TABLE 2.

Summary of in vivo (human clinical and animal) studies assessing the impact of human milk processing on infant digestion

Human milk type & processing					Digestion-Study Design & Results
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
Mother’s milk (Transitional/Mature 8–9 and 21–22 d postpartum) Donor human milk (unknown, presumed mature)	Mother’s milk (preterm); Donor human milk (unknown, presumed term)	HoP1 (Thermal)	Pathogen inactivation	62.5°C, 30 min	Clinical-Crossover (2 interventions, 8–9 wk postpartum; 21–22 d postpartum)	(1) Raw mother’s milk (2) Pasteurized donor human milk	Preterm (30 ± 3 wk GA)	N = 20 (10 male, 10 female) mother-infant pairs	Survival of anti- Bordetella pertussis filamentous hemagglutinin (FHA) and anti-pertussis toxin (PT) antibodies (IgG, IgM, IgA) in gastric samples (30 min after feed) from infants fed mother’s milk or donor human milk.	Spectrophotometric ELISAs of milk, gastric samples (30 min after feed) and stool samples	(1) Anti-PT IgA, anti-PT IgG and anti-FHA IgG in donor human milk was reduced during infant digestion at both postpartum times--anti-PT antibodies stable or increased in mother’s milk.	[29]
											(2) Anti-FHA specific IgA and IgM higher in gastric contents from infants fed mother’s milk vs. donor human milk at 8–9 d.
											(3) Pasteurization of anti-pertussis antibodies may reduce their survival during infant digestion.
											(4) Both donor human milk and mother’s milk contain PT-specific antibodies that can survive digestion and can compensate for lower IgG transplacental transfer in preterm infants compared to term infants.
Mother’s milk (Transitional/Mature 8–9 and 21–22 d postpartum) Donor human milk (unknown, presumed mature)	Mother’s milk(preterm); Donor human milk (unknown, presumed term)	HoP1 (Thermal)	Pathogen inactivation	62.5°C, 30 min	Clinical-Crossover (2 interventions, 8–9 wk postpartum; 21–22 d postpartum)	(1) Raw mother’s milk (2) Pasteurized donor human milk	Preterm (30 ± 3 wk GA)	N= 20 (10 male, 10 female)	Relative abundance of anti H1N1-hemagglutinin and H3N2-specific IgG, IgM, IgA in milk, gastric contents (30 min after feed) and stool	Spectrophotometric ELISAs of milk, gastric samples (30 min after feed) and stool samples	(1) Gastric digestion reduced anti-H3N2 neuraminidase IgG from mother’s milk and from donor human milk at 21–22 d and 8–9 d, respectively.	[30]
											(2) Anti-influenza A-specific IgM was higher in mother’s milk than donor human milk at both postnatal times in feed and gastric samples.
											(3) All influenza A antibodies were detected in stool 24h postfeeding (resisted digestion).
Mother’s milk (Transitional/Mature 8–9 and 21–22 d postpartum) Donor human milk (unknown, presumed mature)	Mother’s milk (preterm); Donor human milk (unknown, presumed term)	HoP1 (Thermal)	Pathogen inactivation	62.5°C, 30 min	Clinical-Crossover (2 interventions, 8–9 wk postpartum; 21–22 d postpartum)	(1) Raw mother’s milk (2) Pasteurized donor human milk	Preterm (30 ± 3 wk GA)	N= 20 (10 male, 10 female), mother infant pairs	Concentration of secretory IgA, total IgA, total IgM, and total IgG in milk and gastric contents (30 min after feed) and stool.	Spectrophotometric ELISAs of milk, gastric samples (30 min after feed) and stool samples	(1) Total IgA, sIgA and total IgM/IgG concentrations were higher in the stomach from preterm infants fed mother’s milk vs. donor human milk.	[28]
											(2) This could be because of initial higher concentration of maternal antibodies in mother’s milk vs. donor human milk.
