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Heat treatment and irradiation reduce anti-bacterial and immune-modulatory properties of bovine colostrum
Journal of Functional Foods 57 (2019) 182–189
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Heat treatment and irradiation reduce anti-bacterial and immunemodulatory properties of bovine colostrum Duc Ninh Nguyena, Andrew J. Currieb, Shuqiang Rena, Stine B. Beringa, Per T. Sangilda,c,
T ⁎
a
Section for Comparative Pediatrics and Nutrition, Department of Veterinary and Animal Sciences, University of Copenhagen, Denmark Medical, Molecular & Forensic Sciences, Murdoch University, Perth, Australia c Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen, Denmark b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobial activity Bacterial growth inhibition Bovine colostrum Preterm infants Pasteurization
Colostrum contains bioactive components protecting the newborn intestine against bacteria. It is unclear how to optimize processing conditions with highest product bioactivity. Non-pasteurized (BC00), standard-pasteurized (72 °C-15 s, BC72), gently-pasteurized bovine colostrums without (63 °C-30 min, BC63) and with gamma-irradiation (BC63g) were tested for effects on bacterial growth inhibition (Escherichia coli, Staphylococcus aureus and S. epidermidis), intestinal epithelial cell (IEC) proliferation and cytokine secretion in vitro. Thermal processing decreased endogenous bacteria and IgG levels. All BCs inhibited bacterial growth 1–2 h after inoculation, but only BC00, BC63 and BC63g retained activity after 4–24 h. After 4 h, the activity against S. epidermidis of BC63g was lower than BC00 but still potent when mixed with formula. All BCs stimulated IEC proliferation, with the most pronounced responses for BC00. Only BC00 and BC63 increased IL-8 secretion in lipopolysaccharide-stimulated IECs. Thermal processing reduced bioactivity and combined gentle pasteurization and gamma-irradiation improved BC sterility and bioactivity, relative to standard pasteurization.
1. Introduction It is well accepted that mother’s own milk is the best choice for newborn infants, in part due to its protective effects against infection and gut inflammation (Chatterton, Nguyen, Bering, & Sangild, 2013; Kelley, 2012). However, many infants, including vulnerable preterm infants (born < 37 weeks of gestation, 15 million every year, 11% of all births), have limited access to mother’s milk, especially during the first weeks of life, and they are often fed or supplemented with infant formula (Lucas & Cole, 1990; Merewood, Brooks, Bauchner, MacAuley, & Mehta, 2006). Feeding infant formula has been documented to be detrimental to infant health with increased prevalence of infection, gut inflammation and neurodevelopmental delays (Strunk et al., 2014). Donor human milk has been considered as an alternative to mother’s own milk for preterm infants, but it is still unclear to which extent it matches mother’s milk and if it is superior to infant formula in protecting against infectious diseases (Corpeleijn et al., 2016). One consideration is the pasteurization process applied to donor milk, which may decrease the levels of multiple bioactive proteins including immunoglobulins (Ig) and lactoferrin (Ewaschuk, Unger, Harvey, O’Connor, & Field, 2011).
Bovine colostrum (BC) may be a better option than infant formula to provide protection against infection and inflammation for newborn infants as it contains a wide spectrum of bioactive proteins and peptides at higher concentrations than in mature milk, e.g. lactoferrin, lactoperoxidase, osteopontin and transforming growth factor β (Chatterton et al., 2013). These components possess antimicrobial and anti-inflammatory activities (Chatterton et al., 2013). For instance, dietary lactoferrin exerts strong in vitro antimicrobial activity against bacteria causing neonatal sepsis and enhances systemic immune responses against neonatal infection in both newborn pigs and infants (Manzoni et al., 2009; Manzoni, Mostert, & Stronati, 2011; Reznikov et al., 2018; Woodman et al., 2018). Supplementation of osteopontin into infant formula has shown to decrease gut inflammation in newborn pigs (Møller et al., 2011) and modulate systemic immune development in newborn infants (West et al., 2017). Using preterm pigs as a model for vulnerable newborn infants, we have documented that feeding gentlypasteurized BC (63 °C for 30 min), promotes gut maturation and protects against gut inflammation and infection (Brunse, Worsøe, Pors, Skovgaard, & Sangild, 2018; Jensen et al., 2013; Shen et al., 2015; Sun, Li, Nguyen, et al., 2018; Sun, Li, Pan, et al., 2018). The BC feeding also modulates expression of innate immune genes associated with anti-
⁎ Corresponding author at: Section for Comparative Pediatrics and Nutrition, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Dyrlægevej 68, DK-1870 Frederiksberg C, Denmark. E-mail address: [email protected] (P.T. Sangild).
https://doi.org/10.1016/j.jff.2019.04.012 Received 7 January 2019; Received in revised form 19 March 2019; Accepted 6 April 2019 1756-4646/ © 2019 Published by Elsevier Ltd.
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inflammatory effects against gut inflammation via epigenetic mechanisms (Pan, Gong, Gao, & Sangild, 2018; Willems et al., 2015). Numerous preclinical studies in preterm pigs with beneficial effects of BC feeding have led to a pilot clinical trial testing BC as a supplement to mother’s own milk during the first two weeks of life in preterm infants with indications of better tolerance to BC than IF (Juhl et al., 2018; Li, Juhl, et al., 2017). Two larger randomized clinical trials in preterm infants with BC as a supplement to mother’s own milk and as a fortifier to donor human milk are ongoing (NCT03085277 and NCT03537365, respectively, clinicaltrials.gov). Although the protective effects of BC against gut inflammation and infection are well documented, the underlying mechanisms remain elusive. Neonatal sepsis may in part result from bacterial translocation across the immature gut barrier into the systemic circulation (Deitch, 2012; MacFie et al., 1999). The high levels of antimicrobial and immune-modulatory proteins in BC may contribute to eliminating pathogenic bacteria in the gut, leading to decreased gut inflammation and gut-derived sepsis. Similar mechanisms may explain the lower incidence of gut inflammation and sepsis in breast-fed, relative to formula-fed newborn infants (Lucas & Cole, 1990; Trend et al., 2015). Further, processing technology (e.g. pasteurization) strongly affects the levels of heat-labile bioactive proteins, e.g. lactoferrin and IgG (Li, Nguyen, et al., 2017; Li et al., 2018, 2013; Nguyen, Sangild, Li, Bering, & Chatterton, 2016; Saldana, Gelsinger, Jones, & Heinrichs, 2019; Tacoma et al., 2017). High-temperature short-time (72 °C, 15 s) pasteurization is a standard procedure for bovine milk whereas donor human milk is often pasteurized at a low-temperature long-time condition (63 °C, 30 min). The later gentler treatment may help to retain higher levels of bioactive components and thereby greater bioactivity for BC. Gamma-irradiation is also a common sterilization method for foodstuffs with reported efficacy to eliminate pathogens in colostrum without affecting Ig activity (Barin-Bastuji, Perrin, Thorel, & Martel, 1990; Vellenga, Wensing, Breukink, & Hagens, 1986), suggesting another potentially gentle treatment to retain higher levels of bioactive components. We hypothesized that the type of heat treatment during BC processing affects the product antimicrobial and immune-modulatory properties in the newborn intestine, with low-temperature, long-time pasteurization and irradiation retaining greater bioactivity than the standard high-temperature short-time process. We generated four BC products from the same raw milk, but with different processing conditions, to test their antimicrobial activity against bacteria often associated with neonatal sepsis as well as their immune-modulatory activities in intestinal epithelial cells (IECs).
Fig. 1. Schematic diagram demonstrating the production flow of four types of colostrum from the same source of raw colostrum to the final powdered products. BC, bovine colostrum; BC00, raw BC without pasteurization; BC63, BC with gentle pasteurization (63 °C, 30 min) and gentle spray drying; BC63g, BC63 with gamma-irradiation; BC72, BC with conventional pasteurization (72 °C, 15 s) and conventionally harsh spray drying.
15 min to remove the fat content. The skimmed fraction was sterilefiltered at 0.22 µm and stored in aliquots at −80 °C until analysis. The concentration of bovine IgG was used as an indicator of bioactive proteins and analyzed after reconstitution of BC samples at 10 g/L in water using ELISA with sheep anti-bovine IgG antibody (A10-118A, Nordic Biosite, Copenhagen, Denmark). 2.2. Microbiological analysis of bovine colostrum The four BCs and the IF were diluted with sterile saline (dilution factors of 100 to 10−5) and spotted in triplicates onto non-selective sheep blood agar plates (PathWest Laboratory Medicine WA Media, Claremont, Australia) and incubated overnight at 37 °C and 5% CO2. Colony forming units (CFU) were counted after overnight incubation to calculate the bacterial contents in the BCs and were used to determine the bacterial identity by MALDI-TOF (Microflex, Bruker). Representative CFUs were also used for Gram-staining and visualized with a light microscope under oil immersion. 2.3. Frozen bacterial stock and antimicrobial activity of bovine colostrum Three bacterial strains isolated from septic preterm infants (kindly provided by King Edward Memorial Hospital, Western Australia, Australia) were used for the assay, including Escherichia coli, Staphylococcus aureus and S. epidermidis. These strains were stored at −80 °C in Protect Beads (Microorganism preservation system, Technical service consultant, UK) and used to prepare mid-log frozen stocks as previously described (Trend et al., 2015; Woodman et al., 2018). Bacteria in beads were grown overnight at 37 °C, 5% CO2 in heart infusion broth (Oxoid, Hampshire, England) and streaked out onto blood agar. Thereafter single isolates were inoculated in heart infusion broth and incubated overnight at the same condition as above. The overnight cultures were adjusted in fresh broth to OD 0.05 at 600 nm (Biophotometer D30, Eppendorf, Hamburg, Germany) and incubated again until mid-log phase (OD600nm ∼ 0.6) and aliquoted for storage at −80 °C (in 10% glycerol for S. aureus and S. epidermidis, and in 10% fetal bovine serum for E. coli) prior to experiments. The viability of mid-log stocks was evaluated by plating out diluted stocks (dilution factors of 10−1–10−6) and counting as described above and the bacterial concentration was used to calculate starting inoculum doses for bacterial challenge in the antimicrobial assays. For the antimicrobial assay, bacterial mid-log culture stocks were thawed, centrifuged at 3200g and resuspended in sterile saline prior to incubation with BC or IF at a calculated dose of 104–106 cells/mL for S. epidermidis and S. aureus or 102–104 cells/mL for E. coli for 4 h at 37 °C
2. Materials and methods 2.1. Processing of bovine colostrum and preparation for in vitro study The same batch of raw BC collected from Danish Holstein cows during the first 3 days after parturition was used to produce four types of BC powder, as depicted schematically in Fig. 1. Raw BC was homogenized and spray-dried without pasteurization (BC00), or pasteurized at 63 °C for 30 min (BC63) or 72 °C for 15 s (BC72) prior to spraydrying. Powdered samples were then packaged under sterile conditions. The packaged BC63 product was also gamma-irradiated (14 kGy dose) to secure a bacteria-free product (BC63g) as used in clinical trials (Juhl et al., 2018; Li, Juhl, et al., 2017). The powdered products contained 52.0% protein, 17.4% carbohydrate and 22.8% fat. For some experiments, PreNAN GOLD ready-to-feed preterm infant formula (IF, Nestle, Vevey, Switzerland) was used as a negative control. To test bacterial growth inhibition in vitro, BC powders were dissolved in sterile water at a concentration of 16.8 g or 81 kcal per 100 mL, similar to preparations of BC in the clinical trials (Juhl et al., 2018; Li, Juhl, et al., 2017), and stored in aliquots of 1 mL at −80 °C. For in vitro IEC stimulation, the BC powder was reconstituted in sterile water at a concentration of 10 g/L, then centrifuged at 10,000g, 4 °C for 183
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Table 1 Levels of bacteria in infant formula (IF) and four differentially heat-treated bovine colostrum products (BC) detected by counting after plating on blood agar plates and identified by MALDI-TOF (n = 3, values are mean ± SD). E. faecalis
B. licheniformis
B.cereus
A. radioresistens
Unidentified
Bacterial count in liquid samples (CFU/mL) IF 0 BC00 1283 ± 210 BC63 0 BC63g 0 BC72 0
0 50 ± 0 0 0 0
0 0 50 ± 0 0 50 ± 0
0 0 0 0 450 ± 161
0 0 167 ± 157 0 0
Bacterial count in powder (CFU/g powder) IF 0 BC00 7637 ± 1250 BC63 0 BC63g 0 BC72 0
0 298 ± 0 0 0 0
0 0 298 ± 0 0 298 ± 0
0 0 0 0 2679 ± 958
0 0 994 ± 935 0 0
and 5% CO2. The final inoculation dose actually achieved in each experiment was determined by plating out and counting the starting inoculum on agar overnight. After incubation, the BC and IF cultures were diluted to 10−1–10−6 dilutions and 20 µL of each dilution was spotted onto one-sixth of a blood agar plate and incubated overnight at 37 °C and 5% CO2. The CFUs were counted the following day to calculate the bacterial growth after 4 h incubation. In several experiments, the kinetics of bacterial growth was evaluated following bacterial incubation with BC or IF for 1–24 h. In addition, mixtures of BC and IF at different ratios were also evaluated to mimic the clinical situation when IF is supplemented with BC. The bacterial inhibitory capacity of BC was calculated as the percentage of bacteria killed by BC relative to total bacteria detected in culture incubated with IF in the same experiment. This was equivalent to 100 × (1 − CBC/CIF), with CBC and CIF being bacterial concentration (CFU/mL) detected after incubation of bacteria with BC and IF, respectively.
institute, USA). The IgG values were compared using ANOVA and posthoc Tukey test. Differences in bacterial growth among BC and IF treatments across 1–24 h stimulations were analyzed by linear mixed models with time and treatment as fixed factors (including interaction) and experimental period as a random factor, followed by post-hoc Tukey tests for different treatments. Bacterial counts at a specific treatment period were compared using linear mixed models with treatment as a fixed factor and experimental period as a random factor, followed by post-hoc Tukey tests. For comparing cell proliferation, data were fitted into linear mixed models with BC treatment (compare BCs at specific concentrations) or concentration (compare concentrations within the same BCs) as a fixed factor and cell passage (experiment) as a random factor, followed by post-hoc Tukey tests. To test the effects of each BC on cytokine secretion, IL-8 data were fitted into a linear mixed model with BC and LPS treatments as fixed factors (including interaction), and cell passage as a random factor, followed by post-hoc test slices. For comparison of different BCs, data at each LPS treatment condition were fitted into a linear mixed model with BC treatment as a fixed factor and cell passage as a random factor followed by post-hoc Tukey tests.
2.4. In vitro intestinal epithelial cell proliferation and cytokine secretion The IEC cell line IPEC-J2 (ACC-701, DSMZ, Braunschweig, Germany), isolated from the jejunum of a newborn pig, was used to evaluate the effects of BC on IEC proliferation and cytokine secretion. The cells were cultured at cell passages 5–15 in Advanced Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F-12) with supplementation of 5% (v/v) heat inactivated fetal bovine serum, 1% (v/v) GlutaMAX and 0.2% (w/v) penicillin-streptomycin (all from Thermo Fisher Scientific, Hvidovre, Denmark) at 37 °C, 5% CO2. For the proliferation assay, the cells were seeded at 50,000 cells/mL in 96 well plates, cultured for 24 h and serum starved for another 24 h, prior to stimulation with different doses of BC (0.01–1 g/L) for 24 h at 37 °C, 5% CO2. Cell proliferation was determined by spectrophotometry at 490 nm following incubation of cultures with the CellTiter 96AQueous One Solution Reagent (Promega, Nacka, Sweden) for 4 h under the same conditions as above. Cell proliferation was quantified as the absorbance values relative to those without BC stimulation (only medium, assigned to 100%). For the cytokine secretion assay, cultured cells at 70–80% confluence were serum-starved for 24 h, followed by stimulation with BC at 0.05 g/L and/or 1 µg/mL lipopolysaccharide (LPS, from E. coli O127:B8, SigmaAldrich, Copenhagen, Denmark) for 24 h at 37 °C, 5% CO2. After stimulation, the supernatants were collected for analysis of IL-8 by ELISA (specific porcine ELISA duoSet kit, R&D Systems, Abingdon, UK). Each assay was performed in quadruplicates at four independent cell passages.
3. Results 3.1. Microbiological quality and IgG levels in bovine colostrum Among the five tested products, only IF and BC63g were sterile. BC00 was found to contain Enterococcus faecalis and Bacillus licheniformis after aerobic culture on blood agar plates and identification by MALDI-TOF (Table 1 and Fig. 2A and B). The two pasteurized products without gamma-irradiation (BC63 and BC72) were free of these two bacterial species but contained Bacillus cereus together with Acinetobacter radioresistens (BC72) or unidentified bacteria (BC63, Table 1 and Fig. 2C and D). The identified B. cereus strain was evaluated to be a potential spore-forming bacterium under Gram-staining conditions, as shown with endospore formation (empty holes, no crystal violet staining) within the bacterial cells (Fig. 2D, blue arrow). Bovine IgG levels were highest in BC00 (31.5 g/100 g protein) and lowest in BC63g and BC72 (21.0–22.4 g/100 g protein, P < 0.05), with intermediate values in BC63 (26.7 g/100 g protein, Fig. 3). 3.2. Bacterial growth-inhibitory effects of bovine colostrum S. epidermidis, E. coli and S. aureus are among the bacteria frequently detected from the blood culture of infants with neonatal sepsis (Trend et al., 2015) and these bacteria have been thought to be translocated from the gut or skin/environment (Duffy, 2000; Madan et al., 2012). We therefore evaluated the effects of BC on the growth and viability of these bacterial strains isolated from blood cultures of septic preterm infants. The incubation time of 4 h was estimated as the approximate
2.5. Statistics Statistical analyses were performed using JMP 13.0 software (SAS 184
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Fig. 2. Microbiological quality of four differentially heat-treated bovine colostrum products (BCs) and infant formula (IF). (A) Bacterial detection after plating in blood agar plates with dilution factors of 100–105 with only IF and BC63 being sterile. (B) Setup of plating experiment with triplicated plating for each dilution from 100 to 105 times. (C) Photomicrographs of bacterial detection in BC00 and BC72. Red arrows: E. faecalis, green arrows: B. licheniformis, blue arrows: B. cereus, aqua blue arrows: B. cereus with spore formation (empty, unstained oval inside the bacteria). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
incubation with BC00, BC63 or BC63g, relative to IF, P < 0.05, Fig. 4G–I). Similar to E. coli, the growth of S. aureus was not affected following incubation with BC72, relative to IF. Collectively, these data suggest that S. epidermidis and E. coli were the most sensitive bacteria to the anti-bacterial effects of BC, whereas S. aureus was more resistant. The inhibitory effects of BC63 and BC63g were greater than that of BC72 and similar to that of BC00 in many treatments, suggesting that higher temperature of pasteurization (BC72) decreases the anti-bacterial activity whereas gamma-irradiation did not affect the activity, relative to the corresponding product without irradiation (BC63 vs. BC63g). 3.3. Kinetics of bacterial growth and inhibitory effects of bovine colostrum Among the tested bacteria and doses, the growth of S. epidermidis at the inoculation of 105 CFU/mL and E. coli at 104 CFU/mL was the most sensitive to BC anti-bacterial effects. Therefore, we sought to further investigate the growth kinetics of these two bacteria isolates at two specific inoculations when incubated with IF or BC for 24 h to further elucidate if the antimicrobial effects of BC were transient or consistent over time. The 24 h growth curves of S. epidermidis and E. coli following incubation with one of four BCs were lower than that following incubation with IF (P < 0.05, Fig. 5). The bacterial growth-inhibitory effects were detected as early as 1–2 h after incubation (P < 0.05, 2- to 30-fold decreases in counts) in a similar manner for all the four BCs. The effects were consistent throughout the 24 h incubation (P < 0.05) for all BCs with E. coli treatment and for BC63g with S. epidermidis treatment. For S. epidermidis, BC00, BC63 and BC63g at 6 h and BC63g at 24 h decreased bacterial counts, relative to IF (25–40 times at 6 h and 103 times at 24 h, P < 0.05), while no effects were exerted by BC72 at 6–24 h (Fig. 5A). For E. coli, BC00 and BC63 were more potent than BC72 in decreasing bacterial count at 24 h of incubation (8–20 times relative to IF, P < 0.05, Fig. 5B). Collectively, the data suggest that all four types of BC exerted short-term (1–2 h) bacterial growth inhibition and that only BC72 lost its ability to inhibit bacterial growth when incubated for longer time (4–24 h).
Fig. 3. IgG levels in four differentially heat-treated bovine colostrum products (BCs). Values (mean ± SEM, n = 3) not sharing the same letters are significantly different.
time between meals in infants and selected based on previous findings evaluating the antimicrobial activity of mother’s milk (Trend et al., 2015). After 4 h of incubation with IF, the S. epidermidis and S. aureus concentrations increased approximately by 100-fold (for inoculation doses of 104-105 CFU/mL) to 500-fold (for inoculation dose of 106 CFU/ mL). E. coli grew much faster, approximately 1–5 × 104-fold increased bacterial counts, at all tested inoculations after 4 h incubation with IF. All four BCs exerted bacterial inhibitory effects against S. epidermidis, with bacterial counts after incubation decreasing 2–100 times relative to bacteria incubated with IF (Fig. 4A–C, P < 0.05). Among the four BCs, BC00 exerted the highest bacterial growth inhibition against S. epidermidis at the inoculations of 104-105 CFU/mL, greater than BC63g or BC72 (P < 0.05, Fig. 4A and B), while the inhibitory effects were similar among the four BCs at the inoculation dose of 106 CFU/mL (Fig. 4C). For E. coli, BC00, BC63 and BC63g had similar potency to inhibit bacterial growth with initial inoculations of 103–104 CFU/mL (13- to 100-fold decreases in bacterial counts after 4 h of incubation, relative to incubation with IF, P < 0.05). BC72 did not exert any inhibitory effects across the three inoculations (Fig. 4D–F). For S. aureus, the bacterial inhibitory effects of BC was weakest among the three tested bacteria (4 to 25-fold decrease in bacterial counts after 4 h of
3.4. Bacterial growth-inhibitory effects of bovine colostrum mixed with infant formula We next tested the effects of spiking BC into IF on the growth of S. epidermidis. We chose BC63g for this experiment because it was the only sterile product among the four tested BC products. S. epidermidis 185
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Fig. 4. Effects of bovine colostrum (BC) on bacterial growth at different initial inoculations. BC and infant formula (IF) were inoculated with S. epidermidis or S. aureus at concentrations of 104–106 CFU/mL, or E. coli at concentrations of 102–104 CFU/mL and incubated for 4 h at 37 °C and 5% CO2.Values (mean ± SE, n = 4) not sharing the same letters are significantly different (P < 0.05).
3.5. Intestinal epithelial cell proliferation and cytokine secretion
concentrations increased 150-fold following incubation with IF for 4 h (Fig. 6A). When S. epidermidis was incubated with a mixture of IF and BC63g at different ratios (3:1 and 1:1), the bacterial counts decreased 3.3-fold, relative to values after incubation with IF alone (P < 0.01, Fig. 6A). This represented 70% inhibitory capacity, reflecting that the BC content in the mixture killed 70% of the bacteria detected in the culture with IF incubation alone (Fig. 6B). There was no difference in the inhibitory effects between the two mixtures (3:1 and 1:1), suggesting potent bacterial inhibition with even small amounts of BC (25%) in the BC-IF mixture.
When stimulating porcine IPEC-J2 cells (isolated from a newborn pig) with four BCs at different concentrations, most of the treatments showed increases in cell proliferation, relative to the control cells without BC stimulation (P < 0.05, Fig. 7A). Among BC treatments, IEC proliferation stimulated by BC00 was greater than the remaining three BCs at concentration of 0.01 g/L, and greater than BC63g at concentration of 0.1 g/L (P < 0.05, Fig. 7A). At these two concentrations, BC63g effects on cell proliferation were the lowest. No differential Fig. 5. Kinetics of S. epidermidis (A) and E. coli (B) growth over time following incubation with infant formula (IF) or differentially heat-treated bovine colostrum products (BCs). Values (mean ± SEM, n = 3) at each incubation time not sharing the same letters are significantly different (P < 0.05). Overall difference in growth curves was depicted by letters next to the legends. Groups not sharing the same letters are significantly different (P < 0.05).
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Fig. 6. Effects of mixing low-heat treated and gamma-irradiated bovine colostrum (BC63g) into infant formula (IF) on the inhibition of S. epidermidis growth (A) and on S. epidermidis inhibitory capacity, relative to IF (0%, B). Values are mean ± SEM, n = 3. **P < 0.01.
concentrations of multiple bioactive proteins and peptides, including lactoferrin and beta defensin 1, 2 and 5, and this is associated with decreased capacity to inhibit growth of S. epidermidis, S. aureus and E. coli (Trend et al., 2015). Of note, only lactoferrin was inhibitory against these bacteria when spiked into IF (Trend et al., 2015), whereas other antimicrobial proteins in human milk such as defensin 1, 2 and 5 were not efficacious (Trend et al., 2015; Woodman et al., 2018). In the current study, we observed that the antimicrobial effects of BCs were more active against E. coli and S. epidermidis than S. aureus (up to 25–100 times decreased bacterial counts, relative to effects of IF control). These effects were very similar to that observed with human milk and lactoferrin-spiked formula (Trend et al., 2015), suggesting a role for lactoferrin in BC bioactivity. It is noteworthy that lactoferrin bioactivity is strongly dependent on its iron saturation content, with high iron levels in the products associated with less potent effects against bacterial growth (Trend et al., 2015; Woodman et al., 2018). Differences in iron content between breast milk and BC may explain the less potent bioactivity of BC against S. aureus. In a recent study characterizing processing effects on native whey proteins, we found higher levels of native lactoferrin, lactoperoxidase, bovine serum albumin, α-lactalbumin and β-lactoglobulin in BC00, than in BC63g and BC72, but not in BC63 (unpublished data). Pasteurization often causes changes in protein tertiary structure, denaturation and aggregation, potentially leading to loss of bioactivity (Nguyen et al., 2016). Lactoferrin is well known for its bacteriostatic effects via iron chelation and bactericidal activities (Ellison & Giehl, 1991), and the decreased bacterial inhibitory effects of BC72, relative to BC00 and BC63, may partly be explained by lower native IgG and lactoferrin levels. Interestingly, the combination of low-temperature pasteurization and gamma-irradiation (BC63g) decreased native levels of whey proteins, proliferative effects and cytokine secretion in IECs but
effects in cell proliferation among four BCs were detected at 1 g/L. For the cytokine secretion assay, BC-incubated cells did not show increased IL-8 secretion, relative to control cells without BC (Fig. 7B). However, when IECs were challenged with LPS for 24 h, secreted IL-8 levels increased 1.8-fold, and the IEC incubation with LPS plus BC00 or LPS plus BC63 led to even higher IL-8 levels than that secreted from IECs challenged with LPS alone (P < 0.05, Fig. 7B). This suggests that the bioactive components in BC00 and BC63 are able to modulate LPS-induced immune responses in IECs. Cell incubation with LPS plus BC63g or LPS plus BC72 did not cause any increased IL-8 secretion, relative to LPS incubation alone. 4. Discussion Breastfeeding is associated with lower incidence of neonatal sepsis, especially in vulnerable preterm infants (Patel et al., 2013). Breast milk may protect against sepsis partly because of its antimicrobial components, facilitating optimal gut microbiota composition and preventing pathogen translocation across the immature gut barrier into the blood stream (Trend et al., 2015). Similar to human milk, BC is rich in multiple bioactive proteins (Chatterton et al., 2013) that may be beneficial to infants with limited access to human milk. In the current in vitro study, we found that BC inhibited the growth of typical sepsis-related pathogens, relative to IF. Low-temperature pasteurization (63 °C, 30 min) was optimal for BC bioactivity and subsequent gamma-irradiation was required to maintain sterility of the final product. We also showed that combined low-temperature pasteurization with gammairradiation could be superior to conventional pasteurization (72 °C, 15 s) in maintaining both microbiological safety and BC product bioactivity. Relative to colostrum, mature human milk shows decreased
Fig. 7. Immune-modulatory effects of bovine colostrum (BC) on intestinal epithelial cell (IEC) line IPEC-J2. (A) BC-induced IEC proliferation; (B) BC and/or LPSinduced IL-8 secretion in IECs. Values are mean ± SEM. *P < 0.05, compared with the control (A) or compared with the corresponding colostrum treatment without LPS (B). Values in A within the same BC concentration not sharing the same letters are significantly different (P < 0.05). 187
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inhibitory activities of bacterial growth and gut immune-modulatory effects. Gamma-irradiation helped to maintain product sterility with minimal influence on IgG levels and bacterial inhibitory activities. However, more studies are required to investigate the minimal irradiation dose to secure sterility with minimal protein damage. Even when mixed into IF, bioactive BC still exerted strong inhibition of bacterial growth, suggesting potential beneficial effects of fortification of human milk and infant formula with BC for infants. Two ongoing clinical trials in preterm infants will help to reveal the clinical effects of BC63g on infant feeding intolerance, sepsis and gut inflammation. In future studies, it is important to assess the in vivo bioactivity of specific BC components in the immature gastrointestinal tract of infants or relevant model animals, including their effects on gut bacteria, bacterial translocation and mucosal immune development.
