Nanosecond pulsed electric field treatment of human milk: Effects on microbiological inactivation, whey proteome and bioactive protein.

In the current study, nanosecond pulsed electric field (nsPEF) was investigated at lab-scale to optimise processing conditions of donor human milk to reduce bacterial counts, and to evaluate its effect on the bioactive proteins in human milk. Response surface methodology was utilized to optimise critical processing parameters. Two optimal nsPEF processing conditions were validated: 15 kV voltage, 6000 pulses at 20 Hz frequency, and 15 kV voltage, 6000 pulses at 50 Hz frequency. Compared to raw human milk, nsPEF processed milk had over 60 % retention of lysozyme, lactoperoxidase and lactoferrin, and 100 % retention of xanthine oxidase and immunoglobulin A. The contents of the five proteins were significantly higher after nsPEF processing when compared with Holder pasteurization. Liquid chromatography-mass spectrometry analysis showed that loss of milk proteins was smaller for samples treated with nsPEF than Holder pasteurization. These results indicated that nsPEF is a promising novel pasteurization method.

The Results of Different Heating Temperatures on Activities of Bioactive Proteins in Human Milk.

BACKGROUND: The most utilized pasteurization method in donor human milk banks is Holder pasteurization (heating 62.5 °C for 30 min). However, many bioactive proteins are heat sensitive and are inactivated. RESEARCH AIM: To determine the results of a range of heating regimes on the activities of xanthine oxidase, lactoperoxidase and lysozyme, the concentrations of immunoglobulin A and lactoferrin, as well as bacterial inactivation. METHOD: This prospective, cross-sectional, intervention study was designed to measure the influence of heating temperatures on bioactive components in donor human milk. Milk samples were processed at 40, 50, 55, 62.5, 75, 127 °C and the activities of the enzymes, and the concentration of immune proteins, were measured. RESULTS: No bacterial colonies were detectable, using standard culture methods, after heating above 50 ºC. All proteins studied retained over 60% concentrations or activities when the pasteurization temperature was 50 ºC or lower, while their concentrations or activities were lost at higher temperatures. For lactoferrin, the residual concentration was above 80% when heating temperature was under 55 °C, while only 20% remained after Holder pasteurization. Both xanthine oxidase and lactoperoxidase had little residual activity when temperatures were above Holder pasteurization. Lysozyme retained a greater proportion of residual activity than other proteins, following heating at all temperatures. CONCLUSIONS: The concentrations or activities of immune proteins and bioactive enzymes decreased when heated above 50 °C. The results of this study can be used to design temperature control guidance during alternative methods of pasteurization.

Comparative proteomic analysis of donor human milk pasteurized by hydrostatic high-pressure.

The aim of this study was to comprehensively investigate the effect of Holder pasteurization (HoP) compared with that of hydrostatic high-pressure (HHP) processing on human milk proteins, including milk fat globule membrane (MFGM) proteins, whey proteins and caseins. Milk fat globules in milk processed by high-pressure were similar to those in raw milk in terms of their size distribution and microstructure, while the globules in milk processed by HoP were aggregated. The protein profiles of milk subjected to HHP processing more closely resembled those of raw milk than HoP milk. Proteins in milk whey were less affected by HoP or HHP than MFGM and casein proteins. The findings indicated a better preservation of the protein profile for HHP compared to HoP of human milk.

Physicochemical Properties and Whey Proteomes of Camel Milk Powders Produced by Different Concentration and Dehydration Processes.

Camel milk powder production is an alternative to preserve the perishable milk for later-date consumption. However, the impacts of dehydration processes on bioactive compounds in camel milk are largely unknown. Hence, the present study attempted to compare the physicochemical properties and protein profiles of camel milk powders produced by different concentration and dehydration processes. Six camel milk powders were produced by freeze- and spray-drying methods in conjunction with two liquid concentration techniques, namely spray dewatering and reverse osmosis. The results of proteomic analysis showed that direct freeze-dried camel milk powder had the least changes in protein profile, followed by direct spray-dried powder. The camel milk powders that underwent concentration processes had more profound changes in their protein profiles. Among the bioactive proteins identified, lactotransferrin and oxidase/peroxidase had the most significant decreases in concentration following processing. On the contrary, glycosylation-dependent cell adhesion molecule 1, peptidoglycan recognition protein 1, and osteopontin increased in concentration. The results revealed that direct freeze drying was the most ideal method for preserving the bioactive proteins during camel milk powder production. However, the freeze-drying technique has cost and scalability constraints, and the current spray-drying technique needs improvement to better retain the bioactivity of camel milk during powder processing.

