Adaptação Laboratorial Evolutiva para obtenção de cepas de bactérias ácido-lácticas de interesse industrial - uma revisão

Tirloni, Luana; Heidrich, Daiane; Souza, Claucia Fernanda Volken de

Acessibilidade / Reportar erro

Brasil

Brazilian Journal of Food Technology

Español English

Brasil

Español English

sumário « anterior atual seguinte »

Sumário

REVIEW ARTICLE • Braz. J. Food Technol. 26 • 2023 • https://doi.org/10.1590/1981-6723.05323 copiar

Adaptação Laboratorial Evolutiva para obtenção de cepas de bactérias ácido-lácticas de interesse industrial - uma revisão

Autoria SCIMAGO INSTITUTIONS RANKINGS

Resumo

O objetivo deste artigo de revisão foi descrever como a adaptação laboratorial evolutiva (ALE) pode proporcionar o melhoramento de cepas de bactérias ácido-lácticas (BAL) para aplicação em processos biotecnológicos industriais. A revisão foi realizada de acordo com a abordagem Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA), empregando as bases de dados ScienceDirect e Scopus. A busca bibliográfica resultou em 4.167 e 27 documentos, respectivamente, os quais reduziram-se para 12 após a aplicação dos critérios de inclusão/exclusão. Os trabalhos utilizaram BAL dos gêneros Lactobacillus, Lactococcus, Leuconostoc e Enterococcus para a ALE, empregando o método batelada ou batelada alimentada ou ambos, e tinham como objetivo a geração de cepas com maior capacidade de produção de ácido láctico, viabilidade celular e tolerância a diferentes condições de estresse. Os estudos demonstraram que a ALE é uma ferramenta eficiente no melhoramento de cepas para os fenótipos desejados, além de não empregar engenharia genética. O conhecimento das respostas celulares e moleculares dos microrganismos ao estresse possibilita a compreensão dos mecanismos de adaptação das cepas de BAL para sobrevivência e maior produção de metabólitos ao longo da ALE.

Palavras-chave:
Melhoramento; Evolução; Metabolismo; Lactobacillus; Lactococcus; Leuconostoc

