Lyophilized cell-free supernatants of Lactobacillus isolates exhibited antibiofilm, antioxidant, and reduces nitric oxide activity in lipopolysaccharide-stimulated RAW 264.7 cells

Abstract

Background Probiotics can release bioactive substances known as postbiotics, which can inhibit pathogenic microorganisms, improve immunomodulation, reduce antioxidant production, and modulate the gut microbiota. Methods In this study, we evaluated the in vitro antimicrobial effects, antioxidant activity, and anti-inflammatory potential of 10 lyophilized cell-free supernatants (LCFS) of Lactobacillus isolates. LCFS was obtained via centrifugation and subsequent lyophilization of the supernatant collected from the culture medium ofeach isolate. The antibacterial and antibiofilm activities of the LCFS were determined using broth microdilution. The antioxidant potential was evaluated by measuring the total phenolic and flavonoid contents and 2,2-Diphennyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS+) radical scavenging activities. Results All the isolates were able to inhibit the four tested pathogens. The isolates exhibited strong antibiofilm activity and eradicated the biofilms formed by Acinetobacter buamannii and Escherichia coli. All the prepared Lactobacillus LCFS contained phenols and flavonoids and exhibited antioxidant activities in the DPPH and ABTS+ radical scavenging assays. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay revealed that LCFS was not cytotoxic to RAW 264.7 cells. In addition, the ten Lactobacillus LCFS decreased the production of nitric oxide. Conclusions All the isolates have beneficial properties. This research sheds light on the role of postbiotics in functional fermented foods and pharmaceutical products. Further research to elucidate the precise molecular mechanisms of action of probiotics is warranted.

Cinnamomum sp. and Pelargonium odoratissimum as the Main Contributors to the Antibacterial Activity of the Medicinal Drink Horchata: A Study Based on the Antibacterial and Chemical Analysis of 21 Plants

“…An overnight culture of the bacterial strains was used to prepare a bacterial suspension with a final concentration of 5 × 10 5 CFU/mL in MHB. Then, 100 µL of the bacterial suspension was added to 96-well flat bottom plates (Tecan Group Ltd., Mannedorf, Switzerland) and supplemented with crude plant extracts at 1× and 2× MIC concentrations [ 55 ]. Plates were then incubated for 24 h at 37 °C under aerobic conditions.…”

“…Subsequently, the media was removed from the wells, and each well was washed three times with Dulbecco’s phosphate buffered saline (PBS, pH 7.4; Sigma-Aldrich, St. Louis, MO, USA), and each biofilm sample in the well was fixed with 200 µL of methanol (99% v / v ; Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Crystal violet staining (0.1% w / v ; Sigma-Aldrich, St. Louis, MO, USA) was performed for 10 min [ 55 , 56 ], followed by four washing steps with distilled water. Ethanol (95% v / v ; Sigma-Aldrich, St. Louis, MO, USA) was used to dissolve the biofilm, and its inhibition was measured by spectrophotometry in the ELISA Elx808 spectrophotometer (BioTek, Winooski, GU, USA) at an optical density of 570 nm [ 55 ] by comparing with positive controls (bacteria growth in media only).…”

“…Crystal violet staining (0.1% w / v ; Sigma-Aldrich, St. Louis, MO, USA) was performed for 10 min [ 55 , 56 ], followed by four washing steps with distilled water. Ethanol (95% v / v ; Sigma-Aldrich, St. Louis, MO, USA) was used to dissolve the biofilm, and its inhibition was measured by spectrophotometry in the ELISA Elx808 spectrophotometer (BioTek, Winooski, GU, USA) at an optical density of 570 nm [ 55 ] by comparing with positive controls (bacteria growth in media only). Finally, the percentage of biofilm inhibition was calculated.…”

“…Subsequently, the MHB was removed from the wells, and a wash with PBS (pH 7.4) was performed. The biofilms were then supplemented with crude plant extracts at 1× and 2× MIC values in flesh media [ 55 ]. Plates were again incubated for 24 h at 37 °C under aerobic conditions.…”

Investigation Of The Antibiofilm Effects Of Cell-free Supernatants Of Probiotic Bacteria Against The Biofilm Formed By Bacteria Obtained From The Ocular Surface

“…Araştırıcılar bunun hücresiz filtrat içinde bulunan antimikrobiyel maddelere P. aeruginosa'nın direncini göstermişlerdir[25] Laktik asit bakterilerinin antimikrobiyel aktivitesi organik asitler, yağ asitleri, hidrojen peroksit, antimikrobiyal peptitler ve bakteriyosinlerin üretimi gibi farklı mekanizmalar yoluyla olmaktadır[12][13][14][15][16][17][18]24,26] Bulguların farklılığı test patojenlerinin farklı kaynaklardan izole edilmiş olmasından kaynaklanabileceği gibi kullanılan filtratın yoğunluğundan da kaynaklanıyor olabilir.Laktik asit bakterilerinin hücresiz filtratlarının antibiyofilm etkileri son dönemde yapılan çalışmalarda gösterilmiştir[27,28]. Fermente palmiye özütünden elde edilen Lactobacillus brevis'in hücresiz filtratının hem A. baumanii hem E. coli'nin oluşturduğu biofilm üzerine yüksek inhibitör etkisi olduğu görülmüştür[27]. Avokado ve salatalıktan izole edilen L. monocytogenes suşlarına karşı fermente mısır gevreğinden izole edilen Lactobacillus brevis ve hazır L. acidophilus ve Lacticaseibacillus rhamnosus suşları kullanılmıştır.…”