Optimization of Justicia gendarussa Burm.f. fermentation by Aspergillus oryzae based on total phenolic, total flavonoid, and antioxidant capacity responses

Syaefudin Suminto; Chandra Daniel Setiawan; Waras Nurcholis; Uswatun Hasanah; Trivadila Trivadila

doi:10.7324/JABB.2025.235051

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Abstract

This study aimed to determine the optimal fermentation conditions for Justicia gendarussa leaves to produce compounds with the highest antioxidant activity. Solid-state fermentation was conducted using Aspergillus oryzae, with treatment variables including incubation time, temperature, moisture content, and inoculum concentration optimized using Design-Expert v.13.0 software. Methanol was used to extract the fermented products, which were analyzed for total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric antioxidant power (FRAP) methods. The results revealed that fermentation generally increased TPC by approximately threefold and antioxidant activity while decreasing TFC by half. Correlation analysis indicated a negative relationship between inoculum concentration and TPC. TFC increased with prolonged fermentation time but decreased at higher inoculum concentrations. Antioxidant activity, as determined by the DPPH method, increased at higher fermentation temperatures, whereas the FRAP method showed higher antioxidant capacity at lower inoculum concentrations. The optimal fermentation conditions for achieving the highest TPC, TFC, and antioxidant activity were an incubation period of 14 d at 45°C, 80% moisture content, and an inoculum concentration of 20%.

Keyword: Aspergillus oryzae Justicia gendarussa Total flavonoid content Total phenolic content Solid-state fermentation

Citation:

Suminto S, Setiawan CD, Nurcholis W, Hasanah U, Trivadila T. Optimization of Justicia gendarussa Burm.f. fermentation by Aspergillus oryzae based on total phenolic, total flavonoid, and antioxidant capacity responses. J App Biol Biotech. 2025. Online First. http://doi.org/10.7324/JABB.2025.235051

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. Souza LG, Almeida SM, Lemos TL, Ribeiro PR, Canuto KM, Braz-Filho R, et al. Brazoides A-D, new alkaloids from Justicia gendarussa burm. F. Species. J Braz Chem Soc 2017;28:1281-7. https://doi.org/10.21577/0103-5053.20160291
2. Suminto S, Takatsuji E, Iguchi A, Kanzaki H, Okuda T, Nitoda T. A new asteltoxin analog with insecticidal activity from Pochonia suchlasporia TAMA 87. J Pest Sci 2020;45:81-5. https://doi.org/10.1584/jpestics.D19-081
3. Soccol CS, Costa ES, Letti LA, Karp SG, Woiciechowski AL, Vandenberghe LP. Recent developments and innovations in solid state fermentation. Biotechnol Res Innov 2017;1:52-71. https://doi.org/10.1016/j.biori.2017.01.002
4. Yafetto L. Application of solid-state fermentation by microbial biotechnology for bioprocessing of agro-industrial wastes from 1970 to 2020: A review and bibliometric analysis. Heliyon 2022;8:e09173. https://doi.org/10.1016/j.heliyon.2022.e09173
5. Punia S, Sandhu KS, Grasso S, Purewal SS, Kaur M, Siroha AK, et al. Aspergillus oryzae fermented rice bran: A byproduct with enhanced bioactive compounds and antioxidant potential. Foods 2020;10:70. https://doi.org/10.3390/foods10010070
6. Sulasiyah S, Sarjono PR, Aminin AL. Antioxidant from turmeric fermentation products (Curcuma longa) by Aspergillus oryzae. J Sci Appl Chem 2018;21:13-8. https://doi.org/10.14710/jksa.21.1.13-18
7. Puspitasari C, Pinsirodom P, Wattanachaisaereekul S. Effect of solid-state fermentation using Aspergillus oryzae and Aspergillus niger on bitter and bioactive compounds of Moringa oleifera seed flour. LWT 2024;207:116616. https://doi.org/10.1016/j.lwt.2024.116616
8. Guo N, Zhu YW, Jiang YW, Li HK, Liu ZM, Wang W, et al. Improvement of flavonoid aglycone and biological activity of mulberry leaves by solid-state fermentation. Ind Crop Prod 2020;148:112287. https://doi.org/10.1016/j.indcrop.2020.112287
9. Suminto S, Huang AA, Hasanah U, Nurcholis W. Optimizing solid-state fermentation for metabolite enrichment by Aspergillus tamarii on rice bran and wheat. J Appl Biol Biotechnol 2024;12:195-202. https://doi.org/10.7324/JABB.2024.179836
10. Gao YL, Wang CS, Zhu QH, Qian GY. Optimization of solid-state fermentation with Lactobacillus brevis and Aspergillus oryzae for trypsin inhibitor degradation in soybean meal. J Integr Agric 2013;12:869-76. https://doi.org/10.1016/S2095-3119(13)60305-6
11. Rashid NY, Jamaluddin A, Ghani AA, Razak DL, Jonit J, Mansor A, et al. Quantification of phenolic compounds changes by Aspergillus oryzae on rice bran fermentation. Food Res 2019;3:133-7. https://doi.org/10.26656/fr.2017.3(2).122
12. Villasante J, Espinosa-Ramírez J, Pérez-Carrillo E, Heredia-Olea E, Almajano M. Extrusion and solid-state fermentation with Aspergillus oryzae on the phenolic compounds and radical scavenging activity of pecan nut (Carya illinoinensis) shell. Br Food J 2021;123:4367-82. https://doi.org/10.1108/BFJ-10-2020-0978
13. Meyrath J. Influence of the size of inoculum on various growth phases in Aspergillus oryzae. Antonie Van Leeuwenhoek 1963;29:57-78. https://doi.org/10.1007/BF02046039
14. Batubara I, Komariah K, Sandrawati A, Nurcholis W. Genotype selection for phytochemical content and pharmacological activities in ethanol extracts of fifteen types of Orthosiphon aristatus (Blume) Miq. Leaves using chemometric analysis. Sci Rep 2020;10:20945. https://doi.org/10.1038/s41598-020-77991-2
15. Turker I, Isleroglu H. Optimization of extraction conditions of bioactive compounds by ultrasonic-assisted extraction from artichoke wastes. Acta Chim Slov 2021;68:658-66. https://doi.org/10.17344/acsi.2021.6679
16. Legesse AB, Emire SA, Tadesse MG, Dadi DW, Kassa SK, Oyinloye TM, et al. Optimization of ultrasound-assisted extraction of Verbascum sinaiticum leaves: Maximal phenolic yield and antioxidant capacity. Foods 2024;13:1255. https://doi.org/10.3390/foods13081255
17. Rafi M, Pertiwi TY, Syaefudin S. Determination of total phenolic content and antioxidant activity of six ornamental plants. J Sci App Chem 2019;22:79-84. https://doi.org/10.14710/jksa.22.3.79-84
18. Pérez M, Dominguez-López I, Lamuela-Raventós RM. The chemistry behind the Folin-Ciocalteu method for the estimation of (poly) phenol content in food: Total phenolic intake in a Mediterranean dietary pattern. J Agric Food Chem 2023;71:17543-53. https://doi.org/10.1021/acs.jafc.3c04022
19. Cao C, Lin D, Zhou Y, Li N, Wang Y, Gong W, et al. Solid-state fermentation of Apocynum venetum L. By Aspergillus niger: Effect on phenolic compounds, antioxidant activities and metabolic syndrome-associated enzymes. Front Nut 2023;10:1125746. https://doi.org/10.3389/fnut.2023.1125746
20. Akbari M, Gómez-Urios C, Razavi SH, Khodaiyan F, Blesa J, Esteve MJ. Optimization of solid-state fermentation conditions to improve phenolic content in corn bran, followed by extraction of bioactive compounds using natural deep eutectic solvents. Innov Food Sci Emerg Technol 2024;93:103621. https://doi.org/10.1016/j.ifset.2024.103621
21. Ozdemir MB, K?l?çarslan E, Demir H, Koca E, Salum P, Berkta? S, et al. Upgrading the bioactive potential of hazelnut oil cake by Aspergillus oryzae under solid-state fermentation. Molecules 2024;29:4237. https://doi.org/10.3390/molecules29174237
22. Kim MJ, John KM, Choi JN, Lee S, Kim AJ, Kim YM et al. Changes in secondary metabolites of green tea during fermentation by Aspergillus oryzae and its effect on antioxidant potential. Food Res Int 2013;53:670-7. https://doi.org/10.1016/j.foodres.2012.12.053
23. Rafiquzzaman SM, Kong IS, Kim JM. Enhancement of antioxidant activity, total phenolic and flavonoid content of Saccharina japonica by submerged fermentation with Aspergillus oryzae. KSBB J 2015;30:27-32. https://doi.org/10.7841/ksbbj.2015.30.1.27
24. Rumpf J, Burger R, Schulze M. Statistical evaluation of DPPH, ABTS, FRAP, and Folin-Ciocalteu assays to assess the antioxidant capacity of lignins. Int J Biol Macromol 2023;233:123470. https://doi.org/10.1016/j.ijbiomac.2023.123470
25. Apak R, Özyürek M, Güçlü K, Çapano?lu E. Antioxidant activity/ capacity measurement. 1. Classification, physicochemical principles, mechanisms, and electron transfer (ET)-based assays. J Agric Food Chem 2016;64:997-1027. https://doi.org/10.1021/acs.jafc.5b04739
26. Nwachukwu ID, Sarteshnizi RA, Udenigwe CC, Aluko RE. A concise review of current in vitro chemical and cell-based antioxidant assay methods. Molecules 2021;26:4865. https://doi.org/10.3390/molecules26164865
27. Munteanu IG, Apetrei C. Analytical methods used in determining antioxidant activity: A review. Int J Mol Sci 2021;22:3380. https://doi.org/10.3390/ijms22073380
28. Aryal S, Baniya MK, Danekhu K, Kunwar P, Gurung R, Koirala N. Total phenolic content, flavonoid content and antioxidant potential of wild vegetables from Western Nepal. Plants (Basel) 2019;8:96. https://doi.org/10.3390/plants8040096
29. Muflihah YM, Gollavelli G, Ling YC. Correlation study of antioxidant activity with phenolic and flavonoid compounds in 12 Indonesian indigenous herbs. Antioxidants (Basel) 2021;10:1530. https://doi.org/10.3390/antiox10101530
30. Milella RA, De Rosso M, Gasparro M, Gigante I, Debiase G, Forleo LR, et al. Correlation between antioxidant and anticancer activity and phenolic profile of new Apulian table grape genotypes (V. vinifera L.). Front Plant Sci 2023;13:1064023. https://doi.org/10.3389/fpls.2022.1064023
31. Kumar S, Sandhir R, Ojha S. Evaluation of antioxidant activity and total phenol in different varieties of Lantana camara leaves. BMC Res Notes 2014;7:560. https://doi.org/10.1186/1756-0500-7-560
32. Chen J, Yang J, Ma L, Li J, Shahzad N, Kim CK. Structure-antioxidant activity relationship of methoxy, phenolic hydroxyl, and carboxylic acid groups of phenolic acids. Sci Rep 2020;10:2611. https://doi.org/10.1038/s41598-020-59451-z
33. Derawi D. Experimental design using response surface methods for palm olein-based hydroxy-ether synthesis. Sains Malays 2016;45:1149-54.
34. Tchabo W, Ma Y, Kwaw E, Zhang H, Li X. Influence of fermentation parameters on phytochemical profile and volatile properties of mulberry (Morus nigra) wine. JIB 2017;123:151-8. https://doi.org/10.1002/jib.401
35. Gomi K. Aspergillus: Aspergillus oryzae. In: Batt CA, Tortorello ML, editors. Encyclopedia of Food Microbiology. New York: Academic Press; 2014. p. 92-6. https://doi.org/10.1016/B978-0-12-384730-0.00011-2
36. Jia Y, Guo S, Hu W, Zhang Q, Wang Y, Zhang Z, et al. Effects of different fermentation temperatures on microbiomes of cigar tobacco leaves. Front Bioeng Biotechnol 2025;13:1550383. https://doi.org/10.3389/fbioe.2025.1550383
37. Jiang C, Ge J, He B, Zhang Z, Hu Z, Li Y, et al. Transcriptomic analysis reveals Aspergillus oryzae responds to temperature stress by regulating sugar metabolism and lipid metabolism. PLoS One 2022;17:e0274394. https://doi.org/10.1371/journal.pone.0274394
38. Castro RJ, Sato HH. Protease from Aspergillus oryzae: Biochemical characterization and application as a potential biocatalyst for production of protein hydrolysates with antioxidant activities. J Food Process 2014;2014:372352. https://doi.org/10.1155/2014/372352
39. Yang L, Shakeel Q, Xu X, Ali L, Chen Z, Mubeen M, et al. Optimized submerged batch fermentation for metabolic switching in Streptomyces yanglinensis 3-10 providing platform for reveromycin A and B biosynthesis, engineering, and production. Front Microbiol 2024;15:1378834. https://doi.org/10.3389/fmicb.2024.1378834
40. Jenzsch M, Gnoth S, Kleinschmidt M, Simutis R, Lübbert A. Improving the batch-to-batch reproducibility in microbial cultures during recombinant protein production by guiding the process along a predefined total biomass profile. Bioprocess Biosyst Eng 2006;29:315-21. https://doi.org/10.1007/s00449-006-0080-1
41. Cruz-Casas DE, Aguilar CN, Ascacio-Valdés JA, Rodríguez-Herrera R, Chávez-González ML, Flores-Gallegos AC. Enzymatic hydrolysis and microbial fermentation: The most favorable biotechnological methods for the release of bioactive peptides. Food Chem (Oxf) 2021;3:100047. https://doi.org/10.1016/j.fochms.2021.100047