											(3) sIgA and total IgM in mother’s milk were partially digested in the stomach, total IgA and IgG in mother’s milk were stable in gastric contents (none were digested).
											4) Lower digestibility of antibodies in donor human milk could be because of changes in Ig structure after pasteurization.
Mother’s milk (raw/pasteurized) (Transitional/Mature)	Preterm	HoP1 (Thermal)	Pathogen inactivation	62.5°C, 30 min	Clinical-Crossover	(1) Raw mother’s milk (2) Pasteurized donor human milk	Preterm (30 ± 1 wk GA) at 27 ± 12 d postpartum	N = 12 (6 males, 6 female)	Lipid analysis (triglycerides, diglycerides, monoglycerides, total fatty acids) and protein composition of milk and gastric contents. Free taurine (marker of meal dilution) Particle size distribution	Lipid analysis (thin layer chromatography and gas chromatography	(1) Pasteurization enhanced the proteolysis of lactoferrin, but reduced proteolysis of α-lactalbumin (after 90 min).	[27]
										-flame ionization detection)	(2) Lipolysis (predigestion) was lower for pasteurized milk than raw milk.
										Protein analysis (SDS-PAGE with semi-quantitation)	(3) Strong emulsion destabilization was observed, with smaller aggregates and a higher specific surface for pasteurized milk.
										Free taurine (Cation exchange chromatography)	(4) Increase in particle size by mode and by volume in raw milk during gastric digestion vs. pasteurized milk. Differences no longer exist during intestinal digestion.
										Particle size distribution (laser light scattering)
Transitional	Preterm	HoP1 (Thermal)	Pathogen inactivation	62.5°C, 30 min	Clinical-Crossover (One-week pasteurized mother’s milk); one-week raw mother’s milk)	(1) Raw mother’s milk (2) Pasteurized mother’s milk	Preterm (27–30 wk)	N=5 (4 females, 1 male)	Percent fat absorption	72h fat balance-fecal fat measured by gravimetric methods-total fat in stool at the end of each intervention week	(1) Fat balance with pasteurized milk resulted in higher fat content in the stool compared with raw milk (p=0.06).	[31]
											(2) The mean net fat absorption coefficient was 17% higher during the balance with raw milk vs. pasteurized milk (88% [80%–92%) vs. 71% [47%–87%] p=0.06).
Mother’s milk and pooled donor human milk (Mature/Transitional)	Preterm	HoP1 (Thermal)	Pathogen inactivation	63°C, 30 min	Clinical-RCT	(1) Raw mother’s milk (2) Pooled pasteurized donor human milk	Preterm (Birthweight 1000–1500 g)	N=68 (33 infants raw mother’s milk, intervention); 35 infants pooled, pasteurized donor human milk control)	Hematological and biochemical measurements (serum albumin, creatinine, sodium, potassium, vitamin E)	Serum albumin, creatinine (Technicon autoanalyzer II); Sodium, potassium (Flame spectrophotometer); Serum vitamin E (microplate, Fabianek et al.)	(1) Serum albumin, creatinine, potassium, and sodium values were similar in the two groups; this declined over the course of the study.	[32]
											(2) Normal vitamin E levels, however; differences at 19 and 33 d after intervention (lower vitamin E in infants fed pasteurized milk vs. raw mother’s milk).
Donor human milk (mature (2–5 mo postpartum) vs. fresh mother’s milk(transitional/mature)	Term (donor human milk); preterm (mother’s milk)	Autoclave sterilization (Thermal)	Pathogen inactivation	100°C, 5 min	Clinical-Case Control (Matched by birthweight, within 100 g and gestational age, within 2 weeks)	(1) Raw mother’s milk (2) Sterilized donor human milk	Preterm (low birth weight, <1300 g)	N=24	Fat, nitrogen, and lactose balance studies- 2 weeks post intervention	72-hour balance studies of fat (colorimetric); nitrogen (Kjeldhal); lactose (kit);	(1) Fat absorption higher in mother’s milk vs. donor human milk (90.4% vs. 69.8%) during the first week; 86% vs. 58.4% in the second week.	[25]
											(2) Nitrogen absorption higher in mother’s milk vs. donor human milk (90.1% vs. 83.8% during first week and 86.6% vs. 82.5% in second week.
											(3) No significant differences in % carbohydrate absorbed.
Donor human milk (mature assumed)	Term (assumed)	(1) HoP1 (Thermal) (2) Flash heat (brought to boil), rapidly cooled (Thermal)	Pathogen inactivation	(1) 62.5°C, 30 min (2) Brought to boil	Clinical-Crossover	(1) Raw human milk (2) Pasteurized human milk (3) Boiled human milk	Preterm (<1300g; 3–6 wk)	N = 7 (4 female, 3 male)	Fat absorption %, nitrogen balance, calcium, phosphorous and sodium balance.	48-hour balance study, testing fecal fat by saponification and fatty acid titration with alkali (Van de Kamer); Sodium via flame emission spectrophotometry; Calcium by atomic absorption spectrophotometry; Phosphorous via colorimetric (molybdate/vanadate reagent)	(1) Fat absorption decreased by 28%–37% in boiled pasteurized and boiled milk, respectively relative to raw milk.	[24]
											(2) No differences in nitrogen, calcium, phosphorus, or sodium balance.
Mother’s milk and pooled donor human milk (mature assumed)	Mother’s milk (preterm); pooled donor human milk (term assumed)	HoP1 (Thermal)	Pathogen inactivation	Series A: 72°C, 5 min Series B: 62°C, 20 min; 97°C, 20 min; 100°C, 3 min	Clinical-Crossover	Series A: (1) Raw mother’s milk (2) Pasteurized milk 72°C, 5 min Series B: (1) Raw pooled milk (2) Pasteurized milk 62°C, 20 min (3) Pasteurized milk 97°C, 20 min (4) Pasteurized milk 100°C, 3 min	Series A: Preterm (960–1960 g) Series B: Preterm (1080–2220 g)	Series A:N = 8 infants (31 fat balances) Series B:N = 24 infants (67 fat balances)	Series A: Fat balances (4–5 d duration) Series B: Fat balances (4–7 d duration)	Series A: Fecal fat determined by Blix and Lindberg method. Series B: Fecal fat determined by Mojonnier extraction method	(1) No significant differences in fat absorption with heating milk (62.5°C, 30 min; 72°C for 5 min or 97°C, 3 min).	[33]
Mature (assumed)	Term (assumed)	(1) HoP1 (Thermal) (2) UV-C2 (Non-thermal)	Pathogen inactivation	(1) 62.5°C, 30 min (2) 4863J/L dose	Animal study (Preterm pigs, Large White Danish X Landrace X Duroc, 106d gestation)	(1) Pooled, Raw donor human milk (2) HoP donor human milk (3) UV-C2 treated milk	Preterm	N=57 (N=18/19 per group)	Effect on mucosal morphology, digestive function (plasma citrulline, marker of intestinal dysfunction), intestinal permeability, NEC, gut microbiota, food transit time.	Mucosal morphology assessed by villus heigh and crypt depth via hematoxylin and eosin-stained paraformaldehyde-fixed histology slice; Plasma citrulline (marker of intestinal dysfunction); Intestinal permeability via lactulose-mannitol bolus; Gut microbiota by 16S rRNA gene MiSeq-based sequencing; Food transit time via chromium-oxide added bolus feed.	(1) No difference in food transit time, intestinal permeability, or diagnosis of necrotizing enterocolitis.	[22]
											(2) Intestinal health was improved in pigs fed UV-C2 treated milk compared with HoP milk as indicated by a higher plasma citrulline concentration (36%) and villus height (38%).