Lactic acid bacteria^* * NS: not shown. LA: lactic acid; SNV: single nucleotide variation; D-LA: D-lactic acid; SHF: separate hydrolysis and fermentation;	Adaptive conditions	ALE method	Parameters	Evaluations	Main findings	References
Lactiplantibacillus pentosus CECT4023T	20 g L^-1 xylose 32 °C pH 7.0	Batch mode	Shaking: 150 rpm Cycles: NS Time between passages: 24 h Generations: 850	Lactic fermentation tests in bioreactor LA yield Xylose consumption	The evolved strain showed 2-fold more production of LA and 1.5-fold more consumption of xylose compared with the original strain.	Cubas-Cano et al., 2019Cubas-Cano, E., González-Fernández, C., & Tomás-Pejó, E. (2019). Evolutionary engineering of Lactobacillus pentosus improves lactic acid productivity from xylose-rich media at low pH. Bioresource Technology, 288, 121540. PMid:31174085. http://dx.doi.org/10.1016/j.biortech.2019.121540 http://dx.doi.org/10.1016/j.biortech.201...
Lacticaseibacillus paracasei 4564	100 g L^-1 sugar beet molasses pH 6.5 41 °C	Batch mode (first phase) Fed-batch mode (second phase) and pulsed fed-batch mode (third phase)	Shaking: 100 rpm Cycles: NS Time between passages: 20-24 h Generations: NS	Antioxidant activity DPPH Free Radical-Scavenging Activity LA-production capability	The evolved strain showed: one-fold higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging rates compared with the original strain. The LA production (89.4 g L^-1) of the evolved strain was superior to the original. The evolved strain showed a 59% higher capability to produce LA.	Mladenović et al., 2019Mladenović, D., Pejin, J., Kocić-Tanackov, S., Djukić-Vuković, A., & Mojović, L. (2019). Enhanced lactic acid production by adaptive evolution of Lactobacillus paracasei on agro-industrial substrate. Applied Biochemistry and Biotechnology, 187(3), 753-769. PMid:30058018. http://dx.doi.org/10.1007/s12010-018-2852-x http://dx.doi.org/10.1007/s12010-018-285...
Lacticaseibacillus casei	37 °C pH 5.5, 5.0, 4.6, and 4.3 75 days	Batch mode	Shaking: NS Cycles: NS Time between passages: NS Generations: NS	Cell counts Intracelular pH Intracelular amino acid determination Lactate and acetate analyses Membrane permeability	The evolved strain showed a 318-fold higher survival rate than the original strain and exhibited a slightly higher production of lactate (13.6%) and 65.6% higher amount of acetate. The intracellular pH of the evolved strain was higher with lower inner membrane permeability. The survival rate of the evolved strain was increase in 1.36-, 2.10-, or 3.42-fold by the addition of 50 mM aspartate, arginine, and both, respectively.	Zhang et al., 2012Zhang, J., Wu, C., Du, G., & Chen, J. (2012). Enhanced acid tolerance in Lactobacillus casei by adaptive evolution and compared stress response during acid stress. Biotechnology and Bioprocess Engineering, 17(2), 283-289. http://dx.doi.org/10.1007/s12257-011-0346-6 http://dx.doi.org/10.1007/s12257-011-034...
Lacticaseibacillus casei Zhang	Amoxicillin (0.5 μg/mL) and Gentamicin (1 μg/mL)	-	Shaking: NS Cycles: NS Time between passages: 24 h Generations: 1200	Evaluation of fitness as represented by minimum inhibitory concentrations (MICs) Identification of new mutations	The MIC for amoxicillin increased to a maximum level (8 μg/mL) for the evolved strains. There was a 4-fold increase in mutation accumulation (including SNVs, short insertions and deletions) in the evolved strains comparing to the control.	Wang et al., 2017Wang, J., Dong, X., Shao, Y., Guo, H., Pan, L., Hui, W., Kwok, L. Y., Zhang, H., & Zhang, W. (2017). Genome adaptive evolution of Lactobacillus casei under long-term antibiotic selection pressures. BMC Genomics, 18(1), 320. PMid:28438179. http://dx.doi.org/10.1186/s12864-017-3710-x http://dx.doi.org/10.1186/s12864-017-371...
Lacticaseibacillus casei	37 °C pH 5.5, 5.0, 4.6 and 4.3 70 days	Batch mode	Shaking: NS Cycles: NS Time between passages: 12 h Generations: 50	Cell counts Bile tolerance Intracelular pH	ALE resulted in 1.6-fold higher biomass in evolved strain and higher resistance to bile salt. The evolved strain showed the capability of maintaining a higher pH compared to that of the original strain.	Ming et al., 2016Ming, H., Xu, D., Guo, Z., & Liu, Y. (2016). Adaptive evolution of Lactobacillus casei under acidic conditions enhances multiple-stress tolerance. Food Science and Technology Research, 22(3), 331-336. http://dx.doi.org/10.3136/fstr.22.331 http://dx.doi.org/10.3136/fstr.22.331...
Lactobacillus delbrueickii Uc-3 (NCIM 5219)	pH 4.5	Fed-batch mode	Shaking: NS Cycles: 40 Time between passages: 48 h Generations: NS	LA concentration, productivity, and yield H+-ATPase activity determination Analysis of H+-ATPase gene expression by one-step quantitative real-time PCR	The evolved strain demonstrated a 1.80-fold increase in LA production, but its viability was reduced due to the acidic pH and/or end-product inhibition There were higher H+- ATPase activity and a 415-fold increase in H+-ATPase gene expression compared to the original strain.	Singhvi et al., 2018Singhvi, M., Zendo, T., & Sonomoto, K. (2018). Free lactic acid production under acidic conditions by lactic acid bacteria strains: Challenges and future prospects. Applied Microbiology and Biotechnology, 102(14), 5911-5924. PMid:29804138. http://dx.doi.org/10.1007/s00253-018-9092-4 http://dx.doi.org/10.1007/s00253-018-909...