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1. Souza LG, Almeida SM, Lemos TL, Ribeiro PR, Canuto KM, Braz-Filho R, et al. Brazoides A-D, new alkaloids from Justicia gendarussa burm. F. Species. J Braz Chem Soc 2017;28:1281-7. https://doi.org/10.21577/0103-5053.20160291

2. Suminto S, Takatsuji E, Iguchi A, Kanzaki H, Okuda T, Nitoda T. A new asteltoxin analog with insecticidal activity from Pochonia suchlasporia TAMA 87. J Pest Sci 2020;45:81-5. https://doi.org/10.1584/jpestics.D19-081

3. Soccol CS, Costa ES, Letti LA, Karp SG, Woiciechowski AL, Vandenberghe LP. Recent developments and innovations in solid state fermentation. Biotechnol Res Innov 2017;1:52-71. https://doi.org/10.1016/j.biori.2017.01.002

4. Yafetto L. Application of solid-state fermentation by microbial biotechnology for bioprocessing of agro-industrial wastes from 1970 to 2020: A review and bibliometric analysis. Heliyon 2022;8:e09173. https://doi.org/10.1016/j.heliyon.2022.e09173

5. Punia S, Sandhu KS, Grasso S, Purewal SS, Kaur M, Siroha AK, et al. Aspergillus oryzae fermented rice bran: A byproduct with enhanced bioactive compounds and antioxidant potential. Foods 2020;10:70. https://doi.org/10.3390/foods10010070

6. Sulasiyah S, Sarjono PR, Aminin AL. Antioxidant from turmeric fermentation products (Curcuma longa) by Aspergillus oryzae. J Sci Appl Chem 2018;21:13-8. https://doi.org/10.14710/jksa.21.1.13-18

7. Puspitasari C, Pinsirodom P, Wattanachaisaereekul S. Effect of solid-state fermentation using Aspergillus oryzae and Aspergillus niger on bitter and bioactive compounds of Moringa oleifera seed flour. LWT 2024;207:116616. https://doi.org/10.1016/j.lwt.2024.116616

8. Guo N, Zhu YW, Jiang YW, Li HK, Liu ZM, Wang W, et al. Improvement of flavonoid aglycone and biological activity of mulberry leaves by solid-state fermentation. Ind Crop Prod 2020;148:112287. https://doi.org/10.1016/j.indcrop.2020.112287

9. Suminto S, Huang AA, Hasanah U, Nurcholis W. Optimizing solid-state fermentation for metabolite enrichment by Aspergillus tamarii on rice bran and wheat. J Appl Biol Biotechnol 2024;12:195-202. https://doi.org/10.7324/JABB.2024.179836

10. Gao YL, Wang CS, Zhu QH, Qian GY. Optimization of solid-state fermentation with Lactobacillus brevis and Aspergillus oryzae for trypsin inhibitor degradation in soybean meal. J Integr Agric 2013;12:869-76. https://doi.org/10.1016/S2095-3119(13)60305-6

11. Rashid NY, Jamaluddin A, Ghani AA, Razak DL, Jonit J, Mansor A, et al. Quantification of phenolic compounds changes by Aspergillus oryzae on rice bran fermentation. Food Res 2019;3:133-7. https://doi.org/10.26656/fr.2017.3(2).122

