TABLE 3.
Summary of simulated static and dynamic in vitro studies assessing the impact of human milk processing on infant digestion
| Human milk type & processing |
Digestion-Study Design |
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|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Lactational Stage (Colostrum, Mature, Transitional) | Preterm/Term milk | Processing Type and Classification | Processing Rationale | Processing parameters | Study type | Groups | Infant type | Sample size (N) | Outcomes assessed | Methods | Key findings | Ref |
| Pooled donor human milk (from 6 mothers) | Term (assumed) | (1) HoP1 (Thermal) (2) HPP2 (Non-thermal) | Pathogen inactivation | (1) 62.5 °C, 30 min | Static In vitro Gastrointestinal Digestion | (1) Raw human milk | Term | N=1 pool (digested in triplicate for each treatment) | Microstructure, particle size distribution and proteolysis | Microstructure via confocal laser scanning microscopy; Protein composition using SDS-PAGE; Degree of protein hydrolysis using the ortho-phthalaldehyde method; free amino acid analysis via HPLC | (1) Raw milk had a major peak size distribution of approximately 5–8 μm vs. approximately 50 μm for treated samples of HoP1 and HPP2. After 60 min of gastric digestion, HPP2 milk had a similar size distribution to raw milk. | [40] |
| (2) 400 MPa, 5 min, 25°C | (2) HoP1 human milk | (2) During gastric digestion, the protein profile of HoP1 milk began to differ from that of raw milk, with the lactoferrin band becoming fainter, while the ß-casein band was resistant to complete digestion. Lactoferrin in Raw and HPP2 milk was resistant to digestion. | ||||||||||
| (3) HPP2-treated milk | (3) Greater digestion of ß-casein in raw and HPP2 treated milk, indicative of increased hydrolysis. | |||||||||||
| (4) Overall, lower protein hydrolysis in stomach vs. intestinal. HPP2 had a slightly higher hydrolysis during the gastric phase; HoP1 milk had the highest hydrolysis during the intestinal phase. | ||||||||||||
| (5) No significant difference between raw milk and treated milk for either individual free amino acid concentrations or the total amino acid concentration after 60 min of intestinal digestion. | ||||||||||||
| Donor human milk (Mature, 5–9 mo postpartum) | Term | HoP1 (Thermal) | Pathogen inactivation | 62.5°C, 30 min | Static in vitro gastrointestinal digestion & Caco-2/TC7 in vitro absorption | (1) Raw, unpasteurized donor human milk | Preterm (4-weeks postpartum) | N=1 pool (Triplicate digestion for each treatment) | Characteristics of lipolysis (Total fatty acid analysis, triglycerides, free fatty acids, cholesterol, diacylglycerides and monoacylglycerides) and in vitro lipid uptake and gene expression | Lipid analysis (thin layer chromatography, gas chromatography-flame ionization detection); In vitro lipid uptake using Caco-2/TC7 cell culture | (1) Lipolysis occurred mainly in the intestinal phase. Triacylglycerides were hydrolysed into free fatty acids, diacylglycerol and monoacylglycerol. The extent of lipolysis was 52% lower in pasteurized milk vs. raw milk justbefore in vitro digestion. No differences were observed during gastric or intestinal digestion. | [45] |
| (2) HoP1 donor human milk | (2) No differences between pasteurized and raw milk with respect to lipid absorption using Caco2 cells. | |||||||||||
| Pooled donor human milk (mature assumed) | Term (assumed) | HoP1 (Thermal) | Pathogen inactivation | 62.5°C, 30 min | Static in vitro gastrointestinal digestion | (1) Raw, unpasteurized donor human milk | Term | N=1 pool (divided in 2) | Release of peptides from human milk proteins | Peptidomic analysis via solid-phase extraction followed by MS/MS. Characterization of peptides by computational methods. | (1) Protein-derived peptides present, most abundantly derived from ß-casein, given its high susceptibility to plasmin-mediated proteolysis. | [42] |
| (2) HoP1 donor human milk | (2) Pasteurization did not appear to alter bioactive peptide release following in vitro digestion of raw and pasteurized human milk-- many peptides from caseins and whey proteins were released. | |||||||||||
| (3) Bioactive peptides released from in vitro digestion include angiotensin I-converting inhibitory peptides, antioxidative peptides and immunomodulatory peptides. | ||||||||||||
| Mature (assumed) | Term (assumed) | HTST3 (Thermal) | Pathogen inactivation | 95°C, 1 min | Static in vitro gastrointestinal digestion | (1) Pooled, Raw donor human milk | Not defined | N=1 pool; digestion in duplicate | Protein degradation | Protein composition via SDS-PAGE and semi-quantitation; Casein micelle size via photon correlation spectroscopy (Zetasizer). | (1) Lactoferrin was digested quickly; high heat treatment of milk resulted in very little difference in protein degradation, except for α-lactoglobulin which showed a 10%–20% higher degradation compared to raw milk. | [39] |