Comprehensive biochemical and proteomic characterization of seasonal Australian camel milk.

Although camel milk is increasingly becoming a popular alternative to bovine milk around the world including Australia, studies of Australian camel milk are still lacking. A comprehensive and systematic analysis of major nutritional components, physical properties, antimicrobial enzymes and whey proteomes of Australian camel milk obtained over four seasons was conducted, for the first time in present study. The composition and physical properties of Australian camel milk varied with season, milking frequency and yield. The highest lactoperoxidase and polyamine oxidase activity was observed in summer and winter, respectively. A total of 97 proteins were quantified, on a relative basis, across all the seasonal bulk milk samples. Summer camel milk contained higher amounts of functional whey proteins, such as lactotransferrin, peptidoglycan recognition protein 1, osteopontin and lactoperoxidase. These results contribute to a better understanding of the Australian camel milk and provide insights into processing of dairy products from this milk.

Comparing the effects of hydrostatic high-pressure processing vs holder pasteurisation on the microbial, biochemical and digestion properties of donor human milk.

In this study, hydrostatic high-pressure processing (HHP), a non-thermal pasteurisation method, was used to achieve the microbiological safety of donor human milk. After HHP, no bacteria were detected in human milk processed at 400 MPa for 5 min. Activities of a selection of bioactive components, including lysozyme, xanthine oxidase, lactoperoxidase, immunoglobulin A, lactoferrin, lipoprotein lipase and bile salt-stimulated lipase, did not decrease significantly. This study further investigated the gastrointestinal digestion kinetics of HoP and HHP milk compared with raw human milk, using an in vitro static infant digestion model. After 60 min of 'gastric digestion', the microstructure and protein profile of HHP milk samples were more similar to raw milk samples than HoP milk samples. Overall, HPP showed a better retention in milk nutrients and closer digestion behavior than that of HoP.

Digestibility of proteins in camel milk in comparison to bovine and human milk using an in vitro infant gastrointestinal digestion system.

The absence of ß-lactoglobulin, high ß-/αs-casein ratio and protective proteins make camel milk a promising alternative protein base for making human infant formulae. In this study, protein digestibility of camel milk was compared with that of bovine and human milk using an in vitro infant gastrointestinal digestion system. A low degree of gastric proteolysis was observed in all three kinds of milk, and a single clot was formed in camel milk. The soluble milk proteins remaining in the gastric digesta were digested rapidly and extensively in the intestinal phase, while the proteins in the camel milk clot were hydrolysed gradually. Despite several similarities, bioactive peptides unique to individual milk were identified in the three intestinal milk digesta. The results suggest that camel milk proteins are equally digestible as bovine and human milk proteins under infant gastrointestinal digestion conditions, and it may be a prospective substitute for infant formula base.

New Insights on Tramadol and Immunomodulation.

PURPOSE OF REVIEW: Opioids are administered to cancer patients although concerns have been raised that they may promote tumour growth or metastasis owing to their ability to suppress anti-cancer immunity. Tramadol has been reported to preserve or promote the immune response and may therefore be preferred to other opioids in cancer patients. We reviewed the literature documenting the immunomodulatory effects of tramadol. RECENT FINDINGS: Recent clinical evidence appears to confirm that tramadol possesses anti-inflammatory properties, and preserves some signalling cascades of the immune system relevant to anti-cancer defence. Tramadol is reported to promote or preserve immunity including natural killer cell activity which is important in anti-cancer defences.

A sensitive and high-throughput fluorescent method for determination of oxidase activities in human, bovine, goat and camel milk.