12. Villasante J, Espinosa-Ramírez J, Pérez-Carrillo E, Heredia-Olea E, Almajano M. Extrusion and solid-state fermentation with Aspergillus oryzae on the phenolic compounds and radical scavenging activity of pecan nut (Carya illinoinensis) shell. Br Food J 2021;123:4367-82. https://doi.org/10.1108/BFJ-10-2020-0978

13. Meyrath J. Influence of the size of inoculum on various growth phases in Aspergillus oryzae. Antonie Van Leeuwenhoek 1963;29:57-78. https://doi.org/10.1007/BF02046039

14. Batubara I, Komariah K, Sandrawati A, Nurcholis W. Genotype selection for phytochemical content and pharmacological activities in ethanol extracts of fifteen types of Orthosiphon aristatus (Blume) Miq. Leaves using chemometric analysis. Sci Rep 2020;10:20945. https://doi.org/10.1038/s41598-020-77991-2

15. Turker I, Isleroglu H. Optimization of extraction conditions of bioactive compounds by ultrasonic-assisted extraction from artichoke wastes. Acta Chim Slov 2021;68:658-66. https://doi.org/10.17344/acsi.2021.6679

16. Legesse AB, Emire SA, Tadesse MG, Dadi DW, Kassa SK, Oyinloye TM, et al. Optimization of ultrasound-assisted extraction of Verbascum sinaiticum leaves: Maximal phenolic yield and antioxidant capacity. Foods 2024;13:1255. https://doi.org/10.3390/foods13081255

17. Rafi M, Pertiwi TY, Syaefudin S. Determination of total phenolic content and antioxidant activity of six ornamental plants. J Sci App Chem 2019;22:79-84. https://doi.org/10.14710/jksa.22.3.79-84

18. Pérez M, Dominguez-López I, Lamuela-Raventós RM. The chemistry behind the Folin-Ciocalteu method for the estimation of (poly) phenol content in food: Total phenolic intake in a Mediterranean dietary pattern. J Agric Food Chem 2023;71:17543-53. https://doi.org/10.1021/acs.jafc.3c04022

19. Cao C, Lin D, Zhou Y, Li N, Wang Y, Gong W, et al. Solid-state fermentation of Apocynum venetum L. By Aspergillus niger: Effect on phenolic compounds, antioxidant activities and metabolic syndrome-associated enzymes. Front Nut 2023;10:1125746. https://doi.org/10.3389/fnut.2023.1125746

20. Akbari M, Gómez-Urios C, Razavi SH, Khodaiyan F, Blesa J, Esteve MJ. Optimization of solid-state fermentation conditions to improve phenolic content in corn bran, followed by extraction of bioactive compounds using natural deep eutectic solvents. Innov Food Sci Emerg Technol 2024;93:103621. https://doi.org/10.1016/j.ifset.2024.103621

21. Ozdemir MB, K?l?çarslan E, Demir H, Koca E, Salum P, Berkta? S, et al. Upgrading the bioactive potential of hazelnut oil cake by Aspergillus oryzae under solid-state fermentation. Molecules 2024;29:4237. https://doi.org/10.3390/molecules29174237

22. Kim MJ, John KM, Choi JN, Lee S, Kim AJ, Kim YM et al. Changes in secondary metabolites of green tea during fermentation by Aspergillus oryzae and its effect on antioxidant potential. Food Res Int 2013;53:670-7. https://doi.org/10.1016/j.foodres.2012.12.053

23. Rafiquzzaman SM, Kong IS, Kim JM. Enhancement of antioxidant activity, total phenolic and flavonoid content of Saccharina japonica by submerged fermentation with Aspergillus oryzae. KSBB J 2015;30:27-32. https://doi.org/10.7841/ksbbj.2015.30.1.27

24. Rumpf J, Burger R, Schulze M. Statistical evaluation of DPPH, ABTS, FRAP, and Folin-Ciocalteu assays to assess the antioxidant capacity of lignins. Int J Biol Macromol 2023;233:123470. https://doi.org/10.1016/j.ijbiomac.2023.123470

25. Apak R, Özyürek M, Güçlü K, Çapano?lu E. Antioxidant activity/ capacity measurement. 1. Classification, physicochemical principles, mechanisms, and electron transfer (ET)-based assays. J Agric Food Chem 2016;64:997-1027. https://doi.org/10.1021/acs.jafc.5b04739

26. Nwachukwu ID, Sarteshnizi RA, Udenigwe CC, Aluko RE. A concise review of current in vitro chemical and cell-based antioxidant assay methods. Molecules 2021;26:4865. https://doi.org/10.3390/molecules26164865

