Effect of protein fortification on heat damage and occurrence of ß-casomorphins in (un)digested donor human milk intended for nutrition of preterm infants.

Pasteurized donor human milk (PDHM) for preterm infant nutrition is fortified with hydrolyzates of cow's milk proteins, which have been poorly investigated in relation to heat-damage and occurrence of the bioactive peptides ß-casomorphins (BCMs). Therefore, thermal protein modifications of three commercial fortifiers were assessed by measuring well-recognized indexes of heat load. The fortifiers did not contain pyrraline, whereas furosine and lysinoalanine levels roughly overlapped the lowest values reported for liquid formulas addressed to term infant nutrition. Bovine BCMs 3 to 7 and human BCMs 3 to 9 were searched. Bovine BCMs 3, 4, 6 and 7 were found in the undigested fortifiers. Following in vitro digestion simulating the digestive conditions of premature infant, bovine BCMs still occurred in fortified PDHM; the human BCMs 3, 7, 8 and 9 formed. Overall, these results better address the nutritional features of protein fortifiers and fortified PDHM intended for nutrition of preterm infants.

Milk A1 ß-casein and health-related outcomes in humans: a systematic review.

CONTEXT: Various epidemiological studies suggest a positive association between exposure to cow's milk A1 ß-casein protein and risk for noncommunicable chronic diseases. The consumption of A2 cow's milk is increasing, likely because A2 milk is postulated to have positive effects on digestive health. OBJECTIVE: A systematic review was conducted to investigate associations between A1 ß-casein and health-related outcomes in humans. DATA SOURCES: Five electronic databases, 3 clinical trial registries, and the internet were searched systematically. STUDY SELECTION: Using predefined inclusion criteria, 2 authors independently selected studies investigating the effect of A1 ß-casein or ß-casomorphin-7 intake/exposure on any health-related outcome in humans. Discrepancies were resolved by consensus. DATA EXTRACTION: Two authors independently extracted data and assessed risk of bias. The certainty of evidence per outcome was evaluated using the GRADE approach. Discrepancies were resolved by consensus. RESULTS: Fifteen randomized controlled trials, 2 case-control studies, and 8 ecological studies were included. Most randomized controlled studies and case-control studies investigating a potential effect on various outcomes were based on intermediate markers and found no significant difference between the 2 milk types. In contrast, most ecological studies reported that population-level A1 ß-casein exposure is associated with adverse health outcomes. The certainty of the evidence for the included outcomes, as assessed by the GRADE approach, was rated as moderate for digestive symptoms and as low to very low for all other outcomes. CONCLUSIONS: Human-based evidence from clinical trials and epidemiological studies published prior to October 2017 provides moderate certainty for adverse digestive health effects of A1 ß-casein compared with A2 ß-casein but low or very low certainty for other health effects. These conclusions may change in the future, given the emergent nature of this topic and the ongoing research in this area. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number CRD42016043795.

Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

Preliminary analysis of allelochemicals produced by the diatom Phaeodactylum tricornutum.

Marine diatom Phaeodactylum tricornutum is known to exude allelochemicals with negative effects on Heterosigma akashiwo according to our previous study, while the information about the allelochemical compounds remains unknown. The present study dealt with isolation and analysis of the active substances released by P. tricornutum into the culture medium. Filtrate of P. tricornutum was extracted using ethyl acetate and chloroform respectively. The anti-algal fractions were isolated using high performance liquid chromatography (HPLC) and screened using activity-guided fraction methods. Results demonstrated that fraction Ⅱ and Ⅵ showed significant allelopathic effect on H. akashiwo growth. Then the anti-algal activity fractions were analyzed preliminary using gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-electrospray time-of-flight mass spectrometry (HPLC-ESI-TOF-MS). An active compound was derived from fraction Ⅵ with the molecular weight of 578 and possible molecular formula of C30H38N6O6, which was speculated to be TYR-PRO-PHE-PRO-GLY-NH2. a kind of glycinamides.

Aptamer-based sensing of ß-casomorphin-7.

ß-Casomorphin-7 (BCM-7), a seven amino acid peptide, is released during digestion of ß-casein A1 variant of milk which is speculated to be associated with certain diseases. Fifteen ssDNA aptamers having high affinity toward BCM-7 were identified from a 72 nt long random library after ten rounds of systematic evolution of ligands by exponential enrichment. Dissociation constant values of selected aptamers were in the range of 7.7-156.7 nM. Seq6 aptamer exhibited the lowest Kd value. Nine aptamers were evaluated for their binding toward BCM-7, BCM-9A1, and BCM-9A2 peptides, and binding was variable. SeqU5 exhibited the lowest binding with BCM-9A1 and BCM-9A2. Aptamer-coated gold nanoparticles (GNPs) resulted in color change of GNPs in the presence of BCM-7, thereby establishing recognition of BCM-7 by aptamers. The enzyme-linked aptamer-sorbent assay (ELASA) was evaluated as an assay of BCM-7 in biological fluids. BCM-7-peroxidase competed with BCM-7 in ELASA, performed with BCM-7 solution and BCM-7 spiked urine pretreated with urease, plasma, and ß-casein digest samples.

Formation and Degradation of Beta-casomorphins in Dairy Processing.

Milk proteins including casein are sources of peptides with bioactivity. One of these peptides is beta-casomorphin (BCM) which belongs to a group of opioid peptides formed from ß-casein variants. Beta-casomorphin 7 (BCM7) has been demonstrated to be enzymatically released from the A1 or B ß-casein variant. Epidemiological evidence suggests the peptide BCM 7 is a risk factor for development of human diseases, including increased risk of type 1 diabetes and cardiovascular diseases but this has not been thoroughly substantiated by research studies. High performance liquid chromatography coupled to UV-Vis and mass spectrometry detection as well as enzyme-linked immunosorbent assay (ELISA) has been used to analyze BCMs in dairy products. BCMs have been detected in raw cow's milk and human milk and a variety of commercial cheeses, but their presence has yet to be confirmed in commercial yoghurts. The finding that BCMs are present in cheese suggests they could also form in yoghurt, but be degraded during yoghurt processing. Whether BCMs do form in yoghurt and the amount of BCM forming or degrading at different processing steps needs further investigation and possibly will depend on the heat treatment and fermentation process used, but it remains an intriguing unknown.

Isotope dilution liquid chromatography-tandem mass spectrometry for simultaneous identification and quantification of beta-casomorphin 5 and beta-casomorphin 7 in yoghurt.

A highly selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous identification and quantification of beta-casomorphin 5 (BCM5) and beta-casomorphin 7 (BCM7) in yoghurt. The method used deuterium labelled BCM5-d10 and BCM7-d10 as surrogate standards for confident identification and accurate and quantification of these analytes in yoghurt. Linear responses for BCM5 and BCM7 (R(2)=0.9985 and 0.9986, respectively) was observed in the range 0.01-10ng/µL. The method limits of detection (MLDs) in yoghurt extracts were found to be 0.5 and 0.25ng/g for BCM5 and BCM7, respectively. Analyses of spiked samples were used to provide confirmation of accuracy and precision of the analytical method. Recoveries relative to the surrogate standards of these spikes were in the range of 95-106% for BCM5 and 103-109% for BCM7. Precision from analysis of spiked samples was expressed as relative standard deviation (%RSD) and values were in the range 1-16% for BCM5 and 1-6% for BCM7. Inter-day reproducibility was between 2.0-6.4% for BCM5 and between 3.2-6.1% for BCM7. The validated isotope dilution LC-MS/MS method was used to measure BCM5 and BCM7 in ten commercial and laboratory prepared samples of yoghurt and milk. Neither BCM5 nor BCM7 was detected in commercial yoghurts. However, they were observed in milk and laboratory prepared yoghurts and interestingly their levels decreased during processing. BCM5 decreased from 1.3ng/g in milk to 1.1ng/g in yoghurt made from that milk at 0day storage and

Sequential release of milk protein-derived bioactive peptides in the jejunum in healthy humans.

BACKGROUND: The digestive hydrolysis of dietary proteins leads to the release of peptides in the intestinal tract, where they may exert a variety of functions, but their characterization and quantification are difficult. OBJECTIVES: We aimed to characterize and determine kinetics of the formation of peptides present in the jejunum of humans who ingested casein or whey proteins by using mass spectrometry and to look for and quantify bioactive peptides. DESIGN: Subjects were equipped with a double-lumen nasogastric tube that migrated to the proximal jejunum. A sample collection was performed for 6 h after the ingestion of 30 g (15)N-labeled casein (n = 7) or whey proteins (WPs; n = 6). Nitrogen flow rates were measured, and peptides were identified by using mass spectrometry. RESULTS: After casein ingestion, medium-size peptides (750-1050 kDa) were released during 6 h, whereas larger peptides (1050-1800 kDa) were released from WPs in the first 3 h. A total of 356 and 146 peptides were detected and sequenced in the jejunum after casein and WP ingestion, respectively. ß-casein was the most important precursor of peptides, including bioactive peptides with various activities. The amounts of ß-casomorphins (ß-casein 57-, 58-, 59-, and 60-66) and ß-casein 108-113 released on the postprandial window were sufficient to elicit the biological action of these peptides (ie, opioid and antihypertensive, respectively). CONCLUSIONS: Clear evidence is shown of the presence of bioactive peptides in the jejunum of healthy humans who ingested casein. Our findings raise the question about the physiologic conditions under which these peptides can express their bioactivity in humans. This trial was registered at clinicaltrials.gov as NCT00862329.

Mapping of alpha-neo-endorphin- and neurokinin B-immunoreactivity in the human brainstem.

We have studied the distribution of alpha-neo-endorphin- or neurokinin B-immunoreactive fibres and cell bodies in the adult human brainstem with no prior history of neurological or psychiatric disease. A low density of alpha-neo-endorphin-immunoreactive cell bodies was only observed in the medullary central gray matter and in the spinal trigeminal nucleus (gelatinosa part). Alpha-neo-endorphin-immunoreactive fibres were moderately distributed throughout the human brainstem. A high density of alpha-neo-endorphin-immunoreactive fibres was found only in the solitary nucleus (caudal part), in the spinal trigeminal nucleus (caudal part), and in the gelatinosa part of the latter nucleus. Neurokinin B-immunoreactive cell bodies (low density) were found in the periventricular central gray matter, the reticular formation of the pons and in the superior colliculus. The distribution of the neurokinin-immunoreactive fibres was restricted. In general, for both neuropeptides the density of the immunoreactive fibres was low. In the human brainstem, the proenkephalin system was more widely distributed than the prodynorphin system, and the preprotachykinin A system (neurokinin A) was more widely distributed than the preprotachykinin B system (neurokinin B).

Milk from cows of different ß-casein genotypes as a source of ß-casomorphin-7.

The aim of this study was to quantify ß-casomorphin-7 in raw, hydrolyzed and processed milk in different stages of the cow lactation. The obtained results lead to the following conclusion: the highest amount of ß-casomorphin-7 released from the hydrolyzed and processed milk is related to the ß-casein A1 allele, irrespective of a lactation period. Some traces of ß-casomorphin-7 in milk from cows with the ß-casein A2 variant are probably a result of the acid hydrolysis of ß-casein during its digestion with pepsin. It has been shown that processing of raw milk at high temperatures affects, in a slight degree, the differences between ß-casomorphins-7 originating from different ß-casein genotypes. The obtained results suggest a possibility to provide a new nutritional factor for milk quality based on the content of ß-casomorphin-7 liberated in vivo from milk digested by a mixture of the gastrointestinal enzymes.

Nanoelectrospray with ion-trap mass spectrometry for the determination of beta-casomorphins in derived milk products.

Beta-casomorphins (b-CMs) are bioactive peptides derived from casein with opioid agonist effects similar to morphine. The use of electrospray (ESI) with quadrupole ion-trap mass spectrometry (QIT-MS) for these compounds in two matrices, cheese and milk, was examined. It was compared to a liquid chromatography (LC) coupled to mass spectrometry (LC-MS), and a "soft" ionisation technique, NanoMate, with selected ion monitoring (SIM), which are unreliable for the determination of trace casomorphins in derived milk products. b-CM mass fragmentation pathways were done for the four most common b-CMs: beta-casomorphin (1-5) bovine (b-CM-5), beta-casomorphin (1-7) bovine (b-CM-7), [D-Ala2, D-Pro4,Tyr5]-beta-casomorphin (1-5) amide (b-CM-10) and beta-casomorphin (1-5) amide [D-Ala2,Hyp4,Tyr5] (b-CM-11). The major product ions obtained in QIT-MS were used to construct fragmentation pathways for b-CMs. The different collision energies using automated nanoelectrospray ion source NanoMate and conventional LC in QIT-MS were studied. Calibration data for b-CMs, using spiked milk or cheese samples (10 g or 10 mL), were: NanoMate/MS (25-1000 microg/L), r(2)=0.998; NanoMate/MS(2) (5-1000 microg/L), r(2)=0.9992; NanoMate/MS(3) (2.5-1000 microg/L), r(2)=0.9998. Reproducibility data (% RSD, N=5) for NanoMate/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L.

Content of beta-casomorphins in milk of women with a history of allergy.

The prevalence of food allergies increased over the past decade. Most symptoms of food allergy appear during the first 2 yr of life. The aim of this study was to determine the beta-casomorphin-5 and -7 (BCMs) in colostrum and milk of 12 breast-feeding women with a history and clinical manifestation of food allergy. The results were compared with the data obtained from a control group of healthy age-matched breast-feeding women. The level of BCM in women with food allergy was constant during lactation, whereas the highest level of opioid peptides was found in colostrums of healthy women with a subsequent rapid decrease in mature milk. These differences in BCMs profile between allergic and healthy breast-feeding women suggest that BCM content in the human milk may be an indicator of allergic conditions.

Histidine decarboxylase (HDC) knock out mouse immune cells have altered expression of ACTH, triiodothyronine and endorphin.

OBJECTIVE: In earlier experiments the immune cells of HDC gene knock-out (HDC-KO) mice contained significantly more serotonin, than those of the wild ones. It was supposed that serotonin, being another biogenic amine, replenishes histamine deficiency. Now we extended our earlier studies to the investigation of the levels of other hormones (adrenocorticotropic hormone [ACTH], beta-endorphin, triiodothyronine [T(3)]) in this knockout model. METHODS: Peritoneal lavage fluid samples and thymuses were gained from HDC-KO and wild type mice. Their cells were prepared for flow cytometry and confocal microscopy by using specific antibodies to the three hormones (1st antibodies) and 2nd antibody to the 1st antibodies. The results of wild type and KO animals were compared. RESULTS: In KO animals the ACTH content in mast cells was significantly reduced and in thymic lymphocytes halved. Endorphin content was reduced both in peritoneal and thymic lymphocytes as well as in mast cells. T(3) content showed a two and a half-fold elevation in the monocyte-macrophage-granulocyte group. The confocal microscopic analysis showed the characteristic picture of HDC-KO mast cells, their cytoplasm being almost free of granules. CONCLUSION: Knock-out of the histidine decarboxylase gene causes a general endocrine imbalance in the hormones which are related to histamine, inside the immune cells. Levels of some hormones are elevated, others decreased.

Changes of beta-casomorphin content in human milk during lactation.