Milk oxidases are an integral part of milk immune system, and good indicators for milk thermal history. Current assay methods for milk oxidases are either insensitive, tedious or not cost-effective. In this study, a high-throughput fluorescence assay method for determination of xanthine oxidase (XO) and polyamine oxidase (PAO) activities in milk samples was developed. The hydrogen peroxide generated by XO catalysed oxidation of hypoxanthine, and PAO catalysed oxidation of spermine, was coupled to horseradish peroxidase conversion of Amplex® Red (1-(3,7-dihydroxyphenoxazin-10-yl)ethanone) to the fluorescent product resorufin. The assay was highly sensitive, with limits of detection of activity in milk being 3 × 10-7 and 7 × 10-7 U/mL for XO and PAO, respectively. Intra-run and inter-run results showed good assay repeatability and reproducibility. The assay was successfully applied to survey the XO and PAO activities in human, bovine, goat and camel milk samples, and it can be readily adapted for measurements of other oxidase activities.

A sensitive, high-throughput fluorescent method for the determination of lactoperoxidase activities in milk and comparison in human, bovine, goat and camel milk.

Lactoperoxidase (LPO) is one of the major antibacterial ingredients in milk and an extensively employed indicator for milk heat treatment. The traditional method for LPO activity measurement using ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate) cannot achieve high sensitivity and is affected by indigenous milk thiocyanate. A more sensitive microplate fluorescent assay was developed by monitoring generation of red-fluorescent resorufin from LPO catalysed oxidation of Amplex® Red (1-(3,7-dihydroxyphenoxazin-10-yl)ethanone) in this study. The assay is particularly suitable for milk LPO activity measurement as it eliminates the influences of indigenous milk hydrogen peroxide and thiocyanate. The method limit of detection was 7.1x10-6 U/mL of LPO in milk and good intra-run and inter-run precision was obtained. The LPO activities ranked as bovine > goat > camel > human in the four types of milk analysed. The high sensitivity and low cost of this assay makes it suitable for LPO activity analyses in both laboratory and commercial scales.

A role for caveola-forming proteins caveolin-1 and CAVIN1 in the pro-invasive response of glioblastoma to osmotic and hydrostatic pressure.

In solid tumours, elevated interstitial fluid pressure (osmotic and hydrostatic pressure) is a barrier to drug delivery and correlates with poor prognosis. Glioblastoma (GBM) further experience compressive force when growing within a space limited by the skull. Caveolae are proposed to play mechanosensing roles, and caveola-forming proteins are overexpressed in GBM. We asked whether caveolae mediate the GBM response to osmotic pressure. We evaluated in vitro the influence of spontaneous or experimental down-regulation of caveola-forming proteins (caveolin-1, CAVIN1) on the proteolytic profile and invasiveness of GBM cells in response to osmotic pressure. In response to osmotic pressure, GBM cell lines expressing caveola-forming proteins up-regulated plasminogen activator (uPA) and/or matrix metalloproteinases (MMPs), some EMT markers and increased their in vitro invasion potential. Down-regulation of caveola-forming proteins impaired this response and prevented hyperosmolarity-induced mRNA expression of the water channel aquaporin 1. CRISPR ablation of caveola-forming proteins further lowered expression of matrix proteases and EMT markers in response to hydrostatic pressure, as a model of mechanical force. GBM respond to pressure by increasing matrix-degrading enzyme production, mesenchymal phenotype and invasion. Caveola-forming proteins mediate, at least in part, the pro-invasive response of GBM to pressure. This may represent a novel target in GBM treatment.

Bio-Guided Fractionation of Papaya Leaf Juice for Delineating the Components Responsible for the Selective Anti-proliferative Effects on Prostate Cancer Cells.