did not decrease its bacterial growth-inhibitory effects, compared with BC63. We postulate that low-temperature pasteurization at 63 °C for 30 min does not markedly change the tertiary structure of bioactive proteins (similar levels of native proteins in BC63 and BC00). Likewise, although the combination of this gentle pasteurization and gamma-irradiation may affect the structure of bioactive proteins, it was probably still less detrimental than the standard pasteurization (72 °C for 15 s) with clear reduction in the bacterial growth inhibition in BC72, relative to BC00, BC63 and BC63g. Further, despite possible irradiation-induced changes in the structure of a component like lactoferrin, this protein may still retain its ability to chelate iron and prevent bacterial nutrient consumption, leading to retained high bacterial growth-inhibitory effects. In this study, we used a relatively high irradiation dose of 14 kGy to secure sterility of the final product, and further investigations are required to elucidate the dose–response effects of irradiation on whey protein bioactivity. Interestingly, all four tested BCs exerted bacterial growth inhibition during the first 1–2 h after incubation, but only unpasteurized or gently pasteurized products (BC00, BC63 and BC63g) had long-lasting effects on bacterial inhibition (6–24 h). This implies that the lower levels of bioactive components in BC72 are consumed fully during the first 2 h of incubation whereas the remaining amounts of bioactive components in the gently treated products are still sufficient to exert bacterial inhibition for up to 24 h of incubation. This may have important implications for limiting infections in newborn infants frequently fed with gently processed BC to continuously prevent gut inflammation derived from bacterial overgrowth (Cole & Ziegler, 2007; Deitch, 1994). A sterile product with long-lasting antimicrobial activity like BC63g may also be important to decrease the risks of bacterial translocation across the immature gut barrier in newborn infants causing infection and sepsis (Duffy, 2000; MacFie et al., 1999). The ability of BC63g to induce gut maturation and decrease sepsis in preterm infants predominantly fed formula or human milk is currently being tested in two randomized clinical trials (NCT03085277, NCT03537365, clinicaltrials.gov). For many newborn infants, particularly preterm infants, nutrient fortification is critical to maintain adequate growth (Sun, Li, Nguyen, et al., 2018). However, fortification of mother’s own milk or donor human milk with infant formula products may also increase the incidence of feeding intolerance and gut inflammation (Sullivan et al., 2010). Using preterm pigs, we recently showed that fortification of donor human milk with BC63g was superior to fortification with a commercial infant formula in facilitating growth and gut maturation and preventing systemic infections (Sun et al., 2018; Sun et al., 2018). In the current study, we mimicked the in vivo conditions by fortifying IF with 25–50% (v/v) of BC63g and still observed potent bacterial growth inhibition when incubating them with S. epidermidis, relative to bacterial incubation with IF alone. The antimicrobial activity against gut pathogens may explain how BC63g fortification of donor human milk decreases systemic infections as observed in preterm pigs, where bacteria detected in infected organs are often found also in the gut (Sun, Li, Nguyen, et al., 2018; Sun, Li, Pan, et al., 2018). In a previous study comparing levels of bioactive proteins among various whey protein concentrates with different processing conditions, we found that gentler processing led to higher levels of IgG and lactoferrin in the final products, which were associated with better capacity to stimulate IEC proliferation and cytokine secretion (Nguyen et al., 2016). Similarly, the current study demonstrated that the effects of conventionally pasteurized product (BC72) on IEC proliferation and cytokine secretion were less potent than that of non-pasteurized and more gently pasteurized products (BC00 and BC63). We speculate that higher levels of bioactive components in BC00 and BC63 (e.g., IgG, lactoferrin) may better modulate immune responses of IECs, and thereby enhancing downstream signaling pathways associated with cell proliferation and cytokine secretion. We conclude that heat treatment during processing decreased the levels of bioactive proteins in the BC products as well as their in vitro
Ethics statements Our study did not include any human subjects and animal experiments. Acknowledgement The authors would like to thank Dr. Kirsty Townsend and Dr. Rebecca Abraham for their support in microbiological experiments. The study was funded by the Innovation Fund Denmark (NEOCOL, grant number 6150-00004B) and Biofiber Damino, Denmark. Biofiber Damino had no impact on the study design, experiments nor data analysis. University of Copenhagen holds a patent on the use of BC for pediatric patients. PTS is listed as a sole inventor but has declined any share of potential revenue arising from commercial exploitation of such a patent. The remaining authors declare no potential conflicts of interest. References Barin-Bastuji, B., Perrin, B., Thorel, M. F., & Martel, J. L. (1990). Evaluation of γ irradiation of cows’ colostrum for Brucella abortus, Escherichia coli K99, Salmonella dublin and Mycobacterium paratuberculosis decontamination. Letters in Applied Microbiology, 11, 163–166. https://doi.org/10.1111/j.1472-765X.1990.tb00150.x. Brunse, A., Worsøe, P., Pors, S. E., Skovgaard, K., & Sangild, P. T. (2018). Oral supplementation with bovine colostrum prevents septic shock and brain barrier disruption during bloodstream infection in preterm newborn pigs. Shock (Augusta, Ga.). https:// doi.org/10.1097/SHK.0000000000001131. Chatterton, D. E. W., Nguyen, D. N., Bering, S. B., & Sangild, P. T. (2013). Anti-inflammatory mechanisms of bioactive milk proteins in the intestine of newborns. The International Journal of Biochemistry & Cell Biology, 45(8), 1730–1747. https://doi. org/10.1016/j.biocel.2013.04.028. Cole, C. R., & Ziegler, T. R. (2007). Small bowel bacterial overgrowth: A negative factor in gut adaptation in pediatric SBS. Current Gastroenterology Reports, 9(6), 456–462. https://doi.org/10.1007/s11894-007-0059-3. Corpeleijn, W. E., de Waard, M., Christmann, V., van Goudoever, J. B., Jansen-van der Weide, M. C., Kooi, E. M. W., ... van Zoeren-Grobben, D. (2016). Effect of donor milk on severe infections and mortality in very low-birth-weight infants: The early nutrition study randomized clinical trial. JAMA Pediatrics, 170(7), 654–661. https://doi. org/10.1001/jamapediatrics.2016.0183. Deitch, E. A. (1994). Role of bacterial translocation in necrotizing enterocolitis. Acta Paediatrica, 83(s396), 33–36. https://doi.org/10.1111/j.1651-2227.1994.tb13239.x. Deitch, E. A. (2012). Gut-origin sepsis: Evolution of a concept. The Surgeon, 10(6), 350–356. https://doi.org/10.1016/j.surge.2012.03.003. Duffy, L. C. (2000). Interactions mediating bacterial translocation in the immature intestine. The Journal of Nutrition, 130(2S Suppl), 432S–436S. https://doi.org/10.1093/ jn/130.2.432S. Ellison, R. T., & Giehl, T. J. (1991). Killing of gram-negative bacteria by lactoferrin and lysozyme. The Journal of Clinical Investigation, 88(4), 1080–1091. https://doi.org/10. 1172/JCI115407. Ewaschuk, J. B., Unger, S., Harvey, S., O’Connor, D. L., & Field, C. J. (2011). Effect of pasteurization on immune components of milk: Implications for feeding preterm infants. Applied Physiology, Nutrition, and Metabolism, 36(2), 175–182. https://doi. org/10.1139/h11-008. Jensen, M. L., Sangild, P. T., Lykke, M., Schmidt, M., Boye, M., Jensen, B. B., & Thymann, T. (2013). Similar efficacy of human banked milk and bovine colostrum to decrease incidence of necrotizing enterocolitis in preterm piglets. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 305(1), R4–R12. https://doi.org/10.1152/ajpregu.00094.2013. Juhl, S. M., Ye, X., Zhou, P., Li, Y., Iyore, E. O., Zhang, L., ... Sangild, P. T. (2018). Bovine
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birth weight infants. Journal of Perinatology: Official Journal of the California Perinatal Association, 33(7), 514–519. https://doi.org/10.1038/jp.2013.2. Reznikov, E. A., Comstock, S. S., Hoeflinger, J. L., Wang, M., Miller, M. J., & Donovan, S. M. (2018). Dietary bovine lactoferrin reduces Staphylococcus aureus in the tissues and modulates the immune response in piglets Systemically Infected with S. aureus. Current Developments Nutrition, 2(4), nzy001. https://doi.org/10.1093/cdn/nzy001. Saldana, D. J., Gelsinger, S. L., Jones, C. M., & Heinrichs, A. J. (2019). Effect of different heating times of high-, medium-, and low-quality colostrum on immunoglobulin G absorption in dairy calves. Journal of Dairy Science, 102(3), 2068–2074. https://doi. org/10.3168/jds.2018-15542. Shen, R. L., Thymann, T., Østergaard, M. V., Støy, A. C. F., Krych, Ł., Nielsen, D. S., ... Sangild, P. T. (2015). Early gradual feeding with bovine colostrum improves gut function and NEC resistance relative to infant formula in preterm pigs. American Journal of Physiology. Gastrointestinal and Liver Physiology, 309(5), G310–323. https:// doi.org/10.1152/ajpgi.00163.2015. Strunk, T., Inder, T., Wang, X., Burgner, D., Mallard, C., & Levy, O. (2014). Infectioninduced inflammation and cerebral injury in preterm infants. The Lancet. Infectious Diseases, 14(8), 751–762. https://doi.org/10.1016/S1473-3099(14)70710-8. Sullivan, S., Schanler, R. J., Kim, J. H., Patel, A. L., Trawöger, R., Kiechl-Kohlendorfer, U., ... Lucas, A. (2010). An exclusively human milk-based diet is associated with a lower rate of necrotizing enterocolitis than a diet of human milk and bovine milk-based products. The Journal of Pediatrics, 156(4), 562–567.e1. https://doi.org/10.1016/j. jpeds.2009.10.040. Sun, J., Li, Y., Nguyen, D. N., Mortensen, M. S., van den Akker, C. H. P., Skeath, T., ... Sangild, P. T. (2018). Nutrient fortification of human donor milk affects intestinal function and protein metabolism in preterm pigs. The Journal of Nutrition, 148(3), 336–347. https://doi.org/10.1093/jn/nxx033. Sun, J., Li, Y., Pan, X., Nguyen, D. N., Brunse, A., Bojesen, A. M., ... Sangild, P. T. (2018). Human milk fortification with bovine colostrum is superior to formula-based fortifiers to prevent gut dysfunction, necrotizing enterocolitis, and systemic infection in preterm pigs. JPEN Journal of Parenteral and Enteral Nutrition. https://doi.org/10. 1002/jpen.1422. Tacoma, R., Gelsinger, S. L., Lam, Y. W., Scuderi, R. A., Ebenstein, D. B., Heinrichs, A. J., & Greenwood, S. L. (2017). Exploration of the bovine colostrum proteome and effects of heat treatment time on colostrum protein profile. Journal of Dairy Science, 100(11), 9392–9401. https://doi.org/10.3168/jds.2017-13211. Trend, S., Strunk, T., Hibbert, J., Kok, C. H., Zhang, G., Doherty, D. A., ... Currie, A. J. (2015). Antimicrobial protein and Peptide concentrations and activity in human breast milk consumed by preterm infants at risk of late-onset neonatal sepsis. PloS One, 10(2), e0117038. https://doi.org/10.1371/journal.pone.0117038. Vellenga, L., Wensing, T., Breukink, H. J., & Hagens, F. H. (1986). Effects of irradiated sow colostrum on some biochemical and haematological measurements in newborn piglets. Research in Veterinary Science, 41(3), 316–318. https://doi.org/10.1016/ S0034-5288(18)30622-2. West, C. E., Kvistgaard, A. S., Peerson, J. M., Donovan, S. M., Peng, Y.-M., & Lönnerdal, B. (2017). Effects of osteopontin-enriched formula on lymphocyte subsets in the first 6 months of life: A randomized controlled trial. Pediatric Research, 82(1), 63–71. https://doi.org/10.1038/pr.2017.77. Willems, R., Krych, L., Rybicki, V., Jiang, P., Sangild, P. T., Shen, R. L., ... Jenke, A. C. (2015). Introducing enteral feeding induces intestinal subclinical inflammation and respective chromatin changes in preterm pigs. Epigenomics, 7(4), 553–565. https:// doi.org/10.2217/epi.15.13. Woodman, T., Strunk, T., Patole, S., Hartmann, B., Simmer, K., & Currie, A. (2018). Effects of lactoferrin on neonatal pathogens and Bifidobacterium breve in human breast milk. PLOS ONE, 13(8), e0201819. https://doi.org/10.1371/journal.pone. 0201819.
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Heat treatment and irradiation reduce anti-bacterial and immunemodulatory properties of bovine colostrum Duc Ninh Nguyena, Andrew J. Currieb, Shuqiang Rena, Stine B. Beringa, Per T. Sangilda,c,
T ⁎
a
Section for Comparative Pediatrics and Nutrition, Department of Veterinary and Animal Sciences, University of Copenhagen, Denmark Medical, Molecular & Forensic Sciences, Murdoch University, Perth, Australia c Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen, Denmark b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobial activity Bacterial growth inhibition Bovine colostrum Preterm infants Pasteurization
Colostrum contains bioactive components protecting the newborn intestine against bacteria. It is unclear how to optimize processing conditions with highest product bioactivity. Non-pasteurized (BC00), standard-pasteurized (72 °C-15 s, BC72), gently-pasteurized bovine colostrums without (63 °C-30 min, BC63) and with gamma-irradiation (BC63g) were tested for effects on bacterial growth inhibition (Escherichia coli, Staphylococcus aureus and S. epidermidis), intestinal epithelial cell (IEC) proliferation and cytokine secretion in vitro. Thermal processing decreased endogenous bacteria and IgG levels. All BCs inhibited bacterial growth 1–2 h after inoculation, but only BC00, BC63 and BC63g retained activity after 4–24 h. After 4 h, the activity against S. epidermidis of BC63g was lower than BC00 but still potent when mixed with formula. All BCs stimulated IEC proliferation, with the most pronounced responses for BC00. Only BC00 and BC63 increased IL-8 secretion in lipopolysaccharide-stimulated IECs. Thermal processing reduced bioactivity and combined gentle pasteurization and gamma-irradiation improved BC sterility and bioactivity, relative to standard pasteurization.
1. Introduction It is well accepted that mother’s own milk is the best choice for newborn infants, in part due to its protective effects against infection and gut inflammation (Chatterton, Nguyen, Bering, & Sangild, 2013; Kelley, 2012). However, many infants, including vulnerable preterm infants (born < 37 weeks of gestation, 15 million every year, 11% of all births), have limited access to mother’s milk, especially during the first weeks of life, and they are often fed or supplemented with infant formula (Lucas & Cole, 1990; Merewood, Brooks, Bauchner, MacAuley, & Mehta, 2006). Feeding infant formula has been documented to be detrimental to infant health with increased prevalence of infection, gut inflammation and neurodevelopmental delays (Strunk et al., 2014). Donor human milk has been considered as an alternative to mother’s own milk for preterm infants, but it is still unclear to which extent it matches mother’s milk and if it is superior to infant formula in protecting against infectious diseases (Corpeleijn et al., 2016). One consideration is the pasteurization process applied to donor milk, which may decrease the levels of multiple bioactive proteins including immunoglobulins (Ig) and lactoferrin (Ewaschuk, Unger, Harvey, O’Connor, & Field, 2011).
Bovine colostrum (BC) may be a better option than infant formula to provide protection against infection and inflammation for newborn infants as it contains a wide spectrum of bioactive proteins and peptides at higher concentrations than in mature milk, e.g. lactoferrin, lactoperoxidase, osteopontin and transforming growth factor β (Chatterton et al., 2013). These components possess antimicrobial and anti-inflammatory activities (Chatterton et al., 2013). For instance, dietary lactoferrin exerts strong in vitro antimicrobial activity against bacteria causing neonatal sepsis and enhances systemic immune responses against neonatal infection in both newborn pigs and infants (Manzoni et al., 2009; Manzoni, Mostert, & Stronati, 2011; Reznikov et al., 2018; Woodman et al., 2018). Supplementation of osteopontin into infant formula has shown to decrease gut inflammation in newborn pigs (Møller et al., 2011) and modulate systemic immune development in newborn infants (West et al., 2017). Using preterm pigs as a model for vulnerable newborn infants, we have documented that feeding gentlypasteurized BC (63 °C for 30 min), promotes gut maturation and protects against gut inflammation and infection (Brunse, Worsøe, Pors, Skovgaard, & Sangild, 2018; Jensen et al., 2013; Shen et al., 2015; Sun, Li, Nguyen, et al., 2018; Sun, Li, Pan, et al., 2018). The BC feeding also modulates expression of innate immune genes associated with anti-
⁎ Corresponding author at: Section for Comparative Pediatrics and Nutrition, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Dyrlægevej 68, DK-1870 Frederiksberg C, Denmark. E-mail address: [email protected] (P.T. Sangild).
https://doi.org/10.1016/j.jff.2019.04.012 Received 7 January 2019; Received in revised form 19 March 2019; Accepted 6 April 2019 1756-4646/ © 2019 Published by Elsevier Ltd.
