Effects of supplementation of citrulline and Lactobacillus helveticus in intestinal tract

Abstract

Citrulline has been considered as a functional amino acid due to its ability to re-generate arginine and nitric oxide (NO) production. Lactobacillus helveticus exhibits probiotic properties to contribute various health-promoting effects. Citrulline and L. helveticus have been reported to show the positive actions in gastrointestinal tract. Additionally, some LAB are found to utilize arginine/citrulline via arginine deiminase (ADI) pathway to produce ATP. The ATP produced may provide extra energy to improve the bacterial growth. An adequate amount of live LAB is important for maintaining their health-promoting effect. Fermented milk (FM) has been used as functional food. However, the survival of LAB in FM decreases during storage. The ATP generated via ADI pathway may be another factor to maintain the survival of LAB in FM. L. helveticus is able to utilize citrulline via ADI pathway, thus, combination of citrulline and L. helveticus would be considered to have a synergistic effect to maximize their beneficial effects. We firstly investigated the effect of addition of citrulline with L. helveticus ACSS 511 (LH511) in FM, and we found that the cell concentration of LH511 was higher than the control when incubated with 4-mM citrulline after 14 days of storage. We concluded that co-incubation with citrulline may preserve the cell concentration of LH511. Secondly, we compared the effect of citrulline for cell growth of different LAB and investigated the anti-bacterial and NO production effects in selected LAB. The results suggested that LH511 had significant effect on NO production and anti-bacterial effects on addition with citrulline than other LAB. The effect of combination of citrulline and LH511 on intestinal epithelial barrier functions was investigated in IPEC-J2 cell lines. The effect of anti-adhesion, trans-epithelial electrical resistance (TEER) and expression of tight-junction (TJ) proteins of arginine, citrulline, LH511 and the combination of LH511+citrulline were examined and compared. It was found that citrulline exhibited greater effect than arginine and the combination of citrulline+LH511 generally contributed the strongest ability among all the treatments. The enriched citrulline FM with LH511 (Cit_LHFM) was applied to the intestinal epithelium, and its protective effect against pathogenic lipopolysaccharides (LPS) infection and the potential mechanisms were investigated. The results showed that Cit_LHFM significantly attenuated the damage in TJ integrity induced by LPS and it modulated the excessive inflammation via regulating the toll-like receptors (TLRs) mediated Akt-signalling pathway. Finally, the effect of Cit_LHFM was examined in a mice model with dextran sulfate sodium (DSS)-induced colitis. When we compared the effect of milk containing citrulline, FM with LH511 and Cit_LHFM on protecting the injury caused by DSS, it was observed that all treatments ameliorated the clinical signs induced by DSS. Importantly, Cit_LHFM showed an outstanding ability in restoring the intestinal barrier integrity and functions as compared with the other treatments.The results demonstrated that Cit_LHFM could enhance the intestinal barrier functions and therefore it is suggested that it could be used as a novel functional food product.