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For bioactive milk peptides to be relevant to infant health, they must be released by gastrointestinal proteolysis and resist further proteolysis until they reach their site of activity. The intestinal tract is the likeliest site for most bioactivities, but it is currently unknown whether bioactive milk peptides are present therein. The purpose of the present study was to identify antimicrobial and bifidogenic peptides in the infant intestinal tract. Milk peptides were extracted from infant intestinal samples, and the activities of the bulk peptide extracts were determined by measuring growth of Escherichia coli, Staphylococcus aureus, and Bifidobacterium longum spp. infantis after incubation with serial dilutions. The peptide profiles of active and inactive samples were determined by peptidomics analysis and compared to identify candidate peptides for bioactivity testing. We extracted peptides from 29 intestinal samples collected from 16 infants. Five samples had antimicrobial activity against S. aureus and six samples had bifidogenic activity for B. infantis. We narrowed down a list of 6645 milk peptides to 11 candidate peptides for synthesis, of which 6 fully inhibited E. coli and S. aureus growth at concentrations of 2500 and 3000 µg/mL. This study provides evidence for the potential bioactivity of milk peptides in the infant intestinal tract.

Oral administration of engineered immunoglobulins has the potential to prevent enteric pathogen-induced diarrhea in infants. To prevent infection, these antibodies need to survive functionally intact in the proteolytic environment of the gastrointestinal tract. This research examined both ex vivo and in vivo the functional survival across infant digestion of palivizumab, a model FDA-approved recombinant antibody against respiratory syncytial virus (RSV) F protein. Palivizumab-fortified feed (formula or human milk), infant gastric, and intestinal samples were incubated to simulate in vivo digestion (ex vivo digestion). Palivizumab-fortified human milk was also fed to infants, followed by collection of gastric and intestinal samples (in vivo digestion). Palivizumab was purified from the samples of digestate using protein G spin columns followed by filtration through molecular weight cut-off membranes (30 kDa). Palivizumab functional survival across ex vivo and in vivo digestion was determined via an anti-idiotype ELISA and an RSV plaque reduction neutralization test. Palivizumab concentration and RSV neutralization capacity both decreased when incubated in intestinal samples (ex vivo study). The concentration and neutralization activity of orally-supplemented palivizumab also decreased across infant digestion (in vivo study). These results indicate that if recombinant IgGs were selected for oral supplementation to prevent enteric infections, appropriate dosing would need to account for degradation occurring in the digestive system. Other antibody formats, structural changes, or encapsulation could enhance survival in the infant gastrointestinal tract.

Background Potentially, orally administered antibodies specific to enteric pathogens could be administered to infants to prevent diarrheal infections, particularly in developing countries where diarrhea is a major problem. However, to prevent infection, such antibodies would need to resist degradation within the gastrointestinal tract. Methods Palivizumab, a recombinant antibody specific to respiratory syncytial virus (RSV), was used in this study as a model for examining the digestion of neutralizing antibodies to enteric pathogens in infants. The survival of this recombinant IgG1 across digestion in 11 infants was assayed via an anti-idiotype ELISA and RSV F protein-specific ELISA. Concentrations were controlled for any dilution or concentration that occurred in the digestive system using mass spectrometry-based quantification of co-administered, orally supplemented, indigestible polyethylene glycol (PEG-28). Results Binding activity of Palivizumab IgG1 decreased (26-99%) across each phase of in vivo digestion as measured by both anti-idiotype and RSV F protein-specific ELISAs. Conclusion Antibodies generated for passive protection of the infant gastrointestinal tract from pathogens will need to be more resistant to digestion than the model antibody fed to infants in this study, or provided in higher doses to be most effective. Impact Binding activity of palivizumab IgG1 decreased (26-99%) across each phase of in vivo infant digestion as measured by both anti-idiotype and RSV F protein-specific ELISAs. Palivizumab was likely degraded by proteases and changes in pH introduced in the gut. Antibodies generated for passive protection of the infant gastrointestinal tract from pathogens will need to be more resistant to digestion than the model antibody fed to infants in this study, or provided in higher doses to be most effective. The monoclonal antibody IgG1 tested was not stable across the infant gastrointestinal tract. The observation of palivizumab reduction was unlikely due to dilution in the gastrointestinal tract. The results of this work hint that provision of antibody could be effective in preventing enteric pathogen infection in infants. Orally delivered recombinant antibodies will need to either be dosed at high levels to compensate for digestive losses or be engineered to better resist digestion. Provision of enteric pathogen-specific recombinant antibodies to at-risk infants could provide a new and previously unexplored pathway to reducing the infection in infants. The strategy of enteric recombinant antibodies deserves more investigation throughout medicine as a novel means for treatment of enteric disease targets.

Oral administration of enteric pathogen-specific immunoglobulins may be an ideal approach for preventing infectious diarrhea in infants and children. For oral administration to be effective, antibodies must survive functionally intact within the highly proteolytic digestive tract. As an initial step toward assessing the viability of this approach, we examined the survival of palivizumab, a recombinant monoclonal antibody (IgG1κ), across infant digestion and its ability to neutralize respiratory syncytial virus (RSV). Human milk and infant digestive samples contain substances known to interfere with the RSV neutralization assay (our selected functional test for antibody survival through digestion), therefore, antibody extraction from the matrix was required prior to performing the assay. The efficacy of various approaches for palivizumab purification from human milk, infant's gastric and intestinal digestates, including casein precipitation, salting out, molecular weight cut-off, and affinity chromatography (protein A and G) were compared. Affinity chromatography using protein G with high-salt elution followed by 30-kDa molecular weight cut-off centrifugal filtration was the most effective technique for purifying palivizumab from human milk and infant digestates with a high yield and reduced background interference for the viral neutralization assay. This work is broadly applicable to the optimal isolation of antibodies from human milk and infant digesta for viral neutralization assays, enables the examination of how digestion affects the viral neutralization capacity of antibodies within milk and digestive samples, and paves the way for assessment of the viability of oral administration of recombinant antibodies as a therapeutic approach to prevent enteric pathogen-induced infectious diarrhea in infants.

Graphical abstract, Highlights • Naturally-occurring antibodies were more resistant to degradation than monoclonal antibodies. • Monoclonal sIgA was more resistant to degradation than IgG and IgA. • Monoclonal antibodies may need to be provided at a higher dose to compensate for digestive losses., To help rationally design an antibody for oral administration, we examined how different isotypes (IgG, IgA and sIgA) with the same variable sequence affect antibody stability across digestion. We compared the degradation of recombinant palivizumab (IgG1), and recombinant IgA and sIgA versions of palivizumab spiked in human milk to the degradation of naturally-occurring anti-respiratory syncytial virus (RSV) sIgA/IgA and IgG in human milk from four donors across gastric and intestinal phases of an in vitro model of infant digestion via a validated RSV F protein ELISA. Palivizumab IgG and IgA formats were less stable than the sIgA version after complete simulated gastrointestinal digestion: palivizumab IgG, IgA and sIgA decreased across complete simulated gastrointestinal digestion by 55%, 48% and 28%, respectively. Naturally-occurring RSV F protein-specific IgG was stable across digestion, whereas naturally-occurring sIgA/IgA was stable in the gastric phase but decreased 33% in the intestinal phase of simulated digestion.

Novel antioxidant and anti-inflammatory peptides were isolated from hydrolysates of Siamese crocodile (Crocodylus siamensis) hemoglobin. C. siamensis hemoglobin hydrolysates (CHHs) were obtained by pepsin digestion at different incubation times (2, 4, 6, and 8 H) at 37 °C and subjected to antioxidant and anti-inflammatory activity assessment. CHH obtained by 2-H hydrolysis (2H-CHH) showed the highest anti-inflammatory activity with respect to decreasing nitric oxide (NO) production, whereas the strongest antioxidant activity was found for 6-H hydrolysis (6H-CHH) against nitric oxide radicals. To evaluate the anti-inflammatory and antioxidant activity of individual peptide components, 2H-CHH and 6H-CHH were purified by semipreparative HPLC. Peptide fraction P57 isolated from 6H-CHH was found to exhibit the highest nitric oxide radical inhibition activity (32.0%). Moreover, purification of 2H-CHH yielded peptide fraction P16, which displayed a high efficacy in decreasing NO production of macrophage RAW 264.7 cells (83.2%) and significantly reduced proinflammatory cytokines and inflammatory mediators interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and prostaglandin-E2 (PGE2) production to about 2.0, 0.3, and 1.9 ng/mL, respectively. Using LTQ orbitrap XL mass spectrometry, active peptide sequences were identified as antioxidant KIYFPHF (KF7), anti-inflammatory SAFNPHEKQ (SQ9), and IIHNEKVQAHGKKVL (IL15). Additionally, CHHs simulated gastric and intestinal in vitro digestion positively contributed to antioxidant and anti-inflammatory activity. Taken collectively, the results of this work demonstrate that CHHs contain several peptides with anti-inflammatory and antioxidant properties, which may prove valuable as treatment or supplement against diseases associated with inflammation and oxidative stress.