Lacticaseibacillus rhamnosus	−30 °C 13 °C 37 °C	Batch mode	Shaking: NS Cycles: 150 Time between passages: 16 h Generations: NS	Viability Growth Dynamics Whole Genome Resequencing	The evolved strain viability increased up to 93.65%. The exponential growth rates were faster, with a 13-min shorter mean doubling time than the original strain. Six gene regions and an intergenic space, related with encoding exopolysaccharide biosynthesis protein that could be responsible for the strain adaptation were discovered.	Kwon et al., 2018Kwon, Y. W., Bae, J. H., Kim, S. A., & Han, N. S. (2018). Development of freeze-thaw tolerant Lactobacillus rhamnosus GG by Adaptive Laboratory Evolution. Frontiers in Microbiology, 9, 2781. PMid:30524399. http://dx.doi.org/10.3389/fmicb.2018.02781 http://dx.doi.org/10.3389/fmicb.2018.027...
Lactobacillus delbrueckii subsp. bulgaricus ET45	37, 40 and 45 °C	Batch mode	Shaking: NS Cycles: NS Time between passages: NS Generations: NS	Enzymatic hydrolysis Production of D-LA by SHF	The enzymatic hydrolysis of the evolved strain showed 8% higher cellulose to D-LA conversion. There was an increase in the LA production by the evolved strain (108 g/L D-LA) and 60% conversion of cellulose to D-LA.	Prasad et al., 2020Prasad, J. V., Sahoo, T. K., Naveen, S., & Jayaraman, G. (2020). Evolutionary engineering of Lactobacillus bulgaricus reduces enzyme usage and enhances conversion of lignocellulosics to D-lactic acid by simultaneous saccharification and fermentation. Biotechnology for Biofuels, 13(1), 171. PMid:33088341. http://dx.doi.org/10.1186/s13068-020-01812-x http://dx.doi.org/10.1186/s13068-020-018...
Lactobacillus delbrueckii subsp. bulgaricus	52 °C	Batch mode	Shaking: NS Cycles: NS Time between passages: 48 h Generations: NS	Morphology Lactic acid production pH and viscosity Amino acids Volatile compounds	Changes were observed in the morphology of the evolved strain after ALE (changed from rod to filament (52 °C) to cocci after frozen storage). The evolved strain did not show LA production. The pH remains 6.84 ± 0.13. There was a 115- and 275-fold higher production of Arginine and Methionine (237.24 ± 5.94 and 98.83 ± 1.78 µg/100 g, respectively) by the evolved strain. The evolved strain showed an increase in diacetyl formation in milk.	Liang et al., 2021Liang, J., Yoo, M. J. Y., Seale, B., & Grazioli, G. (2021). Nutritional and volatile characterisation of milk inoculated with thermo-tolerant Lactobacillus bulgaricus through Adaptive Laboratory Evolution. Foods, 10(12), 2944. PMid:34945497. http://dx.doi.org/10.3390/foods10122944 http://dx.doi.org/10.3390/foods10122944...
Lactococcus lactis TM29	38, 39 and 40 °C	Batch mode	Shaking: NS Cycles: NS Time between passages: NS Generations: 860	Growth rate Physiological and transcriptomic characterization Next-Generation Sequencing	The evolved strain showed a 33% faster growth rate compared to the original strain and a 12% higher specific lactate production. Changes in the expression of single proteins (chaperone; riboflavin transporter) were observed. Two had pleiotropic effects (RNA polymerase) in the evolved strain after ALE. Deletion in 10 genes was also found to affect thermal tolerance significantly.	Chen et al., 2015Chen, J., Shen, J., Hellgren, L. I., Jensen, P. R., & Solem, C. (2015). Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate. Scientific Reports, 5(1), 14199. PMid:26388459. http://dx.doi.org/10.1038/srep14199 http://dx.doi.org/10.1038/srep14199...
Lactococcus lactis	10 AU/mL of Lcn972 30 °C for 8 transfers	-	Shaking: NS Cycles: NS Time between passages: 16 h Generations: 850	Milk Acidification and Production of Lactic Acid Minimal Inhibitory Concentration Nisin Production Resistance to Antimicrobials	The evolved strain was capable to acidify the milk (pH below 5.3). ALE increased the MIC of Lcn972, between 4- and 32-fold. Nisin production was not compromised by ALE. Genes were involved in stress response, detoxification modules, cell envelope biogenesis and/or nucleotide metabolism.	López-González et al., 2018bLópez-González, M. J., Escobedo, S., Rodríguez, A., Neves, A. R., Janzen, T., & Martínez, B. (2018b). Adaptive evolution of industrial Lactococcus lactis under cell envelope stress provides phenotypic diversity. Frontiers in Microbiology, 9, 2654. PMid:30455679. http://dx.doi.org/10.3389/fmicb.2018.02654 http://dx.doi.org/10.3389/fmicb.2018.026...
Lc. lactic ssp. cremoris MG1363	5 mM hydrogen peroxide 30 °C	Batch mode	Shaking: 200 rpm Cycles: NS Time between passages: 72 h Generations: NS	Determination of Bacterial Growth and Survival Acidification Test Vitamin K2 Extraction and Analysis Genome Sequencing Proteomics Analysis	The evolved strain showed a higher survival (dropped to 10⁷ CFU/mL at 48 h from the initial 10⁹ CFU/mL but further decreased to 10⁴ CFU/mL at 72 h). ALE improved the production of vitamin K2 (50-110% increase) by the evolved strain. Common mutations in ldh and gapb genes were found in the evolved strain. Proteomics analysis showed that 16 proteins were differently expressed by the evolved strain: glyceraldehyde 3-phosphate dehydrogenase (A2RIN9), universal stress protein A2 (A2RK64) and form amidopyrimidine-DNA glycosylase.	Liu et al., 2021Liu, Y., de Groot, A., Boeren, S., Abee, T., & Smid, E. J. (2021). Lactococcus lactis mutants obtained from Laboratory Evolution showed elevated vitamin K2 content and enhanced resistance to oxidative stress. Frontiers in Microbiology, 12, 746770. PMid:34721346. http://dx.doi.org/10.3389/fmicb.2021.746770 http://dx.doi.org/10.3389/fmicb.2021.746...

Instituto de Tecnologia de Alimentos - ITAL Av. Brasil, 2880, 13070-178 Campinas - SP / Brasil, Tel 55 19 3743-1762 - Campinas - SP - Brazil
E-mail: bjftsec@ital.sp.gov.br

Acompanhe os números deste periódico no seu leitor de RSS

[1] *Corresponding Author: Claucia Fernanda Volken de Souza, Universidade do Vale do Taquari (Univates), Programa de Pós-graduação em Biotecnologia, Laboratório de Biotecnologia de Alimentos, Avelino Talini, 171, Universitário, CEP: 95914-014, Lajeado/RS - Brasil, e-mail: claucia@univates.br