27. Munteanu IG, Apetrei C. Analytical methods used in determining antioxidant activity: A review. Int J Mol Sci 2021;22:3380. https://doi.org/10.3390/ijms22073380

28. Aryal S, Baniya MK, Danekhu K, Kunwar P, Gurung R, Koirala N. Total phenolic content, flavonoid content and antioxidant potential of wild vegetables from Western Nepal. Plants (Basel) 2019;8:96. https://doi.org/10.3390/plants8040096

29. Muflihah YM, Gollavelli G, Ling YC. Correlation study of antioxidant activity with phenolic and flavonoid compounds in 12 Indonesian indigenous herbs. Antioxidants (Basel) 2021;10:1530. https://doi.org/10.3390/antiox10101530

30. Milella RA, De Rosso M, Gasparro M, Gigante I, Debiase G, Forleo LR, et al. Correlation between antioxidant and anticancer activity and phenolic profile of new Apulian table grape genotypes (V. vinifera L.). Front Plant Sci 2023;13:1064023. https://doi.org/10.3389/fpls.2022.1064023

31. Kumar S, Sandhir R, Ojha S. Evaluation of antioxidant activity and total phenol in different varieties of Lantana camara leaves. BMC Res Notes 2014;7:560. https://doi.org/10.1186/1756-0500-7-560

32. Chen J, Yang J, Ma L, Li J, Shahzad N, Kim CK. Structure-antioxidant activity relationship of methoxy, phenolic hydroxyl, and carboxylic acid groups of phenolic acids. Sci Rep 2020;10:2611. https://doi.org/10.1038/s41598-020-59451-z

33. Derawi D. Experimental design using response surface methods for palm olein-based hydroxy-ether synthesis. Sains Malays 2016;45:1149-54.

34. Tchabo W, Ma Y, Kwaw E, Zhang H, Li X. Influence of fermentation parameters on phytochemical profile and volatile properties of mulberry (Morus nigra) wine. JIB 2017;123:151-8. https://doi.org/10.1002/jib.401

35. Gomi K. Aspergillus: Aspergillus oryzae. In: Batt CA, Tortorello ML, editors. Encyclopedia of Food Microbiology. New York: Academic Press; 2014. p. 92-6. https://doi.org/10.1016/B978-0-12-384730-0.00011-2

36. Jia Y, Guo S, Hu W, Zhang Q, Wang Y, Zhang Z, et al. Effects of different fermentation temperatures on microbiomes of cigar tobacco leaves. Front Bioeng Biotechnol 2025;13:1550383. https://doi.org/10.3389/fbioe.2025.1550383

37. Jiang C, Ge J, He B, Zhang Z, Hu Z, Li Y, et al. Transcriptomic analysis reveals Aspergillus oryzae responds to temperature stress by regulating sugar metabolism and lipid metabolism. PLoS One 2022;17:e0274394. https://doi.org/10.1371/journal.pone.0274394

38. Castro RJ, Sato HH. Protease from Aspergillus oryzae: Biochemical characterization and application as a potential biocatalyst for production of protein hydrolysates with antioxidant activities. J Food Process 2014;2014:372352. https://doi.org/10.1155/2014/372352

39. Yang L, Shakeel Q, Xu X, Ali L, Chen Z, Mubeen M, et al. Optimized submerged batch fermentation for metabolic switching in Streptomyces yanglinensis 3-10 providing platform for reveromycin A and B biosynthesis, engineering, and production. Front Microbiol 2024;15:1378834. https://doi.org/10.3389/fmicb.2024.1378834

40. Jenzsch M, Gnoth S, Kleinschmidt M, Simutis R, Lübbert A. Improving the batch-to-batch reproducibility in microbial cultures during recombinant protein production by guiding the process along a predefined total biomass profile. Bioprocess Biosyst Eng 2006;29:315-21. https://doi.org/10.1007/s00449-006-0080-1

41. Cruz-Casas DE, Aguilar CN, Ascacio-Valdés JA, Rodríguez-Herrera R, Chávez-González ML, Flores-Gallegos AC. Enzymatic hydrolysis and microbial fermentation: The most favorable biotechnological methods for the release of bioactive peptides. Food Chem (Oxf) 2021;3:100047. https://doi.org/10.1016/j.fochms.2021.100047

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