Orally delivered antibodies may be useful for the prevention of enteric pathogen infection, but to be effective they need to survive intact across digestion through the gastrointestinal tract. As a test case, we fed a recombinant human antibody, palivizumab, spiked into human milk to four infants and collected gastric, intestinal and stool samples. We identified a tryptic peptide from palivizumab (LLIYDTSK) that differs from all endogenous human antibodies and used this for quantitation of the intact palivizumab. To account for dilution by digestive fluids, we co-fed a non-digestible, non-absorbable molecule-polyethylene glycol 28-quantified it in each sample and used this value to normalize the observed palivizumab concentration. The palivizumab peptide, a stable isotope-labeled synthetic peptide and polyethylene glycol 28 were quantified via a highly sensitive and selective parallel-reaction monitoring approach using nano-liquid chromatography/Orbitrap mass spectrometry. On average, the survival of intact palivizumab from the feed to the stomach, upper small intestine and stool were 88.4%, 30.0% and 5.2%, respectively. This approach allowed clear determination of the extent to which palivizumab was degraded within the infant digestive tract. This method can be applied with some modifications to study the digestion of any protein.

Hepcidin belongs to the antimicrobial peptide family but has weak activity with regards to bacterial killing. The regulatory function of hepcidin in humans serves to maintain an iron-restricted environment that limits the growth of pathogens; this study explored whether hepcidin affected bacterial iron homeostasis and oxidative stress using the model organism Escherichia coli. Using the Miller assay it was determined that under low iron availability exposure to sub-inhibitory doses of hepcidin (4-12μM) led to 2-fold and 4-fold increases in the expression of ftnA and bfd, respectively (P < 0.05), in both a wild type (WT) and Δfur (ferric uptake regulator) background. Quantitative real-time PCR analysis of oxyR and sodA, treated with 4 or 8 μM of hepcidin showed that expression of these genes was significantly (P < 0.05) increased, whereas expression of lexA was unchanged, indicating that hepcidin likely mediated oxidative stress but did not induce DNA damage.

A novel antibacterial peptide from Crocodylus siamensis haemoglobin hydrolysate (CHH) was characterized for antimicrobial activity.CHHs were hydrolysed for 2 h (2 h-CHH), 4 h (4h-CHH), 6 h (6 h-CHH) and 8 h (8 h-CHH). The 8 h-CHH showed antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa at concentrations of 20, 20, 20 and 10 mg mlThe QAIIHNEKVQAHGKKVL fragment of C. siamensis haemoglobin is antibacterial via a mechanism that likely relies on iron dysregulation and oxidative stress which results in bacterial death.We have described for the first time, a novel peptide derived from C. siamensis haemoglobin hydrolysate that has the potential to be developed as a novel antimicrobial peptide.

Antioxidant and anti-inflammatory activities were found from Crocodylus siamensis (C. siamensis) blood. The 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, nitric oxide scavenging, hydroxyl radical scavenging and linoleic peroxidation assays were used to investigate the antioxidant activities of the crocodile blood. Results show that crocodile blood components had antioxidant activity, especially hemoglobin (40.58 % nitric oxide radical inhibition), crude leukocyte extract (78 % linoleic peroxidation inhibition) and plasma (57.27 % hydroxyl radical inhibition). Additionally, the anti-inflammatory activity of the crocodile blood was studied using murine macrophage (RAW 264.7) as a model. The results show that hemoglobin, crude leukocyte extract and plasma were not toxic to RAW 264.7 cells. Also they showed anti-inflammatory activity by reduced nitric oxide (NO) and interleukin 6 (IL-6) productions from lipopolysaccharide (LPS)-stimulated cells. The NO inhibition percentages of hemoglobin, crude leukocyte extract and plasma were 31.9, 48.24 and 44.27 %, respectively. However, only crude leukocyte extract could inhibit IL-6 production. So, the results of this research directly indicate that hemoglobin, crude leukocyte extract and plasma of C. siamensis blood provide both antioxidant and anti-inflammatory activities, which could be used as a supplementary agent in pharmaceutical products.