Mother’s milk (pasteurized/ pasteurized & homogenized) (Transitional/Mature)	Preterm	(1) HoP1 (Thermal) (2) Ultrasonic homogenization (Non-thermal)	Pathogen inactivation & Fat homogenization	(1) 62.5°C, 30 min (2) 595 W (5min x 3)	Clinical-Crossover	(1) Pasteurized (2) Pasteurized-Homogenized	Preterm infants (<32 weeks GA) 29.5 ± 1.5 weeks	N=8 (3 male, 5 female)	Structural disintegration, lipolysis (fatty acid release) and proteolysis	Particle size via laser light scattering (Mastersizer) and	(1) No significant difference in surface weighted mean (D [3,2]), volumed weighted mean (D [4,3]) and the specific surface for both milks.	[34]
										confocal laser scanning microscopy.	(2) Aggregates in pasteurized milk were formed by proteins and by milk fat globule; in pasteurized and homogenized milk, mixed aggregates were observed with co-localized proteins, lipid droplets and amphiphilic molecules.
										Lipid analysis (thin layer chromatography and gas chromatography with flame ionization detection);	(3) Lipolysis was greater during digestion of pasteurized homogenized milk vs. pasteurized milk during gastric digestion; C16:0 and C18:1 were the predominant fatty acids, likely because of greater surface are for lipase adsorption.
										Protein analysis (SDS-PAGE with semi-quantitation);	(4) Concentrations of major milk proteins (lactoferrin, serum albumin, α-lactalbumin and ß-casein) decreased-- no effect of homogenization. Serum albumin showed an enhanced proteolysis posthomogenization.
Mother’s milk and pooled donor human milk (Mature)	Preterm & donor human milk (Term assumed)	(1) HoP1 (Thermal) (2) Ultrasonic homogenization (Non-thermal)	Pathogen inactivation & Fat homogenization	(1) 62.5°C, 30 min (2) Ultrasonic homogenization (10 min)	Clinical-Crossover (Non-homogenized vs. homogenized)	(1) Non-pasteurized, homogenized (2) Non-pasteurized, non-homogenized (3) Pasteurized, homogenized (4) Pasteurized, non-homogenized	Preterm (28–34 wk; 1000–1500g)	N=18 (N=8 Raw, unpasteurized. N=10, pasteurized mother’s milk + donor human milk)	Fat absorption %	Fecal Fat by saponification and fatty acid titration with alkali (Van de Kamer) from 2x72 h fat balance studies	(1) No difference in total fat ingestion among the groups, however; homogenization increased % fat absorption from 86.2% to 91.7% in raw milk and increased % fat absorption from 78.6% to 86.8% in pasteurized milk.	[26]
											(2) Pasteurization of human milk decreased fat absorption--homogenization of pasteurized human milk yielded a similar fat absorption to non-homogenized raw milk.
Donor human milk (colostrum, transitional, mature)	Term (assumed) from milk bank	(1) HoP1 (Thermal) (2) Physical homogenization (immersion blender) (Non-thermal) (3) Centrifugation (Non-thermal)	(Pathogen inactivation & Fat homogenization	(1) 62.5°C, 30 min (2) 4800 rpm, 3 min (3) 13,000 x g for 10 min	Animal study (Male Wistar rats)	(1) Pasteurized-homogenized (2) Homogenized-pasteurized (3) Skimmed-pasteurized and water control (4) Water-control	Term assumed (21–51-d postpartum rat)	N=32 (8 animals per group)	Nutrient (fat) delivery and indirect measures of absorption/metabolism including total cholesterol, HDL-cholesterol, TAG, alanine aminotransferase, aspartate aminotransferase, brain unsaturated/saturated fatty acid.	Blood biochemistry (Commercial kits and spectrophotometer); Brain fatty acids (Gas chromatography, flame ionization detection)	(1) No significant differences in blood biochemistry.	[23]
											(2) Higher concentration of C20:5n-3 (eicosapentaenoic acid); C22:6n-3 (docosahexaenoic acid) and C24:1n-9 enriched in brain in group receiving homogenized DM compared to non-homogenized.

Note.¹Holder pasteurization, HoP; ²Ultraviolet C irradiation, UV-C.