Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva.

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.

Protein levels and protease activity in milk from mothers of pre-term infants: A prospective longitudinal study of human milk macronutrient composition.

BACKGROUND AND AIMS: The composition and enzymology of human milk changes throughout the lactation period, and differ for mothers who give birth prematurely compared to those who deliver at full-term. Understanding the composition of milk from mothers of very low birth weight premature infants is of great significance, and the objective of this study was to evaluate the composition, protein profile and plasmin activity of milk from mothers who delivered infants at different gestational ages. METHODS: Samples of human milk were donated by women (n = 74) in the Cork, Ireland, area who gave birth to full-term (>37 weeks gestation, FT), pre-term (32-37 weeks, PT) and very pre-term (≤32 weeks, VPT) infants. FT milk was collected at 1, 3, 6 and 10 weeks post-partum (PP), while PT and VPT milk was collected weekly until the FT due date of the infant and subsequently followed the FT protocol. RESULTS: Gestational age did not significantly affect lactose or fat content or total energy content of milk. However, protein content, and levels of some individual proteins, were significantly affected by both gestational age at birth and duration of lactation, with significantly higher protein levels in PT or VPT milk samples at 0-7 days and 1-2 months, respectively. Plasmin activity was significantly higher in VPT milk, indicating differences in proteolytic processing in milk. CONCLUSION: Compositional differences between the milk of mothers of term and pre-term infants were greatest in terms of the protein profile, which showed both qualitative and quantitative differences, as well as difference in proteolytic activity.

Macronutrient Digestion and Absorption in the Preterm Infant.

The human fetus receives oral nutrition through swallowed amniotic fluid and this makes a significant nutritional contribution to the fetus. Postnatally, macronutrient absorption and digestion appear to function well in the preterm infant. Although pancreatic function is relatively poor, the newborn infant has several mechanisms to overcome this. These include a range of digestive enzymes in human milk, novel digestive enzymes involved in fat and protein digestion that do not appear to be present in the older child or adult, and the presence of a Bifidobacterium-rich colonic microbiome that may "scavenge" unabsorbed macronutrients and make them available to the infant.

Selective Proteolysis of α-Lactalbumin by Endogenous Enzymes of Human Milk at Acidic pH.

SCOPE: The use of human milk products is increasing for high-risk infants. Human milk contains endogenous enzymes that comprise a dynamic proteolytic system, yet biological properties of these enzymes and their activities in response to variations including pH within infants are unclear. Human milk has a neutral pH around 7, while infant gastric pH varies from 2 to 6 depending on individual conditions. This study is designed to determine the specificity of enzyme-substrate interactions in human milk as a function of pH. METHODS AND RESULTS: Endogenous proteolysis is characterized by incubating freshly expressed human milk at physiologically relevant pH ranging from 2 to 7 without the addition of exogenous enzymes. Results show that the effects of pH on endogenous proteolysis in human milk are protein-specific. Further, specific interactions between cathepsin D and α-lactalbumin are confirmed. The endogenous enzyme cathepsin D in human milk cleaves α-lactalbumin as the milk pH shifts from 7 to 3. CONCLUSIONS: This study documents that selective proteolysis activated by pH shift is a mechanism for dynamic interactions between human milk and the infant. Controlled proteolysis can guide the use of human milk products based on individual circumstance.

Impact of Maternal Diet on Human Milk Composition Among Lactating Women in Latvia.

