Breast milk contains critical growth factors for the newborn, especially transforming growth factor (TGF)-b2 and epidermal growth factor (EGF). Ingestion of breast milk TGF-b21-3 and EGF4 may promote gut barrier function, immune tolerance, and mucosal repair in the immature neonatal intestine as well as prevent necrotizing enterocolitis (NEC).
Orally ingested TGF-b2 protected mice5 and baboons6 from NEC by suppressing mucosal inflammatory responses. TGF-b2 was more efficient than TGF-b1 or TGF-b3 in suppressing inflammatory responses in the developing intestine and protecting against NEC.5
Decreased TGF-b2 expression and bioactivity in animal and human intestinal tissues were associated with increased NEC.5 Moreover, ingested EGF could normalize the overproduction of pro-inflammatory cytokines in the neonatal liver with NEC (rat model) by reducing intestinal injury.4
Supplementation of EGF in infant formula decreased the incidence of NEC in a neonatal rat model.7 Ingested EGF also reduced NEC by changing the apoptotic gene and protein expressions in the ileum8 and enhancing the intestinal growth in baby rats.9 Based on those studies, TGF-b2 and EGF in breast milk may protect premature infants against NEC and mucosal injury.
Figure 1: The role of transforming growth factor (TGF)-beta2 and epidermal growth factor (EGF) in the neonatal intestinal development and prevention of NEC.
Objective
The aim of this study was to compare the concentrations of native TGF-b2 and EGF and activated TGF-b2 between 20-Calories Benefit product (retort donor breast milk) and raw breast milk from mothers.
Methods
Concentrations of native and activated TGF-b2 and native EGF were determined via ELISA in three 20-Calories Benefit donor products (Medolac Laboratories) and in raw breast milk samples from sixteen mothers.
For the activation of TGF-b2, the breast milk samples were acidified with 1N HCl to adjust to pH 3 for 15 min, neutralized with 1N NaOH, and then immediately tested. Breast milk supernatants were collected after centrifugation at 3,500 x g for 20 min at 4°C and diluted in 2x with blocking buffer. Primary antibodies (monoclonal mouse anti-human TGF-b2 and EGF), secondary antibodies (polyclonal HRP conjugated rabbit anti-human TGF-b2 and EGF) and purified recombinant TGF-b2 and EGF (for standard curves) were purchased from Aviva System Biology. ELISAs were recorded with a microplate reader SpectraMax iD5 (Molecular Devices) Multiple unpaired t-tests were used for statistical analysis.
Results
Concentrations of native TGF-b2 and activated TGF-b2 in 20-Cal donor milk products were 1.9-(p = 0.02) and 2.6-fold (p = 0.004), respectively, higher than that in raw breast milk (Figure 2). EGF concentration did not differ between 20-Cal donor milk product and raw breast milk (p = 0.64).
Figure 2. Concentrations of native transforming growth factor-b2 (TGF-b2), epidermal growth factor (EGF) and activated TGF-b2 in raw breast milk (n = 16 for mothers) and in 20-Calories Benefit donor breast milk products (n = 3 for batches). Values are mean ± SEM. Asterisks show statistically significant differences between variables (** p < 0.01; * p < 0.05) using multiple unpaired t-tests.
Discussion
Several active breast milk components with immune-protective functions are affected during pasteurization, including lactoferrin (57–80% reduction), secretory IgA (22–48% reduction), IgM (98–100% reduction) and IgG (34–41% reduction),10-12 therefore retort processing will also decrease their concentrations. However, TGF-b and EGF were not affected by pasteurization.13,14 No previous study has determined whether TGF-b2 and EGF are affected during the retort processing.
For the first time, we found that native and activated TGF-b2 concentrations in Medolac 20-Cal donor milk breast products were higher than in raw breast milk samples. This increase of concentration in TGF-b2 could be due to the heat-treatment during retort processing and the mixture of raw human whole milk and cream. These results are in agreement with Namachivayam et al. (2013)15 that TGF-b2 bioactivity in both pasteurized donor breast milk and raw breast milk (preterm-delivering at 1 week and 1 month of postpartum) was increased after heat-treatment at 80°C for 5 min. These authors used this heat-treatment to activate latent TGF-b2. Moreover, Reeves et al.16 observed that TGF-b2 concentration in pasteurized donor breast milk fortifier (HDMF) was higher than pasteurized donor breast milk due to its processing; HDMF is made from pooled human milk creams that may concentrate TGF-b2.
Latent TGF-b in breast milk is activated to the immunoreactive isoform by acidification (pH 3), which denatures latency-associated peptide (LAP), a peptide binds to TGF-b.17 Other mechanisms can activate TGF-b, such as proteases and heat-treatment.18,19 Pre-activation of breast milk TGF-b using heat-treatment or acidification/neutralization is particularly critical for premature infants due to their higher gastric pH and lower protease activity in the stomach and intestine compared with term infants.20 The capacity to activate TGF-b during digestion in preterm infants is unknown and future studies are needed to understand the functional activity of TGF-b in the neonatal gut.
Conclusions
Medolac 20-Cal donor breast milk products contain higher TGF-b2 concentration compared with raw breast milk due to the activation of latent TGF-b2 by the retort heat-treatment and concentrated TGF-b2 in product formulation. Higher levels of TGF-b2 in Medolac donor breast milk products may improve the protection of preterm infants against NEC/mucosal injury and enhance the maturation of the gastrointestinal tract.
References
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About the Author
Dr. Veronique Demers-Mathieu is the Senior Research Scientist in the department of Neonatal Immunology and Microbiology at Medolac Laboratories. She did 3-years of postdoctoral training in Neonatal Nutrition under Dr. Dallas at Oregon State University and earned her Ph.D. in Food Microbiology at Laval University. Her expertise focuses on the immune components (including antibodies, immune cells, cytokines, and bioactive proteins) from human milk that protect infants against infectious diseases.