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inflammatory effects against gut inflammation via epigenetic mechanisms (Pan, Gong, Gao, & Sangild, 2018; Willems et al., 2015). Numerous preclinical studies in preterm pigs with beneficial effects of BC feeding have led to a pilot clinical trial testing BC as a supplement to mother’s own milk during the first two weeks of life in preterm infants with indications of better tolerance to BC than IF (Juhl et al., 2018; Li, Juhl, et al., 2017). Two larger randomized clinical trials in preterm infants with BC as a supplement to mother’s own milk and as a fortifier to donor human milk are ongoing (NCT03085277 and NCT03537365, respectively, clinicaltrials.gov). Although the protective effects of BC against gut inflammation and infection are well documented, the underlying mechanisms remain elusive. Neonatal sepsis may in part result from bacterial translocation across the immature gut barrier into the systemic circulation (Deitch, 2012; MacFie et al., 1999). The high levels of antimicrobial and immune-modulatory proteins in BC may contribute to eliminating pathogenic bacteria in the gut, leading to decreased gut inflammation and gut-derived sepsis. Similar mechanisms may explain the lower incidence of gut inflammation and sepsis in breast-fed, relative to formula-fed newborn infants (Lucas & Cole, 1990; Trend et al., 2015). Further, processing technology (e.g. pasteurization) strongly affects the levels of heat-labile bioactive proteins, e.g. lactoferrin and IgG (Li, Nguyen, et al., 2017; Li et al., 2018, 2013; Nguyen, Sangild, Li, Bering, & Chatterton, 2016; Saldana, Gelsinger, Jones, & Heinrichs, 2019; Tacoma et al., 2017). High-temperature short-time (72 °C, 15 s) pasteurization is a standard procedure for bovine milk whereas donor human milk is often pasteurized at a low-temperature long-time condition (63 °C, 30 min). The later gentler treatment may help to retain higher levels of bioactive components and thereby greater bioactivity for BC. Gamma-irradiation is also a common sterilization method for foodstuffs with reported efficacy to eliminate pathogens in colostrum without affecting Ig activity (Barin-Bastuji, Perrin, Thorel, & Martel, 1990; Vellenga, Wensing, Breukink, & Hagens, 1986), suggesting another potentially gentle treatment to retain higher levels of bioactive components. We hypothesized that the type of heat treatment during BC processing affects the product antimicrobial and immune-modulatory properties in the newborn intestine, with low-temperature, long-time pasteurization and irradiation retaining greater bioactivity than the standard high-temperature short-time process. We generated four BC products from the same raw milk, but with different processing conditions, to test their antimicrobial activity against bacteria often associated with neonatal sepsis as well as their immune-modulatory activities in intestinal epithelial cells (IECs).
Fig. 1. Schematic diagram demonstrating the production flow of four types of colostrum from the same source of raw colostrum to the final powdered products. BC, bovine colostrum; BC00, raw BC without pasteurization; BC63, BC with gentle pasteurization (63 °C, 30 min) and gentle spray drying; BC63g, BC63 with gamma-irradiation; BC72, BC with conventional pasteurization (72 °C, 15 s) and conventionally harsh spray drying.
15 min to remove the fat content. The skimmed fraction was sterilefiltered at 0.22 µm and stored in aliquots at −80 °C until analysis. The concentration of bovine IgG was used as an indicator of bioactive proteins and analyzed after reconstitution of BC samples at 10 g/L in water using ELISA with sheep anti-bovine IgG antibody (A10-118A, Nordic Biosite, Copenhagen, Denmark). 2.2. Microbiological analysis of bovine colostrum The four BCs and the IF were diluted with sterile saline (dilution factors of 100 to 10−5) and spotted in triplicates onto non-selective sheep blood agar plates (PathWest Laboratory Medicine WA Media, Claremont, Australia) and incubated overnight at 37 °C and 5% CO2. Colony forming units (CFU) were counted after overnight incubation to calculate the bacterial contents in the BCs and were used to determine the bacterial identity by MALDI-TOF (Microflex, Bruker). Representative CFUs were also used for Gram-staining and visualized with a light microscope under oil immersion. 2.3. Frozen bacterial stock and antimicrobial activity of bovine colostrum Three bacterial strains isolated from septic preterm infants (kindly provided by King Edward Memorial Hospital, Western Australia, Australia) were used for the assay, including Escherichia coli, Staphylococcus aureus and S. epidermidis. These strains were stored at −80 °C in Protect Beads (Microorganism preservation system, Technical service consultant, UK) and used to prepare mid-log frozen stocks as previously described (Trend et al., 2015; Woodman et al., 2018). Bacteria in beads were grown overnight at 37 °C, 5% CO2 in heart infusion broth (Oxoid, Hampshire, England) and streaked out onto blood agar. Thereafter single isolates were inoculated in heart infusion broth and incubated overnight at the same condition as above. The overnight cultures were adjusted in fresh broth to OD 0.05 at 600 nm (Biophotometer D30, Eppendorf, Hamburg, Germany) and incubated again until mid-log phase (OD600nm ∼ 0.6) and aliquoted for storage at −80 °C (in 10% glycerol for S. aureus and S. epidermidis, and in 10% fetal bovine serum for E. coli) prior to experiments. The viability of mid-log stocks was evaluated by plating out diluted stocks (dilution factors of 10−1–10−6) and counting as described above and the bacterial concentration was used to calculate starting inoculum doses for bacterial challenge in the antimicrobial assays. For the antimicrobial assay, bacterial mid-log culture stocks were thawed, centrifuged at 3200g and resuspended in sterile saline prior to incubation with BC or IF at a calculated dose of 104–106 cells/mL for S. epidermidis and S. aureus or 102–104 cells/mL for E. coli for 4 h at 37 °C
2. Materials and methods 2.1. Processing of bovine colostrum and preparation for in vitro study The same batch of raw BC collected from Danish Holstein cows during the first 3 days after parturition was used to produce four types of BC powder, as depicted schematically in Fig. 1. Raw BC was homogenized and spray-dried without pasteurization (BC00), or pasteurized at 63 °C for 30 min (BC63) or 72 °C for 15 s (BC72) prior to spraydrying. Powdered samples were then packaged under sterile conditions. The packaged BC63 product was also gamma-irradiated (14 kGy dose) to secure a bacteria-free product (BC63g) as used in clinical trials (Juhl et al., 2018; Li, Juhl, et al., 2017). The powdered products contained 52.0% protein, 17.4% carbohydrate and 22.8% fat. For some experiments, PreNAN GOLD ready-to-feed preterm infant formula (IF, Nestle, Vevey, Switzerland) was used as a negative control. To test bacterial growth inhibition in vitro, BC powders were dissolved in sterile water at a concentration of 16.8 g or 81 kcal per 100 mL, similar to preparations of BC in the clinical trials (Juhl et al., 2018; Li, Juhl, et al., 2017), and stored in aliquots of 1 mL at −80 °C. For in vitro IEC stimulation, the BC powder was reconstituted in sterile water at a concentration of 10 g/L, then centrifuged at 10,000g, 4 °C for 183
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Table 1 Levels of bacteria in infant formula (IF) and four differentially heat-treated bovine colostrum products (BC) detected by counting after plating on blood agar plates and identified by MALDI-TOF (n = 3, values are mean ± SD). E. faecalis
B. licheniformis
B.cereus
A. radioresistens
Unidentified
Bacterial count in liquid samples (CFU/mL) IF 0 BC00 1283 ± 210 BC63 0 BC63g 0 BC72 0
0 50 ± 0 0 0 0
0 0 50 ± 0 0 50 ± 0
0 0 0 0 450 ± 161
0 0 167 ± 157 0 0
Bacterial count in powder (CFU/g powder) IF 0 BC00 7637 ± 1250 BC63 0 BC63g 0 BC72 0
0 298 ± 0 0 0 0
0 0 298 ± 0 0 298 ± 0
0 0 0 0 2679 ± 958
0 0 994 ± 935 0 0
and 5% CO2. The final inoculation dose actually achieved in each experiment was determined by plating out and counting the starting inoculum on agar overnight. After incubation, the BC and IF cultures were diluted to 10−1–10−6 dilutions and 20 µL of each dilution was spotted onto one-sixth of a blood agar plate and incubated overnight at 37 °C and 5% CO2. The CFUs were counted the following day to calculate the bacterial growth after 4 h incubation. In several experiments, the kinetics of bacterial growth was evaluated following bacterial incubation with BC or IF for 1–24 h. In addition, mixtures of BC and IF at different ratios were also evaluated to mimic the clinical situation when IF is supplemented with BC. The bacterial inhibitory capacity of BC was calculated as the percentage of bacteria killed by BC relative to total bacteria detected in culture incubated with IF in the same experiment. This was equivalent to 100 × (1 − CBC/CIF), with CBC and CIF being bacterial concentration (CFU/mL) detected after incubation of bacteria with BC and IF, respectively.
institute, USA). The IgG values were compared using ANOVA and posthoc Tukey test. Differences in bacterial growth among BC and IF treatments across 1–24 h stimulations were analyzed by linear mixed models with time and treatment as fixed factors (including interaction) and experimental period as a random factor, followed by post-hoc Tukey tests for different treatments. Bacterial counts at a specific treatment period were compared using linear mixed models with treatment as a fixed factor and experimental period as a random factor, followed by post-hoc Tukey tests. For comparing cell proliferation, data were fitted into linear mixed models with BC treatment (compare BCs at specific concentrations) or concentration (compare concentrations within the same BCs) as a fixed factor and cell passage (experiment) as a random factor, followed by post-hoc Tukey tests. To test the effects of each BC on cytokine secretion, IL-8 data were fitted into a linear mixed model with BC and LPS treatments as fixed factors (including interaction), and cell passage as a random factor, followed by post-hoc test slices. For comparison of different BCs, data at each LPS treatment condition were fitted into a linear mixed model with BC treatment as a fixed factor and cell passage as a random factor followed by post-hoc Tukey tests.