Background and objectives: Many studies indicate that the maternal diet is an important factor affecting human milk composition. Human milk composition among lactating women in Latvia, as well as the maternal diet during lactation, has not been sufficiently studied. The aim of this research was to assess dietary habits and macronutrient intake among lactating women in Latvia and to examine the effect of diet on human milk composition. Materials and Methods: Research was conducted between November 2016 and December 2017. Mature human milk samples (n = 61) along with a 72h food diary, a food frequency questionnaire (FFQ), and a questionnaire about maternal and infant characteristics were obtained from voluntary women who were recruited via an invitation published in a social media member group for nursing mothers. Fat content in human milk was determined by LVS ISO 2446:2008, protein content was determined by LVS EN ISO 8968-1:2014, lactose was determined by ISO 22662:2007, and the fatty acid profile was analyzed using gas chromatography. Dietary data were evaluated using the Finnish food composition database Fineli, release 19 (3 March 2018). Results: Median values for fat, protein, and lactose in mature human milk were 4.40%, 1.08%, and 6.52%, respectively. Predominant fatty acids in human milk were oleic acid (C18:1 n9c), palmitic acid (C16:0), and linoleic acid (C18:2 n6c) at 34.60%, 24.00%, and 11.00% of total fatty acids, respectively. The trans elaidic acid (C18:1 n9t) level was <0.10% in all human milk samples. Significant, positive associations (p < 0.05) were found between maternal dietary intake of linoleic, α-linolenic, docosahexaenoic, total cis-monounsaturated, total cis-polyunsaturated, and total n-6 and n-3 polyunsaturated fatty acids, the ratio of n-6/n-3, and the level of these fatty acids in human milk. Total energy and carbohydrate intake among participants were lower, but total fat, saturated fat, and sugar intake were higher than recommended. Protein, linoleic acid, and α-linolenic acid intake were adequate, but docosahexaenoic acid intake was noticeably lower than recommended. Women should be supported with information regarding their nutritional needs during lactation and the possible impact of diet on human milk composition. Conclusion: Macronutrient (fat, protein, and lactose) content in human milk is not affected by maternal diet. Conversely, the human milk fatty acid profile is affected by the immediate diet consumed by the mother. Habitual dietary habits can also impact the fatty acid profile of human milk.

New short-term heat inactivation method of cytomegalovirus (CMV) in breast milk: impact on CMV inactivation, CMV antibodies and enzyme activities.

OBJECTIVES: Breast milk (BM) is the primary source of cytomegalovirus (CMV) transmission to premature infants with potentially harmful consequences. We therefore wanted to evaluate temperature and duration of short-term BM pasteurisation with respect to CMV inactivation, effect on CMV-IgG antibodies and BM enzyme activities. METHODS: 116 artificially CMV-spiked BM and 15 wild-type virus-infected samples were subjected for 5 s to different temperatures (55°C-72°C). CMV-IE-1 expression in fibroblast nuclei was assessed using the milk whey fraction in short-term microculture. BM lipase and alkaline phosphatase (AP) activities and CMV binding using CMV-recomLine immunoblotting and neutralising antibodies using epithelial target cells were analysed before and after heating. RESULTS: A minimum of 5 s above 60°C was necessary for CMV inactivation in both CMV-AD-169 spiked and wild-type infected BM. Lipase was very heat sensitive (activities of 54% at 55°C, 5% at 60°C and 2% at 65°C). AP showed activities of 77%, 88% and 10%, respectively. CMV-p150 IgG antibodies were mostly preserved at 62°C for 5 s. CONCLUSION: Our results show that short-term pasteurisation of BM at 62°C for 5 s might be efficient for CMV inactivation and reduces loss of enzyme activities, as well as CMV binding, and functional CMV antibodies.

Ontogeny of alkaline phosphatase activity in infant intestines and breast milk.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating disease of intestinal inflammation that primarily affects premature infants. A potential risk factor for necrotizing enterocolitis is exposure of the premature neonatal intestine to environmental bacteria and their proinflammatory products such as lipopolysaccharide. The metalloenzyme alkaline phosphatase (ALP) has been shown to reduce lipopolysaccharide-mediated inflammation. Additionally, premature rat pups have reduced alkaline phosphatase activity and expression as compared to full term pups. To explore the possibility that the human premature neonatal intestine has a paucity of alkaline phosphatase activity, we measured endogenously produced intestinal alkaline phosphatase activity in meconium as a function of gestational age. To test whether breast milk could serve as a source of exogenous alkaline phosphatase to the neonatal intestine through ingestion, we measured alkaline phosphatase activity in breast milk across a range of time points post-birth. METHODS: Alkaline phosphatase activity was quantified in 122 meconium samples from infants of gestational ages ranging from 24 to 40 weeks and in 289 breast milk samples collected from 78 individual mothers between days 2-49 post-birth. RESULTS: We observed a strong positive correlation between the meconium alkaline phosphatase activity and gestational age, with preterm infants having lower meconium alkaline phosphatase activities than early term or term infants. Breast milk alkaline phosphatase activity was highest in the first week post-birth, with peak alkaline phosphatase activity at day 2 post-birth, followed by relatively low alkaline phosphatase activity in weeks 2-7. CONCLUSIONS: Our results are consistent with the two major risk factors for necrotizing enterocolitis development, preterm birth and lack of breast milk feeding, both contributing to a paucity of alkaline phosphatase activity and impaired capacity to detoxify proinflammatory bacterial products such as lipopolysaccharide.