Alternative therapies against cancer cells with minimal or no effect on healthy tissues are highly sought after. Prostate cancer (PCa) is the second most frequently diagnosed malignancy in males. The Carica papaya L. leaf extract has been traditionally used by Australian aboriginal people for anticancer properties. In this study, medium polar fraction of papaya leaf extract that had shown anti-proliferative activity in PCa cell lines in vitro, in earlier studies, was further fractionated to 28 fractions by semi-preparative HPLC. Nine of these fractions were identified to possess selective anti-proliferative responses on PCa cells in comparison to non-cancerous cells of prostate gland origin. When these nine sub-fractions were mixed in various combinations, a combination containing six of the specific fractions (FC-3) showed the best potency. FC3 inhibited the growth of BPH-1, PC-3, and LNCaP cells in a concentration-dependent manner with an IC50 value <20 µg/mL, while (unlike paclitaxel, the positive control) minimal effect was observed on the proliferation of non-cancerous, WPMY-1 and RWPE-1cells. Furthermore, synergistic interaction of FC-3 with paclitaxel was observed with combination index values in the range of 0.89-0.98 and 0.85-1.10 on PC-3 and LNCaP cells, respectively. Untargeted qualitative analysis using UHPLC (Ultra High-Performance Liquid Chromatography)-QToF (Quadrupole Time of-Flight) mass spectrometry and screening against the METLIN database indicated presence of multiple known anticancer compounds in the FC-3 extract. These outcomes show that the potent and selective anti-proliferative effects are due to a range of bio-active compounds within the medium polar fraction of papaya leaf juice.

Effect of Perioperative Opioids on Cancer-Relevant Circulating Parameters: Mu Opioid Receptor and Toll-Like Receptor 4 Activation Potential, and Proteolytic Profile.

Purpose: The purpose of this study is to investigate the potential interplay between opioid analgesia and tumor metastasis through modulation of µ-opioid receptor (MOR), Toll-like receptor 4 (TLR4) activation, and matrix degradation potential.Experimental Design: Plasma samples were collected from 60 patients undergoing elective lower limb joint replacement preoperatively and at 3, 6, and 24 hours after surgery; pain scores were documented at the same time points. Opioid administration was recorded and converted into morphine IV equivalents. Plasma samples were also collected from 10 healthy volunteers. Alphascreen cyclic AMP assay and MOR-overexpressing cells were employed to quantify MOR activation. HEK-Blue hTLR4 were utilized to measure TLR4 activation. Circulating matrix metalloprotease and tissue inhibitor of matrix protease activities were assessed by gelatin zymography and reverse zymography, respectively.Results: Postoperative plasma samples displayed the ability to activate MOR and to inhibit lipopolysaccharide (LPS)-induced TLR4 activation. Linear mixed model analysis revealed that MOR activation had a significant effect on inhibition of LPS-induced TLR4 activation. Furthermore, TLR4 had a significant effect to explain pain scores. Postoperative samples also displayed altered circulating matrix-degrading enzymes activity potential, but this was correlated neither to opioid administration nor to MOR activation potential.Conclusions: Our results show for the first time that (i) opioids administered to surgery patients result in modulation of ligand-induced TLR4 activation and (ii) postoperative pain is associated with increased circulating TLR4 activation potential. Our study further promotes the use of MOR activation potential rather than opioid intake in clinical studies measuring opioid exposure at a given time point. Clin Cancer Res; 24(10); 2319-27. ©2018 AACR.

Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion.

Opioids modulate the tumor microenvironment with potential functional consequences for tumor growth and metastasis. We evaluated the effects of morphine administration on the circulating proteolytic profile of tumor-free mice. Serum from morphine-treated (1 or 10 mg/kg, i.p. every 12 h) or saline-treated mice was collected at different time points and tested ex vivo in endothelial, lymphatic endothelial, and breast cancer cell migration assays. Serum from mice that were treated with 10 mg/kg morphine for 3 d displayed reduced chemotactic potential for endothelial and breast cancer cells, and elicited reduced cancer cell invasion through reconstituted basement membrane compared with serum from saline controls. This was associated with decreased circulating matrix metalloproteinase 9 (MMP-9) and increased circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-3/4 as assessed by zymography and reverse zymography. By using quantitative RT-PCR, we confirmed morphine-induced alterations in MMP-9 and TIMP expression and identified organs, including the liver and spleen, in which these changes originated. Pharmacologic inhibition of MMP-9 abrogated the difference in chemotactic attraction between serum from saline-treated and morphine-treated mice, which indicated that reduced proteolytic ability mediated the decreased migration toward serum from morphine-treated mice. This novel mechanism may enable morphine administration to promote an environment that is less conducive to tumor growth, invasion, and metastasis.-Xie, N., Khabbazi, S., Nassar, Z. D., Gregory, K., Vithanage, T., Anand-Apte, B., Cabot, P. J., Sturgess, D., Shaw, P. N., Parat, M.-O. Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion.