2.4. In vitro intestinal epithelial cell proliferation and cytokine secretion The IEC cell line IPEC-J2 (ACC-701, DSMZ, Braunschweig, Germany), isolated from the jejunum of a newborn pig, was used to evaluate the effects of BC on IEC proliferation and cytokine secretion. The cells were cultured at cell passages 5–15 in Advanced Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F-12) with supplementation of 5% (v/v) heat inactivated fetal bovine serum, 1% (v/v) GlutaMAX and 0.2% (w/v) penicillin-streptomycin (all from Thermo Fisher Scientific, Hvidovre, Denmark) at 37 °C, 5% CO2. For the proliferation assay, the cells were seeded at 50,000 cells/mL in 96 well plates, cultured for 24 h and serum starved for another 24 h, prior to stimulation with different doses of BC (0.01–1 g/L) for 24 h at 37 °C, 5% CO2. Cell proliferation was determined by spectrophotometry at 490 nm following incubation of cultures with the CellTiter 96AQueous One Solution Reagent (Promega, Nacka, Sweden) for 4 h under the same conditions as above. Cell proliferation was quantified as the absorbance values relative to those without BC stimulation (only medium, assigned to 100%). For the cytokine secretion assay, cultured cells at 70–80% confluence were serum-starved for 24 h, followed by stimulation with BC at 0.05 g/L and/or 1 µg/mL lipopolysaccharide (LPS, from E. coli O127:B8, SigmaAldrich, Copenhagen, Denmark) for 24 h at 37 °C, 5% CO2. After stimulation, the supernatants were collected for analysis of IL-8 by ELISA (specific porcine ELISA duoSet kit, R&D Systems, Abingdon, UK). Each assay was performed in quadruplicates at four independent cell passages.
3. Results 3.1. Microbiological quality and IgG levels in bovine colostrum Among the five tested products, only IF and BC63g were sterile. BC00 was found to contain Enterococcus faecalis and Bacillus licheniformis after aerobic culture on blood agar plates and identification by MALDI-TOF (Table 1 and Fig. 2A and B). The two pasteurized products without gamma-irradiation (BC63 and BC72) were free of these two bacterial species but contained Bacillus cereus together with Acinetobacter radioresistens (BC72) or unidentified bacteria (BC63, Table 1 and Fig. 2C and D). The identified B. cereus strain was evaluated to be a potential spore-forming bacterium under Gram-staining conditions, as shown with endospore formation (empty holes, no crystal violet staining) within the bacterial cells (Fig. 2D, blue arrow). Bovine IgG levels were highest in BC00 (31.5 g/100 g protein) and lowest in BC63g and BC72 (21.0–22.4 g/100 g protein, P < 0.05), with intermediate values in BC63 (26.7 g/100 g protein, Fig. 3). 3.2. Bacterial growth-inhibitory effects of bovine colostrum S. epidermidis, E. coli and S. aureus are among the bacteria frequently detected from the blood culture of infants with neonatal sepsis (Trend et al., 2015) and these bacteria have been thought to be translocated from the gut or skin/environment (Duffy, 2000; Madan et al., 2012). We therefore evaluated the effects of BC on the growth and viability of these bacterial strains isolated from blood cultures of septic preterm infants. The incubation time of 4 h was estimated as the approximate
2.5. Statistics Statistical analyses were performed using JMP 13.0 software (SAS 184
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Fig. 2. Microbiological quality of four differentially heat-treated bovine colostrum products (BCs) and infant formula (IF). (A) Bacterial detection after plating in blood agar plates with dilution factors of 100–105 with only IF and BC63 being sterile. (B) Setup of plating experiment with triplicated plating for each dilution from 100 to 105 times. (C) Photomicrographs of bacterial detection in BC00 and BC72. Red arrows: E. faecalis, green arrows: B. licheniformis, blue arrows: B. cereus, aqua blue arrows: B. cereus with spore formation (empty, unstained oval inside the bacteria). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
incubation with BC00, BC63 or BC63g, relative to IF, P < 0.05, Fig. 4G–I). Similar to E. coli, the growth of S. aureus was not affected following incubation with BC72, relative to IF. Collectively, these data suggest that S. epidermidis and E. coli were the most sensitive bacteria to the anti-bacterial effects of BC, whereas S. aureus was more resistant. The inhibitory effects of BC63 and BC63g were greater than that of BC72 and similar to that of BC00 in many treatments, suggesting that higher temperature of pasteurization (BC72) decreases the anti-bacterial activity whereas gamma-irradiation did not affect the activity, relative to the corresponding product without irradiation (BC63 vs. BC63g). 3.3. Kinetics of bacterial growth and inhibitory effects of bovine colostrum Among the tested bacteria and doses, the growth of S. epidermidis at the inoculation of 105 CFU/mL and E. coli at 104 CFU/mL was the most sensitive to BC anti-bacterial effects. Therefore, we sought to further investigate the growth kinetics of these two bacteria isolates at two specific inoculations when incubated with IF or BC for 24 h to further elucidate if the antimicrobial effects of BC were transient or consistent over time. The 24 h growth curves of S. epidermidis and E. coli following incubation with one of four BCs were lower than that following incubation with IF (P < 0.05, Fig. 5). The bacterial growth-inhibitory effects were detected as early as 1–2 h after incubation (P < 0.05, 2- to 30-fold decreases in counts) in a similar manner for all the four BCs. The effects were consistent throughout the 24 h incubation (P < 0.05) for all BCs with E. coli treatment and for BC63g with S. epidermidis treatment. For S. epidermidis, BC00, BC63 and BC63g at 6 h and BC63g at 24 h decreased bacterial counts, relative to IF (25–40 times at 6 h and 103 times at 24 h, P < 0.05), while no effects were exerted by BC72 at 6–24 h (Fig. 5A). For E. coli, BC00 and BC63 were more potent than BC72 in decreasing bacterial count at 24 h of incubation (8–20 times relative to IF, P < 0.05, Fig. 5B). Collectively, the data suggest that all four types of BC exerted short-term (1–2 h) bacterial growth inhibition and that only BC72 lost its ability to inhibit bacterial growth when incubated for longer time (4–24 h).
Fig. 3. IgG levels in four differentially heat-treated bovine colostrum products (BCs). Values (mean ± SEM, n = 3) not sharing the same letters are significantly different.
time between meals in infants and selected based on previous findings evaluating the antimicrobial activity of mother’s milk (Trend et al., 2015). After 4 h of incubation with IF, the S. epidermidis and S. aureus concentrations increased approximately by 100-fold (for inoculation doses of 104-105 CFU/mL) to 500-fold (for inoculation dose of 106 CFU/ mL). E. coli grew much faster, approximately 1–5 × 104-fold increased bacterial counts, at all tested inoculations after 4 h incubation with IF. All four BCs exerted bacterial inhibitory effects against S. epidermidis, with bacterial counts after incubation decreasing 2–100 times relative to bacteria incubated with IF (Fig. 4A–C, P < 0.05). Among the four BCs, BC00 exerted the highest bacterial growth inhibition against S. epidermidis at the inoculations of 104-105 CFU/mL, greater than BC63g or BC72 (P < 0.05, Fig. 4A and B), while the inhibitory effects were similar among the four BCs at the inoculation dose of 106 CFU/mL (Fig. 4C). For E. coli, BC00, BC63 and BC63g had similar potency to inhibit bacterial growth with initial inoculations of 103–104 CFU/mL (13- to 100-fold decreases in bacterial counts after 4 h of incubation, relative to incubation with IF, P < 0.05). BC72 did not exert any inhibitory effects across the three inoculations (Fig. 4D–F). For S. aureus, the bacterial inhibitory effects of BC was weakest among the three tested bacteria (4 to 25-fold decrease in bacterial counts after 4 h of
3.4. Bacterial growth-inhibitory effects of bovine colostrum mixed with infant formula We next tested the effects of spiking BC into IF on the growth of S. epidermidis. We chose BC63g for this experiment because it was the only sterile product among the four tested BC products. S. epidermidis 185
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Fig. 4. Effects of bovine colostrum (BC) on bacterial growth at different initial inoculations. BC and infant formula (IF) were inoculated with S. epidermidis or S. aureus at concentrations of 104–106 CFU/mL, or E. coli at concentrations of 102–104 CFU/mL and incubated for 4 h at 37 °C and 5% CO2.Values (mean ± SE, n = 4) not sharing the same letters are significantly different (P < 0.05).
3.5. Intestinal epithelial cell proliferation and cytokine secretion
concentrations increased 150-fold following incubation with IF for 4 h (Fig. 6A). When S. epidermidis was incubated with a mixture of IF and BC63g at different ratios (3:1 and 1:1), the bacterial counts decreased 3.3-fold, relative to values after incubation with IF alone (P < 0.01, Fig. 6A). This represented 70% inhibitory capacity, reflecting that the BC content in the mixture killed 70% of the bacteria detected in the culture with IF incubation alone (Fig. 6B). There was no difference in the inhibitory effects between the two mixtures (3:1 and 1:1), suggesting potent bacterial inhibition with even small amounts of BC (25%) in the BC-IF mixture.