Micronutrient content and antioxidant enzyme activities in human breast milk.

Breast milk contains micronutrients that function as cofactors of antioxidant enzymes. High concentrations of iron (Fe) and copper (Cu) can increase the production of reactive oxygen species (ROS). This study aimed to assess the relationship between the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST)) and the concentration of the micronutrients Fe, Cu and zinc (Zn) in breast milk. Breast milk samples were collected from 108 mothers (7-10 days postpartum, transitional milk). The samples were grouped into three groups according to the number of pregnancies (one, two and three or more pregnancies), also grouped according to the body mass index (BMI) suggested by the World Health Organization (WHO) in underweight, normal weight, overweight and obese. Breast milk Fe, Cu and Zn concentrations were determined by atomic absorption spectrophotometry and the activity of the antioxidant enzymes was determined by spectrophotometry. An increase in GPx, SOD and GST activities in relation to the number of pregnancies was found (p = 0.05, p = 0.04 and p < 0.01, respectively). An inverse relationship between GST activity and BMI was found (p = 0.05). A positive correlation was observed between Cu and Zn concentrations (r = 0.52, p < 0.05). A negative correlation was found between Cu concentration and catalase activity (r = -0.22, p < 0.05); Fe content was negatively correlated with GPx and GST activities (r = -0.32, r = -0.22, respectively, p < 0.05). The activities of antioxidant enzymes (GPx, SOD and GST) may be affected by the number of pregnancies and contribute to prevent oxidation of nutritional molecules in breast milk.

Regional variations in human milk oligosaccharides in Vietnam suggest FucTx activity besides FucT2 and FucT3.

Breastfeeding is the normal way of providing young infants with the nutrients they need for healthy growth and development (WHO). Human milk oligosaccharides (hMOS) constitute a highly important class of nutrients that are attracting strong attention in recent years. Several studies have indicated that hMOS have prebiotic properties, but also are effective in anti-adhesion of pathogens, modulating the immune system and providing nutrients for brain growth and development. Most of the latter functions seem to be linked to the presence of fucose-containing immunodeterminant epitopes, and Neu5Ac-bearing oligosaccharides. Analysis of hMOS isolated from 101 mothers' milk showed regional variation in Lewis- and Secretor based immunodeterminants. Lewis-negative milk groups could be sub-divided into two sub-groups, based on the activity of a third and hitherto unidentified fucosyltransferase enzyme. Analysis of hMOS remaining in faeces showed three sub-groups based on hMOS surviving passage through the gut, full consumption, specific partial consumption and non-specific partial consumption, fitting previous findings.

A novel FADS2 isoform identified in human milk fat globule suppresses FADS2 mediated Δ6-desaturation of omega-3 fatty acids.