Biomolecular changes that occur in the antennal gland of the giant freshwater prawn (Machrobrachium rosenbergii).

In decapod crustaceans, the antennal gland (AnG) is a major primary source of externally secreted biomolecules, and some may act as pheromones that play a major role in aquatic animal communication. In aquatic crustaceans, sex pheromones regulate reproductive behaviours, yet they remain largely unidentified besides the N-acetylglucosamine-1,5-lactone (NAGL) that stimulates male to female attraction. In this study, we used an AnG transcriptome of the female giant freshwater prawn (Macrobrachium rosenbergii) to predict the secretion of 226 proteins, including the most abundantly expressed transcripts encoding the Spaetzle protein, a serine protease inhibitor, and an arthropodial cuticle protein AMP 8.1. A quantitative proteome analysis of the female AnG at intermolt, premolt and postmolt, identified numerous proteins of different abundances, such as the hemocyanin subunit 1 that is most abundant at intermolt. We also show that hemocyanin subunit 1 is present within water surrounding females. Of those metabolites identified, we demonstrate that the NAGL and N-acetylglucosamine (NAG) can bind with high affinity to hemocyanin subunit 1. In summary, this study has revealed components of the female giant freshwater prawn AnG that are released and contribute to further research towards understanding crustacean conspecific signalling.

Selective anti-proliferative activities of Carica papaya leaf juice extracts against prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the leading cause of cancer related deaths in men. Carica papaya is a popular tropical plant that has been traditionally used for its nutritional and medicinal properties. METHODS: We investigated the anti-proliferative responses of papaya leaf juice (LJP) and its various extracts ("biological"- in vitro digested, "physical"- size exclusion, and "chemical"-solvent extraction) on a range of cell lines representing benign hyperplasia, tumorigenic and normal cells of prostate origin. RESULTS: Time course analysis (by 24h, 48h and 72h) of LJP (1-0.1mg/mL) before and after in vitro digestion, and of molecular weight based fractions of LJP showed anti-proliferative responses. The medium polarity fraction of LJP (0.03-0.003mg/mL) after 72h exposure showed potent growth inhibitory (IC50=0.02-0.07mg/mL) and cytotoxic activities on all prostate cells, with the exception of the normal (RWPE-1 and WPMY-1) cells. Flow cytometry analysis showed S phase cell cycle arrest and apoptosis as a possible mechanism for these activities. Medium polar fraction of LJP also inhibited migration and adhesion of metastatic PC-3 cells. CONCLUSION: This is the first report suggesting selective anti-proliferative and anti-metastatic attributes of LJP extract against prostatic diseases, including PCa.

Activation of µ-opioid receptor and Toll-like receptor 4 by plasma from morphine-treated mice.

In this study, we quantified the ability of opioids present in biological samples to activate the µ-opioid receptor and TLR4 using cell-based assays. Each assay was standardised, in the presence of plasma, using morphine, its µ receptor-active metabolite morphine-6 glucuronide (M6G) and its µ receptor-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G). Specificity was verified using antagonists. Morphine- and M6G-spiked plasma samples exhibited µ receptor activation, which M3G-spiked plasma lacked. In contrast, M3G showed moderate but consistent activation of TLR-4. Plasma samples were collected at a number of time points from mice administered morphine (1 or 10mg/kg every 12h for 3days) or saline. Morphine administration led to intermittent µ receptor activation, reversed by µ receptor antagonists, and to TRL4 activation at time points where M3G is measured in plasma. Interestingly, this protocol of morphine administration also led to TLR4-independent NF-κB activation, at time points where M3G was not detected, presumably via elevation of circulating cytokines including, but not limited to, TNFα. Circulating TNFα was increased after three days of morphine administration, and TNFα mRNA elevated in the spleen of morphine-treated mice.

Traditional Aboriginal Preparation Alters the Chemical Profile of Carica papaya Leaves and Impacts on Cytotoxicity towards Human Squamous Cell Carcinoma.

Carica papaya leaf decoction, an Australian Aboriginal remedy, has been used widely for its healing capabilities against cancer, with numerous anecdotal reports. In this study we investigated its in vitro cytotoxicity on human squamous cell carcinoma cells followed by metabolomic profiling of Carica papaya leaf decoction and leaf juice/brewed leaf juice to determine the effects imparted by the long heating process typical of the Aboriginal remedy preparation. MTT assay results showed that in comparison with the decoction, the leaf juice not only exhibited a stronger cytotoxic effect on SCC25 cancer cells, but also produced a significant cancer-selective effect as shown by tests on non-cancerous human keratinocyte HaCaT cells. Furthermore, evidence from testing brewed leaf juice on these two cell lines suggested that the brewing process markedly reduced the selective effect of Carica papaya leaf on SCC25 cancer cells. To tentatively identify the compounds that contribute to the distinct selective anticancer activity of leaf juice, an untargeted metabolomic approach employing Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry followed by multivariate data analysis was applied. Some 90 and 104 peaks in positive and negative mode respectively were selected as discriminatory features from the chemical profile of leaf juice and >1500 putative compound IDs were obtained via database searching. Direct comparison of chromatographic and tandem mass spectral data to available reference compounds confirmed one feature as a match with its proposed authentic standard, namely pheophorbide A. However, despite pheophorbide A exhibiting cytotoxic activity on SCC25 cancer cells, it did not prove to be the compound contributing principally to the selective activity of leaf juice. With promising results suggesting stronger and more selective anticancer effects when compared to the Aboriginal remedy, Carica papaya leaf juice warrants further study to explore its activity on other cancer cell lines, as well as investigation to confirm the identity of compounds contributing to its selective effect, particularly those compounds altered by the long heating process applied during the traditional Aboriginal remedy preparation.

Chemical Characterization and in Vitro Cytotoxicity on Squamous Cell Carcinoma Cells of Carica papaya Leaf Extracts.

In traditional medicine, Carica papaya leaf has been used for a wide range of therapeutic applications including skin diseases and cancer. In this study, we investigated the in vitro cytotoxicity of aqueous and ethanolic extracts of Carica papaya leaves on the human oral squamous cell carcinoma SCC25 cell line in parallel with non-cancerous human keratinocyte HaCaT cells. Two out of four extracts showed a significantly selective effect towards the cancer cells and were found to contain high levels of phenolic and flavonoid compounds. The chromatographic and mass spectrometric profiles of the extracts obtained with Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry were used to tentatively identify the bioactive compounds using comparative analysis. The principal compounds identified were flavonoids or flavonoid glycosides, particularly compounds from the kaempferol and quercetin families, of which several have previously been reported to possess anticancer activities. These results confirm that papaya leaf is a potential source of anticancer compounds and warrant further scientific investigation to validate the traditional use of papaya leaf to treat cancer.

Breastmilk-Saliva Interactions Boost Innate Immunity by Regulating the Oral Microbiome in Early Infancy.

INTRODUCTION: Xanthine oxidase (XO) is distributed in mammals largely in the liver and small intestine, but also is highly active in milk where it generates hydrogen peroxide (H2O2). Adult human saliva is low in hypoxanthine and xanthine, the substrates of XO, and high in the lactoperoxidase substrate thiocyanate, but saliva of neonates has not been examined. RESULTS: Median concentrations of hypoxanthine and xanthine in neonatal saliva (27 and 19 µM respectively) were ten-fold higher than in adult saliva (2.1 and 1.7 µM). Fresh breastmilk contained 27.3 ± 12.2 µM H2O2 but mixing baby saliva with breastmilk additionally generated >40 µM H2O2, sufficient to inhibit growth of the opportunistic pathogens Staphylococcus aureus and Salmonella spp. Oral peroxidase activity in neonatal saliva was variable but low (median 7 U/L, range 2-449) compared to adults (620 U/L, 48-1348), while peroxidase substrate thiocyanate in neonatal saliva was surprisingly high. Baby but not adult saliva also contained nucleosides and nucleobases that encouraged growth of the commensal bacteria Lactobacillus, but inhibited opportunistic pathogens; these nucleosides/bases may also promote growth of immature gut cells. Transition from neonatal to adult saliva pattern occurred during the weaning period. A survey of saliva from domesticated mammals revealed wide variation in nucleoside/base patterns. DISCUSSION AND CONCLUSION: During breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors. Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral-and hence gut-microbiota. Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.