Fermentation optimization and kinetic model for high cell density culture of a probiotic microorganism: Lactobacillus rhamnosus LS-8

  • Tao Wang
  • Yingying Lu
  • Hong Yan
  • Xin Li
  • Xin Wang
  • Yuanyuan Shan
  • Yanglei Yi
  • Bianfang Liu
  • Yuan Zhou
  • Xin LüEmail author
Research Paper


To develop a practical food-grade medium and optimal fermentation process for the cost-effective fermentation of Lactobacillus rhamnosus LS-8, both culture medium and conditions were optimized by combining single-factor experimental design, Plackett–Burman design and Box–Behnken design. The medium was simplified to five ingredients (g/L): whey powder (62.5), maltose syrup (50), corn steep liquor (55), NaCl (1) and lysine (0.05), and the optimal culture conditions were initial pH (6.28), constant fermentation pH (4.7), neutralizing agent (NaOH), aeration rate (0.2 v/v/min) and stirrer speed (200 rpm). After culturing in this optimized medium and conditions, the cell density of L. rhamnosus LS-8 was improved to 4.5 × 109 CFU/mL, which was elevated about 9 times higher than that obtained in MRS medium. Moreover, cell growth and substrate consumption kinetic constants were determined by the logistic equation and Luedeking–Piret model, and the R2 values from the model equation were 0.9900 and 0.9971, respectively, indicating that these models were able to simulate the growth and substrate consumption of L. rhamnosus LS-8 accurately. In addition, a high-efficient production process of L. rhamnosus LS-8 was developed by repeated-batch operation, which was verified by five cycles of fermentation with good stability and repeatability. In conclusion, the efficiency of L. rhamnosus LS-8 fermentation was greatly improved as well as the reduction of the cost using the medium and process developed in the present study.


Lactobacillus rhamnosus LS-8 Plackett–Burman design Box–Behnken design Kinetic model Repeated-batch operation 



This work was financially supported by Special Fund for Agro-scientific Research in the Public Interest [Grant No. 201503135].

Compliance with ethical standards

Conflicts of interest

The authors have declared no conflicts of interest.

Supplementary material

449_2019_2246_MOESM1_ESM.doc (1.8 mb)
Supplementary file1 (DOC 1814 kb)


