Technological Aspects for Development of an Anti-Inflammatory Product of Lactobacillus reuteri CRL1098

LUCIANA GEREZ; GRACIELA FONT DE VALDEZ; MILAGROS GRIET; ANA VIRGINIA RODRIGUEZ

San Miguel de Tucumán

Simposio; V Internacional Symposium on Lactic Acid Bacteria; 2016

CERELA-CONICET

Several studies have demonstrated that probiotic lactic acid bacteria (LAB) can exert their beneficial effect through their immunomudulatory activity. Some specific soluble factors secreted by probiotic LAB are responsible for these beneficial effects. Our previous studies demonstrated that a Lactobacillus reuteri CRL1098 soluble factor identified as a 5785 Da peptide (LrS) modulates the inflammatory response triggered by lipopolysaccharide (LPS) in vitro and in a mice model. The microbial biomass and bioactive soluble factors are considered raw material for probiotic product development. Therefore, the cost of the culture media for industrial production of biomass and metabolites is a determinant factor. Previously, to reduce the cost of the growth medium, we formulated a minimum medium named PETG using a fractional factorial model. The aim of this work was to evaluate different factors, such as: agitation, pH and growth phase on biomass production and anti-inflammatory activity of L. reuteri CRL1098 using PETG medium. The batch fermentation was carried out in PETG medium without pH adjustment under static and agitating conditions using bottles (100 mL). Further, the effect of growth and pH was evaluated in a laboratory bioreactor (1.5 L) with continuous agitation (150 rpm) under free and controlled pH (5.5). In all conditions, viability and anti-inflammatory activity were evaluated at both early and late bacterial exponential growth phase. The biomass production was estimated by cell counting in MRS agar. The LrS anti-inflammatory activity produced from this biomass was evaluated using a model of mouse macrophages cell line (RAW 264.7) under non-inflammatory conditions and after the pro-inflammatory challenge with LPS. As inflammatory markers, TNF-α and nitric oxide (NO) were determined in the supernatant culture by ELISA and Griess method, respectively. The highest biomass (8 Log cfu/mL) was similar at both free and controlled pH conditions. This value was achieved earlier at controlled pH conditions (4 h) and after 6 h at free pH. However, the best anti-inflammatory response was observed in LrS obtained from bacteria cultures in early and late exponential growth phase at free pH: NO decrease by 54% and 52%, respectively. These results represent an advance in the search of the most suitable strategies for the production of anti-inflammatory product from a LAB.