Milk is the best, complete food important for the development and nourishment of a neonate. Except for nutrients, milk contains biologically active opioid peptides derived from beta-casein, named beta-casomorphins (BCMs), which can exert effects in the gastrointestinal tract as well as in the whole body of neonates. The content of beta-casomorphins in human milk during maturation phases has not been studied so far. The aim of this study was to determine the content of beta-casomorphin-5 and -7 in human milk in different phases of lactation. A significantly higher concentration of both beta-casomorphins was found in colostrum than in mature milk. The concentration of beta-casomorphin in milk collected in the second month of lactation was similar to the level obtained in the fourth month of lactation. The content of beta-casomorphins in human milk was observed with the period of lactation. The level of opioid peptides may depend on the function of these peptides in neonate's body and may be associated with the maturation process.

Changes in the endorphin and serotonin content of rat immune cells during adulthood following maternal exposure to ethanol during pregnancy and lactation.

Lactating and lactating/pregnant rat dams consumed either 3% (vol/vol) ethanol (as the sole source of fluid) between the 1st and 21st days after delivery or 15% (vol/vol) ethanol for 24h on the 3rd day after delivery. Offspring of ethanol-consuming dams were compared with offspring of untreated control dams. In other groups, offspring of mothers given 3% ethanol during pregnancy were also compared to untreated controls. When the offspring were 2 months of age, endorphin and serotonin contents of immune cells (lymphocytes, granulocytes and monocytes, mast cells of the peritoneal fluid, and lymphocytes of the thymus) were determined by hormone-specific antibodies and flow cytometric as well as confocal microscopic analysis. In rats exposed to ethanol through breast-feeding, endorphin content significantly decreased in thymic cells independent of the alcohol concentration (and duration) during treatment. Each type of peritoneal cell contained significantly more serotonin after 3% alcohol treatment. For the prenatally exposed offspring, serotonin content significantly decreased for both ethanol treatment conditions during pregnancy. Remarkably, one day of exposure to 15% ethanol on the third day of pregnancy was sufficient to induce this enduring change in serotonin content of immune cells of offspring. Considering that endorphin and serotonin are important immunomodulators, these alcohol-induced changes could produce enduring influences on immune function.

Influence of paraformaldehyde and EDAC fixation on the demonstrability of hormones (histamine, endorphin, triiodothyronine) in rat immune cells: an immunocytochemical comparative analysis.

The amount and localization of three hormones (histamine, endorphin and triiodothyronine [T(3)]) was measured in male and female rat peritoneal cells (lymphocytes, mast cells, monocyte-macrophage-granulocyte group [mo-gran]) using flow cytometry as well as confocal microscopy after paraformaldehyde (PFA) or EDAC fixation. In the EDAC fixed lymphocytes and mo-gran of female animals two-magnitude higher levels of histamine were measured after EDAC fixation and one magitude higher in mast cells. The amount of T(3) was almost four-fold in lymphocytes and 2.5-4-fold in mast cells and mo-gran. Endorphin content was not altered by the type of fixation. In each cell type in males one magnitude higher levels of histamine and T(3) were measured after EDAC fixation and a small, but significant, elevation of endorphin. Confocal microscopy supports the quantitative data. The results show that (1) the fixation with the crosslinking molecule, EDAC, is more suitable for immunocytochemical studies of amino-acid type hormones in immune cells, (2) more histamine and T(3) are present in the immune cells than it was supposed previously when studying PFA-fixed preparations, (3) the estimation of the amount of peptide hormones seems to be accurate after PFA fixation, (4) there is a quantitative difference comparing the results of PFA and EDAC fixation between males and females.

[The effects of artificial analogue peptide TSKY isolated from the brain of hibernating ground squirrels in rats and mice].

Elevation of the i.c.v. injection dose of TSKY from 4 to 8 microg increased the movement activity of rats; in EEG theta- and beta-rhythms were enhanced and alpha-rhythm was suppressed. On the contrary, after treatment of 15 microg the rats fell into sleepy-like state; theta- and beta2-rhythms suppression, delta-, alpha- and beta1-rhythms were increased. Exposure under hypoxia-hypercapnia conditions reduced body temperature of mice to 18-19 degrees C, and maintain this state about 3-4 h after transferring into conventional gas medium. Preliminary cooling mice were administrated with TSKY that at dose 100 microg intraperitonally induced a prolonged hypothermia up to 12 h. Analogous injection without cooling raised mice temperature by 1.2 degrees C during about 2 h.