| (2) Heat-pasteurized donor human milk | ||||||||||||
| Mature (1–3 mo postpartum) | Term | (1) HoP1 (Thermal) | Pathogen inactivation | (1) 62.5°C, 30 min | Dynamic in vitro gastrointestinal digestion system (DIDGI) | (1) Pooled, Raw donor human milk | Preterm (4 weeks postpartum) | N=1 pool; triplicate digestion for each treatment | Identification, quantification, and biochemical characteristics of peptides | Peptide identification via mass spectrometry. Computational tools for peptide characterization & quantification via label-free MS. | (1) Before digestion, identified peptides were derived from ß-casein. | [37] |
| (2) HTST3 (Thermal) | (2) 72°C, 15 sec | (2) HoP1 donor human milk | (2) Gastric digestion of HoP1 resulted in a greater number and more abundant ß-casein specific peptides. A delayed release of peptides was observed in raw milk during the intestinal phase. The effect of pasteurization was predominant during the intestinal phase--irrespective of what technology was used. | |||||||||
| (3) HTST3 Pasteurized Milk | (3) Higher intestinal digestion of lactoferrin (barely detectable in the pasteurized samples after 30 min of intestinal digestion). | |||||||||||
| (4) HTST3 pasteurization (at a gastric level), can retain a closer peptide profile compared to raw, than HoP1. Higher abundance of peptides after HTST3 digestion vs. HoP1. | ||||||||||||
| Mature (1–3 mo postpartum) | Term | (1) HoP1 (Thermal) | Pathogen inactivation | (1) 62.5°C, 30 min | Dynamic in vitro gastrointestinal digestion system (DIDGI) | (1) Pooled, Raw donor human milk | Preterm (4-weeks postpartum) | N=1 pool (digested in triplicate per treatment) | Particle size distribution, degree of protein hydrolysis, protein composition, bio-accessibility of amino acids, lipid analysis (lipid classes) | Particle size via laser light scattering (Mastersizer) and confocal laser scanning microscopy; Lipid analysis (think layer chromatography and gas chromatography with flame ionization detection); Protein analysis (SDS-PAGE); | (1) During gastric phase, formation of large aggregates in raw milk compared to HTST3 and HoP1) –Pasteurized samples did not show any modification of the particle size during gastric digestion. | [38] |
| (2) HTST3 (Thermal) | (2) 72°C, 15 sec | (2) HoP1 donor human milk | (2) Pasteurized human milk samples resulted in faster gastric proteolysis of high-molecular-weight protein bands, including lactoferrin vs. raw milk. | |||||||||
| (3) HTST3 pasteurized Milk | (3) Faster intestinal proteolysis in both pasteurized samples for high-molecular-weight bands, including native lactoferrin. Pasteurization increased the resistance of serum albumin to digestion vs. raw. No major differences between HoP1 and HTST3. | |||||||||||
| 4) No differences in release of free amino acids or triglyceride hydrolysis. | ||||||||||||
| Mature (6.6 weeks ± 2.4 weeks postpartum) | Preterm (29.8 ± 3.0 weeks) | HoP1 (Thermal) | Pathogen inactivation | 62.5°C, 30 min | Dynamic in vitro gastrointestinal digestion system (DIDGI) | (1) Raw breast milk | Preterm (28 weeks GA, 4 weeks post-natal) | N=3 per treatment group (triplicate) | Peptides released and free amino | Peptidomics (LCMS analysis w/ label-free peptide quantification); Free amino (fluorescent microplate analysis using ortho-phthalaldehyde (OPA) and ß-mercaptoethanol) | (1) Certain clusters of peptides showed a lower abundance for pasteurized vs. raw milk during the gastric phase and intestinal phase. | [44] |
| (2) Pasteurized breast milk | (2) Pasteurization impacted the human milk peptidome before digestion (from ß-casein) and induced different kinetics of peptide release during gastro-intestinal digestion mainly for heat-denatured proteins (bile salt–stimulated lipase and lactoferrin). | |||||||||||
| (3) Pasteurization impacted some peptide release during digestion (No impact on free amino) but clinical relevancy needs to be determined. | ||||||||||||
| Mature (6–14 wk postpartum) | Term | HoP1 (Thermal) | Pathogen inactivation | 62.5°C, 30 min | Dynamic in vitro gastrointestinal digestion system (DIDGI) | (1) Raw breast milk | Term (4-weeks) | N=2 for raw milk; N=3 for pasteurized milk | Peptides released and free amino | Peptidomics (LCMS analysis w/ label-free peptide quantification); Free amino (fluorescent microplate analysis using ortho-phthalaldehyde and ß-mercaptoethanol) | (1) Pasteurization impacted selectively gastric and intestinal kinetics of peptide release. | [43] |
| (2) Pasteurized breast milk | (2) Pasteurization increased the number of peptides and abundance of peptides common to both raw and pasteurized milk. Origin of peptides predominantly from ß-casein and not from other abundant proteins (lactoferrin, α-lactalbumin). | |||||||||||