INTRODUCTION: The only known non-pharmacological means to alter long chain polyunsaturated fatty acid (LCPUFA) abundance in mammalian tissue is by altering substrate fatty acid ratios. Alternative mRNA splicing is increasingly recognized as a modulator of protein structure and function. Here we report identification of a novel alternative transcript (AT) of fatty acid desaturase 2 (FADS2) that inhibits production of omega-3 but not omega-6 LCPUFA, discovered during study of ATs in human milk fat globules (MFG). METHODS: Human breastmilk collected from a single donor was used to isolate MFG. An mRNA-sequencing library was constructed from the total RNA isolated from the MFG. The constructed library was sequenced using an Illumina HiSeq instrument operating in high output mode. Expression levels of evolutionary conserved FADSAT were measured using cDNA from MFG by semi-quantitative RT-PCR assay. RESULTS: RNA sequencing revealed >15,000 transcripts, including moderate expression of the FADS2 classical transcript (CS). A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was discovered. When FADS2AT2 was transiently transfected into MCF7 cells stably expressing FADS2, delta-6 desaturation (D6D) of alpha-linolenic acid 18:3n-3 → 18:4n-3 was suppressed as were downstream products 20:4n-3 and 20:5n-3. In contrast, no significant effect on D6D of linoleic acid 18:2n-6 → 18:3n-6 or downstream products was observed. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG. FADS1, FADS3AT3, and FADS3AT5 are undetectable. CONCLUSION: The novel, noncatalytic FADS2AT2 regulates FADS2CS-mediated Δ6-desaturation of omega-3 but not omega-6 PUFA biosynthesis. This spliced isoform mediated interaction is the first molecular mechanism by which desaturation of one PUFA family but not the other is modulated.

Influence of a Concurrent Exercise Training Program During Pregnancy on Colostrum and Mature Human Milk Inflammatory Markers: Findings From the GESTAFIT Project.

BACKGROUND: Although exercise reduces systemic inflammation, information regarding its influence on human milk is scarce or inexistent. Research Aim: The aim of this study was to investigate the influence of an exercise intervention during pregnancy on colostrum and mature human milk inflammatory markers. METHODS: The authors conducted a pseudorandomized controlled trial. The exercise group followed a concurrent aerobic and strength training, three 60-minutes sessions per week, from the 17th gestational week until delivery. For the specific aims of this study, only women able to produce enough milk were included for data analyses, resulting in 24 exercise and 23 control women. Colostrum and mature human milk proinflammatory and anti-inflammatory cytokines (fractalkine, interleukin [IL]-1ß, IL-6, IL-8, IL-10, interferon [IFN]-γ, and tumor necrosis factor [TNF]-α) were measured using Luminex xMAP technology. RESULTS: The mothers who followed the exercise program had 36% lower IL-8 and 27% lower TNF-α concentrations in their colostrum than those in the control group ( p < .05 and p < .01, respectively). The colostrum from mothers who followed the exercise program also presented borderline significant 22% lower IL-6 ( p < .100). The mature milk from mothers who followed the exercise program had 30% greater fractalkine ( p = .05) and borderline significant 20% higher IL-10 ( p = .100). The exercise intervention did not affect IFN-γ concentrations. CONCLUSIONS: This concurrent exercise program promoted a less proinflammatory profile in human milk, especially in colostrum. Moreover, it might increase mature human milk fractalkine, which could induce a greater neurodevelopment and neuroprotection in the newborn. This trial was registered at ClinicalTrials.gov (NCT02582567) on October 20, 2015.

Hot topic: Holder pasteurization of human milk affects some bioactive proteins.

The aim of this research was to investigate the effect of Holder pasteurization (HoP; 62.5°C, 30 min) on the protein profile and activities of glutathione peroxidase (GPx) and lysozyme (LZ) in human milk. Over 6 mo of lactation, human milk samples were analyzed before (raw) and after HoP for GPx and LZ activity and electrophoresis protein profile. Holder pasteurization reduced human milk lactoferrin, immunoglobulin fractions, and GPx activity. In addition, GPx activity, which is high in colostrum and transitional milk, was naturally reduced over the 6-mo lactation period. In contrast, HoP did not affect human milk LZ activity. Besides its critical cellular antioxidant role in protecting the organism from oxidative damage, GPx decreases the redox potential of milk, stimulating the growth of anaerobic microorganisms, such as the probiotic Bifidobacterium. Considering the role of lactoferrin in infant health, we conclude that an important part of its function has been inactivated by pasteurization. These compounds should be replaced by human milk banks after the HoP step to recover lost functionality. Otherwise, an alternative technology to HoP that better retains human milk properties should be used by milk banks to eliminate the risk of transmission of infectious agents.

Relation between polyphenols, malondialdehyde, antioxidant capacity, lactate dehydrogenase and toxic elements in human colostrum milk.

The present study investigated the interrelationships between the concentration of total polyphenols (TP), malondialdehyde (MDA), total antioxidant capacity (TAC), lactate dehydrogenase activity (LDH) and content of toxic elements (Al, As, Cd, Ni, Pb) in human colostrum milk (n = 75), and further assessed their potential association with maternal lifestyle characteristics. As and Cd were always below detection limits while Al, Ni and Pb were found at the level of 89.7, 6.2 and 1.3 µg L-1, respectively. Concentrations of TP and MDA, in the studied group were 46.91 ± 21.25 mg GAE L-1 and 0.66 ± 0.27 nmol mL-1, respectively, and were inversely correlated (Rs = -0.32; p < 0.01). TP and TAC increased significantly with maternal consumption of vegetables (Rs = 0.25 and Rs = 0.37, respectively; p < 0.05). Concentration of Al was positively correlated with MDA (Rs = 0.21; p < 0.01) and negatively with TP (Rs = -0.28; p < 0.01). Positive correlation was also found between Pb and MDA (Rs = 0.32; p < 0.01). No association with place of living (urban/rural), women's age and former smoking were found for any studied milk parameter. The results add to the general understanding of factors influencing redox balance in milk and potentially affecting its quality.

Different physiological properties of human-residential and non-human-residential bifidobacteria in human health.

Bifidobacteria have increasingly been shown to exert positive health benefits to humans, which are clearly reflected by their application in various commercialised dairy products and supplements. Bifidobacteria naturally inhabit a range of ecological niches and display substantial differences in their ecological adaptation among species. In general, bifidobacteria could be categorised into two major groups; bifidobacterial species of human origins as human-residential bifidobacteria (HRB) while other species which are the natural inhabitants of animals or environment as non-HRB. Current research has focused on the differential physiological features of HRB and non-HRB, such as metabolic capabilities, whilst comparative and functional genomic investigations have revealed the genetic attributes of bifidobacteria that may explain their colonisation affinities in human gut. It is becoming more apparent that distinct residential origins of bifidobacteria are likely contributed to their comparable adaptive health attributes on human host. Notably, debate still remains about the nature of bifidobacteria for use as human probiotics. Clinical evaluations involving supplementation of bifidobacteria of different origins point out the superiority of HRB in human host. Evidence also suggests that HRB especially infant-type HRB may exert better health-promoting effects and therefore serve as a better probiotic candidate for infant use. In this review, we aim to provide an overview of the genotypic and physiological differences of bifidobacteria associated with different residential origins and to shed light on the practical considerations for selection of bifidobacteria as probiotics in order to establish a healthy gut microbial community in humans.

Breast Milk from Smokers Contains Less Cholesterol and Protein and Smaller Size of Apolipoprotein A-I Resulting in Lower Zebrafish Embryo Survivability.

BACKGROUND: To determine the quality of breast milk (BM), we compared the functions of BM from ex-smokers and nonsmokers. SUBJECTS AND METHODS: We analyzed the contents of lipids, glucose, and protein in BM from ex-smokers (10 cigarettes/day for 13 ± 3 years) as well as infant formula. RESULTS: Nonsmokers' BM showed 2.4- and 1.4-fold higher cholesterol and protein contents, respectively, than BM from smokers. Infant formula contained almost no cholesterol, but did show remarkably higher glucose and triglyceride levels than BM. Microinjection of BM (50 nL) from nonsmokers and smokers into zebrafish embryos resulted in 59% and 44% survival, respectively, whereas formula injection resulted in 31% survival. The higher cholesterol and protein contents of BM were directly correlated with higher embryo survivability, suggesting that cholesterol content is directly and critically associated with growth of neonate infants. Smokers' BM contained smaller-sized apolipoproteinA-I (apoA-I) (24.4 ± 0.2 kDa) than BM from nonsmokers (26.7 ± 0.4 kDa), suggesting that putative modification and cleavage occurred in apoA-I. BM containing higher molecular weight apoA-I resulted in higher embryo survivability. CONCLUSIONS: Smoking before pregnancy can affect the composition and quality of BM, resulting in almost complete loss of cholesterol and protein, especially lactoferrin, lactalbumin, and apoA-I, accompanied by proteolytic degradation. These impairment effects of BM are associated with elevation of oxidative stress and lower embryo survivability.

Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age.

Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown.Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age.Methods: Milk samples were collected from 18 mothers aged 26-40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24-26 wk GA)-delivering mothers and 9 from late GA (LGA) (27-32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age.Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL-1 · d-1 and 0.454 µg · mL-1 · d-1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples.Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not vary substantially across GA and postnatal age.

Imidazolium-labeled glycosides as probes to harness glycosyltransferase activity in human breast milk.

Imidazolium-labeled (ITag-) glycosides are used to harness the glycosyltransferase activity directly from human breast milk. The covalently attached ionic labels provide a bifunctional chemical handle that is used to monitor reaction progress by MS, as well as aid in product purification from complex mixtures. The technology is exemplified in the synthesis of biologically relevant oligosaccharide analogs, LacNAc-ITag, ITag-Lewisx and ITag-Lewisa, in a matter of days from human breast milk without having to isolate specific enzymes.

Enzymes in Human Milk.

Milk proteins are a complex and diverse source of biological activities. Beyond their function, intact milk proteins also act as carriers of encrypted functional sequences that, when released as peptides, exert biological functions, including antimicrobial and immunomodulatory activity, which could contribute to the infant's competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself. A complex array of proteases produced in mother's milk has been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant's stomach, possibly even to a larger extent than the infant's own proteases. As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease-based peptide release system.

Pasteurization Procedures for Donor Human Milk Affect Body Growth, Intestinal Structure, and Resistance against Bacterial Infections in Preterm Pigs.

Background: Holder pasteurization (HP) destroys multiple bioactive factors in donor human milk (DM), and UV-C irradiation (UVC) is potentially a gentler method for pasteurizing DM for preterm infants.Objective: We investigated whether UVC-treated DM improves gut maturation and resistance toward bacterial infections relative to HP-treated DM.Methods: Bacteria, selected bioactive components, and markers of antioxidant capacity were measured in unpasteurized donor milk (UP), HP-treated milk, and UVC-treated milk (all from the same DM pool). Fifty-seven cesarean-delivered preterm pigs (91% gestation; ratio of males to females, 30:27) received decreasing volumes of parental nutrition (average 69 mL · kg-1 · d-1) and increasing volumes of the 3 DM diets (n = 19 each, average 89 mL · kg-1 · d-1) for 8-9 d. Body growth, gut structure and function, and systemic bacterial infection were evaluated.Results: A high bacterial load in the UP (6×105 colony forming units/mL) was eliminated similarly by HP and UVC treatments. Relative to HP-treated milk, both UVC-treated milk and UP showed greater activities of lipase and alkaline phosphatase and concentrations of lactoferrin, secretory immunoglobulin A, xanthine dehydrogenase, and some antioxidant markers (all P < 0.05). The pigs fed UVC-treated milk and pigs fed UP showed higher relative weight gain than pigs fed HP-treated milk (5.4% and 3.5%), and fewer pigs fed UVC-treated milk had positive bacterial cultures in the bone marrow (28%) than pigs fed HP-treated milk (68%) (P < 0.05). Intestinal health was also improved in pigs fed UVC-treated milk compared with those fed HP-treated milk as indicated by a higher plasma citrulline concentration (36%) and villus height (38%) (P < 0.05) and a tendency for higher aminopeptidase N (48%) and claudin-4 (26%) concentrations in the distal intestine (P < 0.08). The gut microbiota composition was similar among groups except for greater proportions of Enterococcus in pigs fed UVC-treated milk than in pigs fed UP and those fed HP-treated milk in both cecum contents (20% and 10%) and distal intestinal mucosa (24% and 20%) (all P < 0.05).Conclusions: UVC is better than HP treatment in preserving bioactive factors in DM. UVC-treated milk may induce better weight gain, intestinal health, and resistance against bacterial infections as shown in preterm pigs as a model for DM-fed preterm infants.