Effect of endorphin exposure at weaning on the endorphin and serotonin content of white blood cells and mast cells in adult rat.

Hormonal imprinting takes place perinatally at the first encounter between the developing receptor and its target hormone, resulting in the accomplishment of normal receptor development. In the presence of an excess of target hormone or the absence of it, or an excess of related molecules which can be bound by the receptor, faulty imprinting develops with life-long consequences. In previous experiments neonatal endorphin exposure caused a decrease in endorphin and serotonin content of peritoneal mast cells of adult animals. In the present experiment 25-day-old (weaned) female rats received 2 microg endorphin, and the endorphin as well as serotonin content of adult mast cells and white blood cells was studied by flow cytometry and confocal microscopy. Peritoneal lymphocytes and blood monocytes contained significantly (p<0.01) less endorphin and peritoneal mast cells less serotonin (p<0.07, i.e. of questionable significance) than the untreated control. The results bring attention to the possibility of durable imprinting of differentiating cells later in life and to the durable (possibly life-long) effect of an endorphin excess (perhaps caused by injury) manifested in the change of endorphin and serotonin content of immune cells.

Endorphin content of white blood cells and peritoneal cells in neonatally benzpyrene treated adult rats.

White blood cells of rats (lymphocytes, monocytes, macrophages, granulocytes and mast cells) contain beta-endorphin. Two months after a single neonatal benzpyrene treatment (imprinting) there is an elevated level of immunoreactive endorphin in the blood and peritoneal cells of female animals and blood cells of males. The endorphin content decreased in the peritoneal cells of males. In the blood, the granulocytes of female, and the lymphocytes of male rats contained the highest amount of endorphin. In the peritoneal fluid also the granulocytes of females contained the highest amount of endorphin, in contrast to males, where the endorphin content of cells decreased and the lowest level of it was present in the lymphocytes. The experiments justify that benzpyrene treatment can durably influence endorphin levels of white blood cells and gives new data to the already known lifelong health destroying effects of perinatal benzpyrene exposition (alterations of hormone receptor binding capacity and sexual behavior).

Dynorphin B is an agonist of nuclear opioid receptors coupling nuclear protein kinase C activation to the transcription of cardiogenic genes in GTR1 embryonic stem cells.

The cardiac differentiation of embryonic stem (ES) cells was found to involve prodynorphin gene and dynorphin B expression and was associated with the interaction of secreted dynorphin B with cell surface opioid receptors coupled with protein kinase C (PKC) signaling and complex subcellular redistribution patterning of selected PKC isozymes. Here, confocal microscopy revealed the presence of immunoreactive dynorphin B-like material in GTR1 ES cells, suggesting that dynorphin peptides may also act intracellularly. Opioid binding sites were identified in ES cell nuclei, with a single dissociation constant in the low nanomolar range. A significant increase in Bmax for a kappa opioid receptor ligand was observed in nuclei isolated from ES-derived cardiomyocytes compared with nuclei from undifferentiated cells. Direct exposure of nuclei isolated from undifferentiated ES cells to dynorphin B or U-50,488H, a synthetic kappa opioid receptor agonist, time- and dose-dependently activated the transcription of GATA-4 and Nkx-2.5, 2 cardiac lineage-promoting genes. Nuclear exposure to dynorphin B also enhanced the rate of prodynorphin gene transcription. These responses were abolished in a stereospecific fashion by the incubation of isolated nuclei with selective opioid receptor antagonists. Nuclei isolated from undifferentiated cells were able to phosphorylate the acrylodan-labeled MARCKS peptide, a high-affinity fluorescent PKC substrate. Exposure of isolated nuclei to dynorphin B induced a remarkable increase in nuclear PKC activity, which was suppressed by opioid receptor antagonists. Nuclear treatment with PKC inhibitors abolished the capability of dynorphin B to prime the transcription of cardiogenic genes.