subsequent research, we will utilize factorial design and response surface analysis to explore the impact of multiple factors, including fermentation conditions (T, pH, O2, speed, seed) and composition of the fermentation medium (carbon source, nitrogen source, metal ion, disperser), on GABA production to obtain the optimum fermentation parameters.
Point 7: the chemical characterization of the material used as an alternative nitrogen source is absent. The centesimal composition and more refined analysis could be improving the quality of results and yield more impressive results.
Response 7: Thank you for your suggestion. Researchers have detected the detailed chemical composition of silkworm pupae. We have added this section and the corresponding reference in the Introduction and Results. Silkworm pupae (Lepidopyera, Bombycidae, Bombyx mori L) are a byproduct of the silk reeling industry. It is rich in lipids (30%), protein (60%), and ash (10%). The lipids of silkworm pupae (China) include saturated fatty acids (22.04% C16:0 and 6.84% C18:0), monounsaturated fatty acids (0.92% C16:1 and 33.91% C18:1), and polyunsaturated fatty acids (5.48% C18:2 and 30.81% C18:3). Silkworm pupae protein (SPP) is rich in 18 different amino acids, of which 8 are essential amino acids for human beings, accounting for over 42% of the total amino acid content. Silkworm pupae also contain 3-4% chitosan and other necessary trace elements (Cu, Zn, Fe, Se). These trace elements include iron (9.54 mg), zinc (17.75 mg), potassium (1826.59 mg), sodium (274.57 mg), calcium (102.31 mg), phosphorus (1369.94 mg), magnesium (287.96 mg), and manganese (2.49 mg). Silkworm pupae are abundant in vitamins A, E, D, B1, B2, and B3. (Line 78-90, 368-370)
Comments on the Quality of English Language
Minor editing of English language required
Thank you for your suggestion. We have checked and edited the language by a colleague fluent in English writing.
Round 2
Reviewer 1 Report
All comments have been done
No comments
Author Response
Point: All comments have been done
Response: Many thank you for your comments and review.
Reviewer 2 Report
Thank you for having given me the opportunity to review the manuscript again. The authors appropriately responded to the comments and the critical improvements were performed in the manuscript. I believe that the manuscript is attractive and informative and represents a contribution to the field, however, I have some considerations at this moment of the editorial process:
(1) is unclear where the t-test was used in the manuscript. All the comparisons were performed among more than two experimental conditions.
(2) the chemical characterization of the material used as an alternative nitrogen source still is absent. The authors did not submit the material used in their investigation to any chemical analysis.
(3) in figure 2, is missing information in the caption, after “, p<0.05, **, p<0.01, ***, p<0.001”. It is unclear what groups are being compared with these p-values.
Minor editing of English language required
Author Response
Response to Reviewer 2 Comments
Point 1: is unclear where the t-test was used in the manuscript. All the comparisons were performed among more than two experimental conditions.
Response 1: Thank you for your suggestion. We have added information on the T test in the statistical analysis in the revised manuscript. The significant difference in gene (gadR, gadC and gadB) expression levels was analyzed by an independent sample T test at the same time. The control group used yeast extract and tryptone as nitrogen sources. (Line 201-203)
Point 2: the chemical characterization of the material used as an alternative nitrogen source still is absent. The authors did not submit the material used in their investigation to any chemical analysis.
Response 2: Thank you for your suggestion. We have added the chemical characterization and picture of the silkworm pupae meal. The silkworm pupae meal was dried at 60°C for 4 h, crushed and sifted to make a 1~2 mm powder. The dried silkworm pupae meal is light brown in color, with a consistent appearance (Supplementary Fig. S1). They do not have any mold and have a distinct odor. They partially dissolve in water, ethanol, chloroform, and ether. The crude protein content of silkworm pupae meal is between 65% and 70%, the crude fat content is between 20% and 30%, the crude ash content is 4% to 5%, the crude fiber content is 4% to 5%, and the moisture content is less than 12%. The silkworm pupae meal can lead to acidification and oxidation at high temperatures or room temperature with prolonged storage. It is recommended to store at low (<4°C) temperatures. (Line 104-112)
Fig. S1 Pictures of dried silkworm pupae meal
Point 3: in figure 2, is missing information in the caption, after “, p<0.05, **, p<0.01, ***, p<0.001”. It is unclear what groups are being compared with these p-values.
Response 3: Thank you for your comment. The significant difference in the data in Fig. 2 was determined by an independent-sample T test. The control group used yeast extract and tryptone as nitrogen sources at the same time. We have added the relevant information in the caption of Fig. 2. The significant difference between these two groups was determined by an independent-samples T test at the same time. *, p<0.05, **, p<0.01, ***, p<0.001. The control group used yeast extract and tryptone as nitrogen sources. (Line 272-274)
Comments on the Quality of English Language
Minor editing of English language required
Thank you for your comments. We have checked and edited the language in the revised manuscript.