  1. 1.
    Zhang L, Wang L, Yi L, Wang X, Zhang Y, Liu J, Guo X, Liu L, Shao C, Lu X (2017) A novel antimicrobial substance produced by Lactobacillus rhamnous LS-8. Food Control 73:754–760CrossRefGoogle Scholar
  2. 2.
    Shafi A, Farooq U, Akram K, Jaskani M, Siddique F, Tanveer A (2014) Antidiarrheal effect of food fermented by various strains of Lactobacillus. Compr Rev Food Sci Food Saf 13:229–239CrossRefGoogle Scholar
  3. 3.
    Desrouilleres K, Millette M, Jamshidian M, Maherani B, Fortin O, Lacroix M (2016) Cancer preventive effect of a specific probiotic fermented milk components and cell walls extracted from a biomass containing L. acidophilus CL1285, L. casei LBC8OR, and L. rhamnosus CLR2 on male F344 rats treated with 1,2-dimethyihydrazine. J Funct Foods 26:373–384CrossRefGoogle Scholar
  4. 4.
    Arnold JW, Simpson JB, Roach J, Bruno-Barcena JM, Azcarate-Peril MA (2018) Prebiotics for lactose intolerance: variability in galacto-oligosaccharide utilization by intestinal Lactobacillus rhamnosus. Nutrients 10:1517–1533PubMedCentralCrossRefGoogle Scholar
  5. 5.
    Hajavi J, Esmaeili SA, Varasteh AR, Vazini H, Atabati H, Mardani F, Momtazi-Borojeni AA, Hashemi M, Sankian M, Sahebkar A (2019) The immunomodulatory role of probiotics in allergy therapy. J Cell Physiol 234:2386–2398PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Gamallat Y, Ren XM, Meyiah A, Li MQ, Ren XX, Jamalat Y, Song SY, Xie LH, Ahmad B, Shopit A, Mousa H, Ma YF, Xin Y, Ding DP (2019) The immune-modulation and gut microbiome structure modification associated with long-term dietary supplementation of Lactobacillus rhamnosus using 16S rRNA sequencing analysis. J Funct Foods 53:227–236CrossRefGoogle Scholar
  7. 7.
    Waśko A, Monika KW, Podleśny M, Magdalena PB, Targoński Z, Agnieszka KK (2010) The Plackett–Burman design in optimization of media components for biomass production of Lactobacillus rhamnosus OXY. Acta Biol Hung 61:344–355PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Fitzpatrick JJ, O'Keeffe U (2001) Influence of whey protein hydrolysate addition to whey permeate batch fermentations for producing lactic acid. Process Biochem 37:183–186CrossRefGoogle Scholar
  9. 9.
    Liew SL, Ariff AB, Raha AR, Ho YW (2005) Optimization of medium composition for the production of a probiotic microorganism, Lactobacillus rhamnosus, using response surface methodology. Int J Food Microbiol 102:137–142PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Nor NM, Mohamed MS, Loh TC, Foo HL, Rahim RA, Tan JS, Mohamad R (2017) Comparative analyses on medium optimization using one-factor-at-a-time, response surface methodology, and artificial neural network for lysine–methionine biosynthesis by Pediococcus pentosaceus RF-1. Biotechnology & Biotechnological Equipment, 935–947.CrossRefGoogle Scholar
  11. 11.
    Lima CJBD, Coelho LF, Contiero J (2010) The use of response surface methodology in optimization of lactic acid production: focus on medium supplementation, temperature and pH control. Food Technol Biotechnol 48:175–181Google Scholar
  12. 12.
    Coelho LF, de Lima CJB, Piassi MB, Contiero J (2010) Medium composition and optimization of Lactic acid production by Lactobacillus plantarum Lmism-6 grown in molasses. J Biotechnol 150:S511–S511CrossRefGoogle Scholar
  13. 13.
    Racine FM, Saha BC (2007) Production of mannitol by Lactobacillus intermedius NRRL B-3693 in fed-batch and continuous cell-recycle fermentations. Process Biochem 42:1609–1613CrossRefGoogle Scholar
  14. 14.
    Maddipati P, Atiyeh HK, Bellmer DD, Huhnke RL (2011) Ethanol production from syngas by Clostridium strain P11 using corn steep liquor as a nutrient replacement to yeast extract. Biores Technol 102:6494–6501CrossRefGoogle Scholar
  15. 15.
    Da Cunha MC, Masotti MT, Mondragon-Bernal OL, Alves J (2018) Highly efficient production of L (+)-lactic acid using medium with potato, corn steep liquor and calcium carbonate by Lactobacillus rhamnosus ATCC 9595. Braz J Chem Eng 35:887–899CrossRefGoogle Scholar
  16. 16.
    Taiwo AE, Madzimbamuto TN, Ojumu TV (2018) Optimization of corn steep liquor dosage and other fermentation parameters for ethanol production by Saccharomyces cerevisiae type 1 and anchor instant yeast. Energies 11:1740–1759CrossRefGoogle Scholar
  17. 17.