When stimulating porcine IPEC-J2 cells (isolated from a newborn pig) with four BCs at different concentrations, most of the treatments showed increases in cell proliferation, relative to the control cells without BC stimulation (P < 0.05, Fig. 7A). Among BC treatments, IEC proliferation stimulated by BC00 was greater than the remaining three BCs at concentration of 0.01 g/L, and greater than BC63g at concentration of 0.1 g/L (P < 0.05, Fig. 7A). At these two concentrations, BC63g effects on cell proliferation were the lowest. No differential Fig. 5. Kinetics of S. epidermidis (A) and E. coli (B) growth over time following incubation with infant formula (IF) or differentially heat-treated bovine colostrum products (BCs). Values (mean ± SEM, n = 3) at each incubation time not sharing the same letters are significantly different (P < 0.05). Overall difference in growth curves was depicted by letters next to the legends. Groups not sharing the same letters are significantly different (P < 0.05).
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Fig. 6. Effects of mixing low-heat treated and gamma-irradiated bovine colostrum (BC63g) into infant formula (IF) on the inhibition of S. epidermidis growth (A) and on S. epidermidis inhibitory capacity, relative to IF (0%, B). Values are mean ± SEM, n = 3. **P < 0.01.
concentrations of multiple bioactive proteins and peptides, including lactoferrin and beta defensin 1, 2 and 5, and this is associated with decreased capacity to inhibit growth of S. epidermidis, S. aureus and E. coli (Trend et al., 2015). Of note, only lactoferrin was inhibitory against these bacteria when spiked into IF (Trend et al., 2015), whereas other antimicrobial proteins in human milk such as defensin 1, 2 and 5 were not efficacious (Trend et al., 2015; Woodman et al., 2018). In the current study, we observed that the antimicrobial effects of BCs were more active against E. coli and S. epidermidis than S. aureus (up to 25–100 times decreased bacterial counts, relative to effects of IF control). These effects were very similar to that observed with human milk and lactoferrin-spiked formula (Trend et al., 2015), suggesting a role for lactoferrin in BC bioactivity. It is noteworthy that lactoferrin bioactivity is strongly dependent on its iron saturation content, with high iron levels in the products associated with less potent effects against bacterial growth (Trend et al., 2015; Woodman et al., 2018). Differences in iron content between breast milk and BC may explain the less potent bioactivity of BC against S. aureus. In a recent study characterizing processing effects on native whey proteins, we found higher levels of native lactoferrin, lactoperoxidase, bovine serum albumin, α-lactalbumin and β-lactoglobulin in BC00, than in BC63g and BC72, but not in BC63 (unpublished data). Pasteurization often causes changes in protein tertiary structure, denaturation and aggregation, potentially leading to loss of bioactivity (Nguyen et al., 2016). Lactoferrin is well known for its bacteriostatic effects via iron chelation and bactericidal activities (Ellison & Giehl, 1991), and the decreased bacterial inhibitory effects of BC72, relative to BC00 and BC63, may partly be explained by lower native IgG and lactoferrin levels. Interestingly, the combination of low-temperature pasteurization and gamma-irradiation (BC63g) decreased native levels of whey proteins, proliferative effects and cytokine secretion in IECs but
effects in cell proliferation among four BCs were detected at 1 g/L. For the cytokine secretion assay, BC-incubated cells did not show increased IL-8 secretion, relative to control cells without BC (Fig. 7B). However, when IECs were challenged with LPS for 24 h, secreted IL-8 levels increased 1.8-fold, and the IEC incubation with LPS plus BC00 or LPS plus BC63 led to even higher IL-8 levels than that secreted from IECs challenged with LPS alone (P < 0.05, Fig. 7B). This suggests that the bioactive components in BC00 and BC63 are able to modulate LPS-induced immune responses in IECs. Cell incubation with LPS plus BC63g or LPS plus BC72 did not cause any increased IL-8 secretion, relative to LPS incubation alone. 4. Discussion Breastfeeding is associated with lower incidence of neonatal sepsis, especially in vulnerable preterm infants (Patel et al., 2013). Breast milk may protect against sepsis partly because of its antimicrobial components, facilitating optimal gut microbiota composition and preventing pathogen translocation across the immature gut barrier into the blood stream (Trend et al., 2015). Similar to human milk, BC is rich in multiple bioactive proteins (Chatterton et al., 2013) that may be beneficial to infants with limited access to human milk. In the current in vitro study, we found that BC inhibited the growth of typical sepsis-related pathogens, relative to IF. Low-temperature pasteurization (63 °C, 30 min) was optimal for BC bioactivity and subsequent gamma-irradiation was required to maintain sterility of the final product. We also showed that combined low-temperature pasteurization with gammairradiation could be superior to conventional pasteurization (72 °C, 15 s) in maintaining both microbiological safety and BC product bioactivity. Relative to colostrum, mature human milk shows decreased
Fig. 7. Immune-modulatory effects of bovine colostrum (BC) on intestinal epithelial cell (IEC) line IPEC-J2. (A) BC-induced IEC proliferation; (B) BC and/or LPSinduced IL-8 secretion in IECs. Values are mean ± SEM. *P < 0.05, compared with the control (A) or compared with the corresponding colostrum treatment without LPS (B). Values in A within the same BC concentration not sharing the same letters are significantly different (P < 0.05). 187
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inhibitory activities of bacterial growth and gut immune-modulatory effects. Gamma-irradiation helped to maintain product sterility with minimal influence on IgG levels and bacterial inhibitory activities. However, more studies are required to investigate the minimal irradiation dose to secure sterility with minimal protein damage. Even when mixed into IF, bioactive BC still exerted strong inhibition of bacterial growth, suggesting potential beneficial effects of fortification of human milk and infant formula with BC for infants. Two ongoing clinical trials in preterm infants will help to reveal the clinical effects of BC63g on infant feeding intolerance, sepsis and gut inflammation. In future studies, it is important to assess the in vivo bioactivity of specific BC components in the immature gastrointestinal tract of infants or relevant model animals, including their effects on gut bacteria, bacterial translocation and mucosal immune development.
did not decrease its bacterial growth-inhibitory effects, compared with BC63. We postulate that low-temperature pasteurization at 63 °C for 30 min does not markedly change the tertiary structure of bioactive proteins (similar levels of native proteins in BC63 and BC00). Likewise, although the combination of this gentle pasteurization and gamma-irradiation may affect the structure of bioactive proteins, it was probably still less detrimental than the standard pasteurization (72 °C for 15 s) with clear reduction in the bacterial growth inhibition in BC72, relative to BC00, BC63 and BC63g. Further, despite possible irradiation-induced changes in the structure of a component like lactoferrin, this protein may still retain its ability to chelate iron and prevent bacterial nutrient consumption, leading to retained high bacterial growth-inhibitory effects. In this study, we used a relatively high irradiation dose of 14 kGy to secure sterility of the final product, and further investigations are required to elucidate the dose–response effects of irradiation on whey protein bioactivity. Interestingly, all four tested BCs exerted bacterial growth inhibition during the first 1–2 h after incubation, but only unpasteurized or gently pasteurized products (BC00, BC63 and BC63g) had long-lasting effects on bacterial inhibition (6–24 h). This implies that the lower levels of bioactive components in BC72 are consumed fully during the first 2 h of incubation whereas the remaining amounts of bioactive components in the gently treated products are still sufficient to exert bacterial inhibition for up to 24 h of incubation. This may have important implications for limiting infections in newborn infants frequently fed with gently processed BC to continuously prevent gut inflammation derived from bacterial overgrowth (Cole & Ziegler, 2007; Deitch, 1994). A sterile product with long-lasting antimicrobial activity like BC63g may also be important to decrease the risks of bacterial translocation across the immature gut barrier in newborn infants causing infection and sepsis (Duffy, 2000; MacFie et al., 1999). The ability of BC63g to induce gut maturation and decrease sepsis in preterm infants predominantly fed formula or human milk is currently being tested in two randomized clinical trials (NCT03085277, NCT03537365, clinicaltrials.gov). For many newborn infants, particularly preterm infants, nutrient fortification is critical to maintain adequate growth (Sun, Li, Nguyen, et al., 2018). However, fortification of mother’s own milk or donor human milk with infant formula products may also increase the incidence of feeding intolerance and gut inflammation (Sullivan et al., 2010). Using preterm pigs, we recently showed that fortification of donor human milk with BC63g was superior to fortification with a commercial infant formula in facilitating growth and gut maturation and preventing systemic infections (Sun et al., 2018; Sun et al., 2018). In the current study, we mimicked the in vivo conditions by fortifying IF with 25–50% (v/v) of BC63g and still observed potent bacterial growth inhibition when incubating them with S. epidermidis, relative to bacterial incubation with IF alone. The antimicrobial activity against gut pathogens may explain how BC63g fortification of donor human milk decreases systemic infections as observed in preterm pigs, where bacteria detected in infected organs are often found also in the gut (Sun, Li, Nguyen, et al., 2018; Sun, Li, Pan, et al., 2018). In a previous study comparing levels of bioactive proteins among various whey protein concentrates with different processing conditions, we found that gentler processing led to higher levels of IgG and lactoferrin in the final products, which were associated with better capacity to stimulate IEC proliferation and cytokine secretion (Nguyen et al., 2016). Similarly, the current study demonstrated that the effects of conventionally pasteurized product (BC72) on IEC proliferation and cytokine secretion were less potent than that of non-pasteurized and more gently pasteurized products (BC00 and BC63). We speculate that higher levels of bioactive components in BC00 and BC63 (e.g., IgG, lactoferrin) may better modulate immune responses of IECs, and thereby enhancing downstream signaling pathways associated with cell proliferation and cytokine secretion. We conclude that heat treatment during processing decreased the levels of bioactive proteins in the BC products as well as their in vitro
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