Whey protein was digested with one of seven kinds of proteases at 37 degrees C (trypsin, proteinase K, actinase E, thermolysin, or papain) or at 25 degrees C (pepsin or chymotrypsin) for 24 h. The digested samples were assayed for the inhibitory activity of angiotensin-converting enzyme and for changes in the systolic blood pressure caused in spontaneously hypertensive rats after gastric intubation. The strongest depressive effect on the systolic blood pressure (-55 mm Hg) was observed at 6 h after gastric intubation of the whey protein that was digested by proteinase K. Finally, six peptides were chromatographically isolated from the proteinase K digest by a combination of hydrophobic reversed-phase HPLC and gel filtration. The amino acid sequences and their origins were clarified as follows: Val-Tyr-Pro-Phe-Pro-Gly [beta-casein (CN); f 59-64], Gly-Lys-Pro (beta 2-microglobulin; f 18-20), Ile-Pro-Ala (beta-lactoglobulin; f 78-80), Phe-Pro (serum albumin; f 221-222; beta-CN, f 62-63, f 157-158, and f 205-206), Val-Tyr-Pro (beta-CN; f 59-61), and Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro (beta-CN; f 80-90). Chemical synthesis of these six peptides confirmed that all peptides, except an undecapeptide, have antihypertensive activity in spontaneously hypertensive rats. The synthetic tripeptide Ile-Pro-Ala, originating from beta-lactoglobulin, showed the strongest antihypertensive activity (-31 mm Hg).

Background: Some studies have shown that nasally administered immunobiotics had the potential to improve the outcome of influenza virus infection. However, the capacity of immunobiotics to improve protection against respiratory syncytial virus (RSV) infection was not investigated before. Objective: The aims of this study were: a) to evaluate whether the nasal administration of Lactobacillus rhamnosus CRL1505 (Lr05) and L. rhamnosus CRL1506 (Lr06) are able to improve respiratory antiviral defenses and beneficially modulate the immune response triggered by TLR3/RIG-I activation; b) to investigate whether viability of Lr05 or Lr06 is indispensable to modulate respiratory immunity and; c) to evaluate the capacity of Lr05 and Lr06 to improve the resistance of infant mice against RSV infection. Results: Nasally administered Lr05 and Lr06 differentially modulated the TLR3/RIG-I-triggered antiviral respiratory immune response. Lr06 administration significantly modulated the production of IFN-α, IFN-β and IL-6 in the response to poly(I:C) challenge, while nasal priming with Lr05 was more effective to improve levels of IFN-γ and IL-10. Both viable Lr05 and Lr06 strains increased the resistance of infant mice to RSV infection while only heat-killed Lr05 showed a protective effect similar to those observed with viable strains. Conclusions: The present work demonstrated that nasal administration of immunobiotics is able to beneficially modulate the immune response triggered by TLR3/RIG-I activation in the respiratory tract and to increase the resistance of mice to the challenge with RSV. Comparative studies using two Lactobacillus rhamnosus strains of the same origin and with similar technological properties showed that each strain has an specific immunoregulatory effect in the respiratory tract and that they differentially modulate the immune response after poly(I:C) or RSV challenges, conferring different degree of protection and using distinct immune mechanisms. We also demonstrated in this work that it is possible to beneficially modulate the respiratory defenses against RSV by using heat-killed immunobiotics.

The effect ofLactobacillus jenseniiTL2937 on the inflammatory immune response triggered by enterotoxigenicEscherichia coli(ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated thatL. jenseniiTL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated thatL. jenseniiTL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action.L. jenseniiTL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.

Aims: To characterize the adhesion molecule of Lactobacillus plantarum LA 318 that shows high adhesion to human colonic mucin (HCM). Methods and Results: The adhesion test used the BIACORE assay where PBS‐washed bacterial cells showed a significant decrease in adherence to HCM than distilled water‐washed cells. A component in the PBS wash fraction adhered to the HCM and a main protein was detected as a c. 40‐kDa band using SDS‐PAGE. Using homology comparisons of the N‐terminal amino acid sequences compared with sequence databases, this protein was identified as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). The DNA sequence of LA 318 GAPDH was 100% identical to the GAPDH (gapB) of L. plantarum WCFS1. The purified GAPDH adhered to HCM. Conclusions: We found the adhesin of L. plantarum LA 318 to HCM in its culture PBS wash fraction. The molecule was identified as GAPDH. Because LA 318 possesses the same adhesin as many pathogens, the lactobacilli GAPDH may compete with pathogens infecting the intestine. Significance and Impact of the Study: This is the first report showing GAPDH expressed on the cell surface of lactobacilli adheres to mucin suggesting L. plantarum LA 318 adheres to HCM using GAPDH binding activity to colonize the human intestinal mucosa.