    Zhang HT, Zhan XB, Wu JR, English N, Yu XB, Lin CC (2012) Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of Agrobacterium sp. ATCC 31749. Appl Microbiol Biotechnol 93:367–379PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Li RF, Xu Y (2011) Fermentation optimization to improve production of antagonistic metabolites by Bacillus subtilis strain BS501a. J Cent South Univ Technol 18:1047–1053CrossRefGoogle Scholar
  19. 19.
    Singh V, Haque S, Niwas R, Srivastava A, Pasupuleti M, Tripathi CK (2016) Strategies for fermentation medium optimization: an in-depth review. Front Microbiol 7:2087–2102PubMedPubMedCentralGoogle Scholar
  20. 20.
    Al-madboly L, Ali S (2017) Optimization of reduced glutathione production by a Lactobacillus plantarum isolate using Plackett–Burman and Box–Behnken designs. Front Microbiol 8:772–780PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Neera PMM, Ramana KV, Bawa AS (2013) Statistical optimization of bacteriocin production by pediococcus acidilactici in a simple food-grade medium. J Food Process Preserv 37:179–187CrossRefGoogle Scholar
  22. 22.
    Tiwari SK, Srivastava S (2008) Statistical optimization of culture components for enhanced bacteriocin production by Lactobacillus plantarum LR/14. Food Biotechnol 22:64–77CrossRefGoogle Scholar
  23. 23.
    Iyer R, Tomar SK, Singh AK (2010) Response surface optimization of the cultivation conditions and medium components for the production of folate by Streptococcus thermophilus. J Dairy Res 77:350–356PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Arbia W, Adour L, Amrane A, Lounici H (2013) Optimization of medium composition for enhanced chitin extraction from Parapenaeus longirostris by Lactobacillus helveticus using response surface methodology. Food Hydrocolloids 31:392–403CrossRefGoogle Scholar
  25. 25.
    Boontim N, Khanongnuch C, Pathom-aree W, Niamsup P, Lumyong S (2018) Production of L-lactic acid by thermotorelant lactic acid bacteria. Chiang Mai J Sci 45:68–76Google Scholar
  26. 26.
    Srivastava AK, Tripathi AD, Jha A, Poonia A, Sharma N (2015) Production, optimization and characterization of lactic acid by Lactobacillus delbrueckii NCIM 2025 from utilizing agro-industrial byproduct (cane molasses). J Food Sci Technol Mysore 52:3571–3578Google Scholar
  27. 27.
    Joshi VK, Chauhan A, Devi S, Kumar V (2015) Application of response surface methodology in optimization of lactic acid fermentation of radish: effect of addition of salt, additives and growth stimulators. J Food Sci Technol Mysore 52:4935–4944CrossRefGoogle Scholar
  28. 28.
    Neysens P, Messens W, De Vuyst L (2003) Effect of sodium chloride on growth and bacteriocin production by Lactobacillus amylovorus DCE 471. Int J Food Microbiol 88:29–39PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Korkeala H, Alanko T, Tiusanen T (1992) Effect of sodium nitrite and sodium chloride on growth of lactic acid baceria. Acta Vet Scand 33:27–32PubMedPubMedCentralGoogle Scholar
  30. 30.
    Buruleanu CL, Avram D, Manea I, Bratu MG (2012) Effects of yeast extract and different amino acids on the dynamics of some components in cabbage juice during fermentation with Bifidobacterium lactis BB-12. Food Sci Biotechnol 21:691–699CrossRefGoogle Scholar
  31. 31.
    Ferreira SLC, Bruns RE, Ferreira HS, Matos GD, David JM, Brandao GC, da Silva EGP, Portugal LA, Reis PS, Souza AS, dos Santos WNL (2007) Box–Behnken design: an alternative for the optimization of analytical methods. Anal Chim Acta 597:179–186PubMedCrossRefGoogle Scholar
  32. 32.
    Mohamad NL, Kamal SMM, Mokhtar MN, Husain SA, Abdullah N (2016) Dynamic mathematical modelling of reaction kinetics for xylitol fermentation using Candida tropicalis. Biochem Eng J 111:10–17CrossRefGoogle Scholar
  33. 33.
    Trigueros DEG, Fiorese ML, Kroumov AD, Hinterholz CL, Nadai BL, Assunção GM (2016) Medium optimization and kinetics modeling for the fermentation of hydrolyzed cheese whey permeate as a substrate for Saccharomyces cerevisiae var. boulardii. Biochem Eng J 110:71–83CrossRefGoogle Scholar
  34. 34.
    Mounsef JR, Salameh D, Louka N, Brandam C, Lteif R (2015) The effect of aeration conditions, characterized by the volumetric mass transfer coefficient K L a, on the fermentation kinetics of Bacillus thuringiensis kurstaki. J Biotechnol 210:100–106PubMedCrossRefGoogle Scholar
  35. 35.
    Shuler ML, Kargı F (2002) Bioprocess engineering: basic concepts. Int Plan Parent Federation 180:1822–1823Google Scholar
  36. 36.
