NUTRITION RESEARCH 83 (2020) 15– 29

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Review Article

Digestion of human milk fat in healthy infants

Xuan He a, b, Shannon M cClorry a, Olle Hernell c, