بهینه‌سازی تولید زیست‌توده باکتری پروبیوتیک لاکتوباسیلوس رامنوسوس در سطح نیمه‌صنعتی
محورهای موضوعی : زیست فناوری میکروبی
مریم آرمند
1
(گروه میکروبیولوژی، دانشکده علوم پایه ،دانشگاه آزاد اسلامی واحدلاهیجان،لاهیجان،ایران)
محمد فائزی قاسمی
2
(گروه میکروبیولوژی، دانشکده علوم ،پایه دانشگاه آزاد اسلامی واحدلاهیجان،لاهیجان،ایران)
محمدرضا فاضلی
3
(گروه کنترل دارو و غذا، دانشکده داروسازی، دانشگاه علوم پزشکی تهران، تهران، ایران)
میرساسان میرپور
4
(گروه میکروبیولوژی، دانشکده علوم ،پایه دانشگاه آزاد اسلامی واحدلاهیجان،لاهیجان،ایران)
کلید واژه: بهینه‌سازی, پروبیوتیک, لاکتوباسیلوس رامنوسوس, طراحی پلاکت- برمن, طراحی آزمون,
چکیده مقاله :
سابقه و هدف: باکتری‌های پروبیوتیک نقش بسیار مهمی در بهبود فلور طبیعی روده داشته و مانع رشد باکتری‌های مضر در دستگاه گوارش شده و ازنظر اهداف دارویی و درمانی دارای اهمیت می‌باشند. هدف از این مطالعه بهینه‌سازی تولید زیست‌توده باکتری لاکتوباسیلوس رامنوسوس GG ATCC53103 ) Lactobacillus rhamnosus (با استفاده از روش طراحی آزمون است.مواد و روش‌ها: در این تحقیق از باکتری پروبیوتیک لاکتوباسیلوس رامنوسوس استفاده شد. به‌منظور بهینه‌سازی از روش طراحی پلاکت- برمن استفاده گردید. تمام کشت های محیط پایه و بهینه‌شده در فرمانتور 1300 لیتری شرکت پارس پاد انجام شد.یافته‌ها: نتایج نشان داد که منابعملاس چغندرقند، گلوکز و کازئین بیشترین اثر را در تولید زیست‌توده لاکتوباسیلوس رامنوسوس دارند. گلوکز با کازئین و ملاس چغندرقند اثر هم‌افزایی داشته و موجب افزایش تولید زیست‌توده می‌شوند. پس از بهینه‌سازی، محیط کشت دارای مقادیر ترکیبات زیر به ازای گرم در لیتر: گلوکز 50/112، ملاس چغندرقند 25/56، کازئین 75/18، عصاره مخمر 75/18، K2HPO4 13/13، تویین 80 88/1، 7H2O.MgSO43750/0، MnSO4. 4H2O 0750/0، CaCl2. 2H2O 1875/0 و سایمتیکن 25/1 به‌منظور تولید بهترین زیست‌توده توسط لاکتوباسیلوس رامنوسوس مشخص گردید. میزان زیست‌توده بیشتر از دو برابر نسبت به شرایط محیط کشت پایه افزایش پیدا کرد.نتیجه‌گیری: با توجه به تولید زیست‌توده باکتری لاکتوباسیلوس رامنوسوس در سطح نیمه‌صنعتی درفرمانتور 1300 لیتری ، امکان تولید صنعتی زیست‌توده لاکتوباسیلوس رامنوسوس وجود خواهد داشت.همچنین کشت این باکتری به‌صورت صنعتی در شرایط کشت ناپیوسته تغذیه شونده و متداوم به‌عنوان یک پروبیوتیک تجاری پیشنهاد می‌شود.
Background & objectives: Probiotics play a very important role in improving the normal intestinal flora and inhibit the growth of harmful bacteria in the gastrointestinal tract and are also important for therapeutic purposes. This study aimed to optimize biomass production by Lactobacillus rhamnosus ATCC53103 (GG) using the experimental design process. Materials and Methods: In this study, the probiotic bacterium Lactobacillus rhamnosus was used. Plackett-Burman design method was used for the optimization. Fermentations in basal and optimized cultures were performed in 1300 liter Parspad Company's fermenters. Results: The results showed that beet molasses, glucose, and casein have the greatest effect on biomass production by Lactobacillus rhamnosus. Glucose with casein and beet molasses have a synergistic effect and increasing the concentration of glucose with increasing the concentration of two other factors increases the production of the biomass. Based on the results obtained, after optimization, the optimal culture medium for biomass production by Lactobacillus rhamnosus has the following compounds g/L-1: glucose 112.50, beet molasses 56.25, casein 18.75, yeast extract 18.75, K2HPO4 13.13, Tween 80 1.88, MgSO4. 7H2O 0.3750, MnSO4. 4H2O 0.0750, CaCl2. 2H2O 0.1875 and Simethicone1.25. The biomass production in optimized conditions was increased more than 2-folds higher than the basal medium. Conclusion: Biomass production by Lactobacillus rhamnosus on a semi-industrial scale was carried out in 1300 liters fermentors. Therefore, the results of this study can be used in the industrial production of Lactobacillus rhamnosus biomass. Also, commercial production under fed-batch and continuous culture conditions is recommended.
