This paper presents an in vitro evaluation of antitumor properties through producing short-chain fatty acids and inducing interleukin 12. In addition, it offers the most important and functional probiotic properties of 24 Lactobacillus gasseri, Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Limosilactobacillus fermentum strains isolated from humans, foods, and fermented foods. To this end, survival in an acidic environment (pH = 2.5), tolerance in bile salt, viability in the presence of pepsin-pancreatin, adhesion percentage, antibiotic resistance, auto-aggregation, and potential percentage of co-aggregation are studied in contact with three human intestinal pathogens. These pathogens are Escherichia coli O157: H7 NCTC 12900, Salmonella enterica subsp. enterica ATCC 13076, and Listeria monocytogenes ATTC 7644. Also, in vitro induction amount of IL-12 in mouse splenocytes is investigated to evaluate antitumor properties by 19 strains of L. gasseri and L. plantarum along with the development of short-chain fatty acids (SCFA) by 5 strains of L. fermentum and L. acidophilus. Gas Chromatography Flame Ionization Detector (GC-FID) and enzyme-linked immunosorbent assay (ELISA) were used to measure short-chain fatty acids and IL-12, respectively. All strains had high viability under acidic conditions. The highest levels of pancreatin and pepsin resistance were found in strains LF56, LF57, LF55, OF, and F and strains LF56, LF57, and A7, respectively. All strains except LF56 had high resistance to bile salts. L. gasseri 54C had the highest average adhesion score (hydrophobicity) of 62.9 % among 19 strains. Despite the susceptibility of different strains of L. plantarum to the tested antibiotics, M8 and M11, S2G, A7, LF55, LF57, and 5G were resistant to kanamycin and chloramphenicol, respectively. Also, 21G was resistant to ampicillin, LF56 to tetracycline and M8, and M11, LF56, and 21G to Erythromycin. In addition, L. gasseri showed moderate resistance to ampicillin, erythromycin, and tetracycline, while L. fermentum ATCC 9338 showed good resistance to ampicillin, erythromycin, and chloramphenicol. In this respect, L. plantarum LF56 and gasseri 54C had the highest average auto-aggregation and co-aggregation against three pathogenic bacteria, respectively. The highest and lowest levels of acetic acid as short-chain fatty acids were produced by L. fermentum 19SH isolated from Horre 41.62 and L. fermentum 21SH from fermented seeds 27.047, respectively. Moreover, L. fermentum, with the OF code of traditional-fermented food origin, produced the most isobutyric acid, butyric acid, and valeric acid, with values of 0.6828, 0.74165, and 0.49915 mmol, respectively. L. fermentum isolated from the human origin with code F produced the most isovaleric acid of 1.1874 mmol. All the tested strains produced good propionic acid except L. fermentum 21SH from fermented seeds. Among strains, L. plantarum M11 isolated from milk and L. gasseri 52B from humans had the highest in vitro induction of IL-12, which is probably related to their cell wall compositions and structure.

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