    Laopaiboon L, Phukoetphim N, Laopaiboon P (2016) Logistic function and modified Gompertz models for batch ethanol fermentation kinetics from sweet sorghum juice under high gravity condition. New Biotechnol 33:S86–S87CrossRefGoogle Scholar
  37. 37.
    Phukoetphim N, Salakkam A, Laopaiboon P, Laopaiboon L (2016) Kinetic models for batch ethanol production from sweet sorghum juice under normal and high gravity fermentations: Logistic and modified Gompertz models. J Biotechnol 243:69–75PubMedCrossRefGoogle Scholar
  38. 38.
    Bule MV, Singhal RS (2011) Fermentation kinetics of production of ubiquinone-10 by Paracoccus dinitrificans NRRL B-3785: effect of type and concentration of carbon and nitrogen sources. Food Sci Biotechnol 20:607–613CrossRefGoogle Scholar
  39. 39.
    Nassourou MA, Noubissie TJB, Njintang YN, Bell JM (2017) Diallel analyses of soluble sugar content in cowpea (Vigna unguiculata L. Walp.). Crop Journal 5:553–559CrossRefGoogle Scholar
  40. 40.
    Dimitrovski D, Velickova E, Langerholc T, Winkelhausen E (2015) Apple juice as a medium for fermentation by the probiotic Lactobacillus plantarum PCS 26 strain. Ann Microbiol 65:2161–2170CrossRefGoogle Scholar
  41. 41.
    Xia W, Chen W, Peng W-f, Li K-t (2015) Industrial vitamin B-12 production by Pseudomonas denitrificans using maltose syrup and corn steep liquor as the cost-effective fermentation substrates. Bioprocess Biosyst Eng 38:1065–1073PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Zeng W, Zhang B, Li MX, Ding S, Chen GG, Liang ZQ (2019) Development and benefit evaluation of fermentation strategies for poly (malic acid) production from malt syrup by Aureobasidium melanogenum GXZ-6. Biores Technol 274:479–487CrossRefGoogle Scholar
  43. 43.
    Fonteles TV, Costa MGM, de Jesus ALT, Rodrigues S (2012) Optimization of the Fermentation of Cantaloupe Juice by Lactobacillus casei NRRL B-442. Food Bioprocess Technol 5:2819–2826CrossRefGoogle Scholar
  44. 44.
    Wang X, Shao C, Liu L, Guo X, Xu Y, Lu X (2017) Optimization, partial characterization and antioxidant activity of an exopolysaccharide from Lactobacillus plantarum KX041. Int J Biol Macromol 103:1173–1184PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Condon S (1987) Responses of lactic acid bacteria to oxygen ☆. FEMS Microbiol Lett 46:269–280CrossRefGoogle Scholar
  46. 46.
    Duwat P, Sourice S, Cesselin B, Lamberet G, Vido K, Gaudu P, Loir Y, Le VF, Loubière P, Gruss A (2001) Respiration capacity of the fermenting bacterium Lactococcus lactis and its positive effects on growth and survival. J Bacteriol 183:4509–4516PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Niel EWJ, Van Karin H, Bärbel HHG (2002) Formation and conversion of oxygen metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under different growth conditions. Appl Environ Microbiol 68:4350–4356PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Polak-Berecka M, Wasko A, Kordowska-Wiater M, Podlesny M, Targonski Z, Kubik-Komar A (2010) Optimization of Medium Composition for enhancing growth of Lactobacillus rhamnosus PEN using response surface methodology. Pol J Microbiol 59:113–118PubMedPubMedCentralGoogle Scholar
  49. 49.
    Chang CP, Liew SL (2013) Growth medium optimization for biomass production of a probiotic bacterium, Lactobacillus rhamnosus ATCC 7469. J Food Biochem 37:536–543Google Scholar
  50. 50.
    Bajpai-Dikshit J, Suresh AK, Venkatesh KV (2003) An optimal model for representing the kinetics of growth and product formation by Lactobacillus rhamnosus on multiple substrates. J Biosci Bioeng 96:481–486PubMedCrossRefPubMedCentralGoogle Scholar
  51. 51.
    Choi M, Al-Zahrani SM, Sang YL (2014) Kinetic model-based feed-forward controlled fed-batch fermentation of Lactobacillus rhamnosus for the production of lactic acid from Arabic date juice. Bioprocess Biosyst Eng 37:1007–1015PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Dong Z, Gu L, Zhang J, Wang M, Du G, Chen J, Li H (2014) Optimisation for high cell density cultivation of Lactobacillus salivarius BBE 09–18 with response surface methodology. Int Dairy J 34:230–236CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Tao Wang
    • 1
  • Yingying Lu
    • 1
  • Hong Yan
    • 1
  • Xin Li
    • 1
  • Xin Wang
    • 1
  • Yuanyuan Shan
    • 1
  • Yanglei Yi
    • 1
  • Bianfang Liu
    • 1
  • Yuan Zhou
    • 1
  • Xin Lü
    • 1
    Email author
  1. 1.College of Food Science and EngineeringNorthwest A&F UniversityYanglingChina

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