varieties of Iranian native olives and investigation of their antimicrobial activity against two
pathogenic members of Entrobacteriaceae. Biological Journal of Microorganism. 2015;4(13).
2. Lebeer S, anderleyden J, De Keersmaecker SC. Genes and molecules of lactobacilli
supporting probiotic action. Microbiology and Molecular Biology eviews. 2008;72(4):
728-64.
3. Lebeer S, anderleyden J, De Keersmaecker SC. Host interactions of probiotic bacterial
surface molecules: comparison with commensals and pathogens. Nature eviews
Microbiology. 2010;8(3):171-84.
4. Corr SC, Hill C, Gahan CG. Understanding the mechanisms by which probiotics inhibit
gastrointestinal pathogens. Advances in food and nutrition research. 2009;56:1-15.
5. Krishna ao , Samak G. Protection and restitution of gut barrier by probiotics: nutritional
and clinical implications. Current Nutrition Food Science. 2013;9(2):99-107.
6. Mack D, Ahrné S, Hyde L, Wei S, Hollingsworth MA. Extracellular MUC3 mucin secretion
follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro. Gut. 2003;52
(6):827-33.
7. Abreu MT. Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition
shapes intestinal function. Nature eviews Immunology. 2010;10(2):131-44.
8. Wells JM, editor Immunomodulatory mechanisms of lactobacilli. Microbial cell factories;
2011: BioMed Central.
9. Doron S, Snydman D , Gorbach SL. Lactobacillus GG: bacteriology and clinical applications. Gastroenterology Clinics. 2005;34(3):483-98.
10. Segers ME, Lebeer S, editors. Towards a better understanding of Lactobacillus rhamnosus
GG-host interactions. Microbial cell factories; 2014: BioMed Central.
11. Kankainen M, Paulin L, Tynkkynen S, von Ossowski I, eunanen J, Partanen P, et al.
Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a
human-mucus binding protein. Proceedings of the National Academy of Sciences. 2009;106
(40):17193-8.
12. Tuomola EM, Ouwehand AC, Salminen SJ. The effect of probiotic bacteria on the adhesion
of pathogens to human intestinal mucus. FEMS Immunology Medical Microbiology.
1999;26(2):137-42.
13. McGuire S. US department of agriculture and US department of health and human services,
dietary guidelines for americans, 2010. Washington, DC: US government printing office, January 2011. Oxford University Press; 2011.
14. athore A, Bhambure , Ghare . Process analytical technology (PAT) for
biopharmaceutical products. Analytical and bioanalytical chemistry. 2010;398(1):137-54.
15. He S, Wang H, Wu B, hou H, hu P, Yang , et al. esponse surface methodology
optimization of fermentation conditions for rapid and efficient accumulation of macrolactin A
by marine Bacillus amyloliquefaciens ESB-2. Molecules. 2013;18(1):408-17.
16. Lei H, hao H, Yu , hao M. Effects of wort gravity and nitrogen level on fermentation
performance of brewer’s yeast and the formation of flavor volatiles. Applied biochemistry and
biotechnology. 2012;166(6):1562-74.
17. Salihu A, Bala M, Bala SM. Application of Plackett-Burman experimental design for lipase
production by Aspergillus niger using shea butter cake. International Scholarly esearch
Notices. 2013.
18. Alvarez M, Aguirre-Ezkauriatza E, amírez-Medrano A, odríguez-S nchez Á. Kinetic
analysis and mathematical modeling of growth and lactic acid production of Lactobacillus
casei var. rhamnosus in milk whey. Journal of dairy science. 2010;93(12):5552-60.
19. Khoshayand F, Goodarzi S, Shahverdi A , Khoshayand M . Optimization of culture
conditions for fermentation of soymilk using Lactobacillus casei by response surface
methodology. Probiotics and antimicrobial proteins. 2011;3(3-4):159-67.