| (3) Lower release of free amino for pasteurized vs.raw milk at 120 min of gastric digestion. | ||||||||||||
| (4) Unknown if changes in peptide digestion kinetics in pasteurized milk is clinically relevant. | ||||||||||||
| Donor human milk (mature, 6.6 ± 2.4 weeks postpartum) | Preterm | HoP1 (Thermal) | Pathogen inactivation | 62.5°C, 30 min | Dynamic in vitro gastrointestinal digestion system (DIDGI) | (1) Pooled, Raw donor human milk | Preterm (4-weeks postpartum) | N=1 pool (digested in triplicate per treatment) | Microstructure and particle size distribution; Lipid analysis (triglycerides, diglycerides, monoglycerides and total fatty acids) and protein composition of milk and gastric contents | Particle size via laser light scattering (Mastersizer) and confocal laser scanning microscopy; Lipid analysis (thin layer chromatography and gas chromatography with flame ionization detection); Protein analysis (SDS-PAGE with semi-quantitation). | (1) During gastric phase, formation of large aggregates in raw milk and increase in main mode diameter and in D [4,3]. Aggregates which appear postpasteurization disappeared at the beginning of gastric digestion. | [7] |
| (2) Pasteurized donor human milk | (2) Larger aggregates formed for pasteurized milk vs. raw milk during the intestinal phase. | |||||||||||
| (3) No differences in gastric lipolysis; higher lipolysis in raw vs. pasteurized milk during intestinal phase. | ||||||||||||
| (4) Gastric phase proteolysis involved predominantly lactoferrin and ß-casein, no differences by treatment. | ||||||||||||
| (5) Released phenylalanine, tyrosine, and arginine higher in pasteurized vs. raw; lower release of serine. | ||||||||||||
| Mature (11 weeks postpartum) | Term | HoP1 (Thermal) | Pathogen inactivation | 62.5°C, 30 min | Dynamic in vitro gastrointestinal digestion system (DIDGI) | (1) Pooled, Raw donor human milk | Term | N=1 pool; raw digested in duplicate; pasteurized digested in triplicate | Microstructure and particle size distribution; Lipid analysis (triglycerides, diglycerides, monoglycerides and total fatty acids) and protein composition of milk and gastric contents | Particle size via laser light scattering (Mastersizer) and confocal laser scanning microscopy; Lipid analysis (thin layer chromatography and gas chromatography with flame ionization detection); Protein analysis (SDS-PAGE with semi-quantitation). | (1) Gastric proteolysis of lactoferrin and ß-casein tended to be faster for pasteurized milk compared to raw. | [41] |
| (2) Pasteurized donor human milk | (2) Lactoferrin and ß-casein were more extensively digested than serum albumin and α-lactalbumin during gastric digestion (regardless of treatment). | |||||||||||
| (3) Pasteurization affected the intestinal release of some amino acids; effect differed according to the amino acid. Lipolysis was also lower in pasteurized milk vs. raw but no difference in fatty acid release. | ||||||||||||
| 4) Raw human milk presented a structural destabilization after 60 min of gastric digestion; no differences in particle size during intestinal phase by pasteurization. | ||||||||||||
| Pooled donor human milk (8 women, mature assumed) | Term (assumed) | Freeze-Thaw (Thermal) | Other |
Freezing: (1) Fresh milk (2) -18°C, 30 d (3) -60°C, 30 d Thawing: (1) Slow thaw (4°C, 10 h) (2) Intermediate thaw (25°C, 1 h) (3) Rapid Thaw (45°C, 1 min) |
Static in vitro gastrointestinal digestion | (1) Fresh milk (2) -18°C milk, slow thaw (3) -18°C milk, intermediate thaw (4) -18°C milk, rapid thaw (5) -60°C milk, slow thaw (6) -60°C milk, intermediate thaw (7) -60°C milk, rapid thaw |
Term | N= 1 pool (1 digestion per treatment) | Particle size distribution, microstructure, protein composition and identification, peptide molecular weight. | Particle size distribution via laser particle size analyzer; microstructure via confocal laser scanning microscope; protein composition by SDS-PAGE; protein band identification by Maldi-TOF/TOF mass spectrometry. | (1) Decrease of human milk fat globule particle size in -60°C, 45°C thaw samples during gastric digestion like fresh human milk. After intestinal digestion, more human milk fat globule particles decreased in size. | [46] |
| (2) Caseins in freeze-thaw milk were digested even faster than fresh. The 6 freeze-thawed samples showed a reduction of hydrolysis compared with fresh human milk. | ||||||||||||
| (3) Human milk frozen at -60°C and thawed at 45°C is most like fresh, including the molecular weight distribution of peptides after digestion. | ||||||||||||
Note.1Holder pasteurization, HoP; 2High pressure processing, HPP; 3High-temperature short time, HTST.