20. Pedram N, Ataei S. Optimization of a Modified GS Medium for a Probiotic Strain
(L. acido hilus ATCC4356). 2014.
21. Hwang C-F, Chang J-H, Houng J-Y, Tsai C-C, Lin C-K, Tsen H-Y. Optimization of medium
composition for improving biomass production of Lactobacillus lantarum Pi06 using the
Taguchi array design and the Box-Behnken method. Biotechnology and Bioprocess
Engineering. 2012;17(4):827-34.
22. Chang C, Liew S. Growth Medium Optimization for Biomass Production of a Probiotic
Bacterium, L. actobacillus rhamnosus ATCC 746. Journal of Food Biochemistry. 2013;37
(5):536-
23. Bern rdez PF, Amado I , Castro LP, Guerra NP. Production of a potentially probiotic culture
of Lactobacillus casei subsp. casei CECT 4043 in whey. International Dairy Journal. 2008;18
(10-11):1057-65.
24. Aguirre-Ezkauriatza E, Aguilar-Y ñez J, amírez-Medrano A, Alvarez M. Production of
probiotic biomass (Lactobacillus casei) in goat milk whey: Comparison of batch, continuous
and fed-batch cultures. Bioresource Technology. 2010;101(8):2837-44.
25. Ming LC, Halim M, Abd ahim , Wan HY, Ariff AB. Strategies in fed-batch cultivation on
the production performance of Lactobacillus salivarius I 24 viable cells. Food science and
biotechnology. 2016;25(5):1393-8.
26. Krzywonos M, Eberhard T. High density process to cultivate Lactobacillus lantarum
biomass using wheat stillage and sugar beet molasses. Electronic Journal of Biotechnology.
2011;14(2):6-.
27. hang B, Shu G, Bao C, Cao J, Tan Y. Optimization of Culture Medium for Lactobacillus
bulgaricus using Box-Behnken Design. Acta Universitatis Cibiniensis Series E: Food
Technology. 2017;21(1):3-10.
28. Coelho L, De Lima C, odovalho C, Bernardo M, Contiero J. Lactic acid production by new
Lactobacillus lantarum LMISM6 grown in molasses: optimization of medium composition.
Brazilian Journal of Chemical Engineering. 2011;28(1):27-36.
29. Beitel SM, Coelho LF, Contiero J. Efficient Conversion of Agroindustrial Waste into D (-)
Lactic Acid by Lactobacillus delbrueckii Using Fed-Batch Fermentation. BioMed esearch
International.;2020.
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varieties of Iranian native olives and investigation of their antimicrobial activity against two
pathogenic members of Entrobacteriaceae. Biological Journal of Microorganism. 2015;4(13).
2. Lebeer S, anderleyden J, De Keersmaecker SC. Genes and molecules of lactobacilli
supporting probiotic action. Microbiology and Molecular Biology eviews. 2008;72(4):
728-64.
3. Lebeer S, anderleyden J, De Keersmaecker SC. Host interactions of probiotic bacterial
surface molecules: comparison with commensals and pathogens. Nature eviews
Microbiology. 2010;8(3):171-84.
4. Corr SC, Hill C, Gahan CG. Understanding the mechanisms by which probiotics inhibit
gastrointestinal pathogens. Advances in food and nutrition research. 2009;56:1-15.
5. Krishna ao , Samak G. Protection and restitution of gut barrier by probiotics: nutritional
and clinical implications. Current Nutrition Food Science. 2013;9(2):99-107.
6. Mack D, Ahrné S, Hyde L, Wei S, Hollingsworth MA. Extracellular MUC3 mucin secretion
follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro. Gut. 2003;52
(6):827-33.
7. Abreu MT. Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition
shapes intestinal function. Nature eviews Immunology. 2010;10(2):131-44.
8. Wells JM, editor Immunomodulatory mechanisms of lactobacilli. Microbial cell factories;
2011: BioMed Central.
9. Doron S, Snydman D , Gorbach SL. Lactobacillus GG: bacteriology and clinical applications. Gastroenterology Clinics. 2005;34(3):483-98.
10. Segers ME, Lebeer S, editors. Towards a better understanding of Lactobacillus rhamnosus
GG-host interactions. Microbial cell factories; 2014: BioMed Central.
11. Kankainen M, Paulin L, Tynkkynen S, von Ossowski I, eunanen J, Partanen P, et al.
Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a
human-mucus binding protein. Proceedings of the National Academy of Sciences. 2009;106
(40):17193-8.
12. Tuomola EM, Ouwehand AC, Salminen SJ. The effect of probiotic bacteria on the adhesion
of pathogens to human intestinal mucus. FEMS Immunology Medical Microbiology.
1999;26(2):137-42.
13. McGuire S. US department of agriculture and US department of health and human services,
dietary guidelines for americans, 2010. Washington, DC: US government printing office, January 2011. Oxford University Press; 2011.
14. athore A, Bhambure , Ghare . Process analytical technology (PAT) for
biopharmaceutical products. Analytical and bioanalytical chemistry. 2010;398(1):137-54.
15. He S, Wang H, Wu B, hou H, hu P, Yang , et al. esponse surface methodology
optimization of fermentation conditions for rapid and efficient accumulation of macrolactin A
by marine Bacillus amyloliquefaciens ESB-2. Molecules. 2013;18(1):408-17.
16. Lei H, hao H, Yu , hao M. Effects of wort gravity and nitrogen level on fermentation
performance of brewer’s yeast and the formation of flavor volatiles. Applied biochemistry and
biotechnology. 2012;166(6):1562-74.
17. Salihu A, Bala M, Bala SM. Application of Plackett-Burman experimental design for lipase
production by Aspergillus niger using shea butter cake. International Scholarly esearch
Notices. 2013.
18. Alvarez M, Aguirre-Ezkauriatza E, amírez-Medrano A, odríguez-S nchez Á. Kinetic
analysis and mathematical modeling of growth and lactic acid production of Lactobacillus
casei var. rhamnosus in milk whey. Journal of dairy science. 2010;93(12):5552-60.
19. Khoshayand F, Goodarzi S, Shahverdi A , Khoshayand M . Optimization of culture
conditions for fermentation of soymilk using Lactobacillus casei by response surface
methodology. Probiotics and antimicrobial proteins. 2011;3(3-4):159-67.
20. Pedram N, Ataei S. Optimization of a Modified GS Medium for a Probiotic Strain
(L. acido hilus ATCC4356). 2014.
21. Hwang C-F, Chang J-H, Houng J-Y, Tsai C-C, Lin C-K, Tsen H-Y. Optimization of medium
composition for improving biomass production of Lactobacillus lantarum Pi06 using the
Taguchi array design and the Box-Behnken method. Biotechnology and Bioprocess
Engineering. 2012;17(4):827-34.
22. Chang C, Liew S. Growth Medium Optimization for Biomass Production of a Probiotic
Bacterium, L. actobacillus rhamnosus ATCC 746. Journal of Food Biochemistry. 2013;37
(5):536-
23. Bern rdez PF, Amado I , Castro LP, Guerra NP. Production of a potentially probiotic culture
of Lactobacillus casei subsp. casei CECT 4043 in whey. International Dairy Journal. 2008;18
(10-11):1057-65.
24. Aguirre-Ezkauriatza E, Aguilar-Y ñez J, amírez-Medrano A, Alvarez M. Production of
probiotic biomass (Lactobacillus casei) in goat milk whey: Comparison of batch, continuous
and fed-batch cultures. Bioresource Technology. 2010;101(8):2837-44.
25. Ming LC, Halim M, Abd ahim , Wan HY, Ariff AB. Strategies in fed-batch cultivation on
the production performance of Lactobacillus salivarius I 24 viable cells. Food science and
biotechnology. 2016;25(5):1393-8.
26. Krzywonos M, Eberhard T. High density process to cultivate Lactobacillus lantarum
biomass using wheat stillage and sugar beet molasses. Electronic Journal of Biotechnology.
2011;14(2):6-.
27. hang B, Shu G, Bao C, Cao J, Tan Y. Optimization of Culture Medium for Lactobacillus
bulgaricus using Box-Behnken Design. Acta Universitatis Cibiniensis Series E: Food
Technology. 2017;21(1):3-10.
28. Coelho L, De Lima C, odovalho C, Bernardo M, Contiero J. Lactic acid production by new
Lactobacillus lantarum LMISM6 grown in molasses: optimization of medium composition.
Brazilian Journal of Chemical Engineering. 2011;28(1):27-36.
29. Beitel SM, Coelho LF, Contiero J. Efficient Conversion of Agroindustrial Waste into D (-)
Lactic Acid by Lactobacillus delbrueckii Using Fed-Batch Fermentation. BioMed esearch
International.;2020.
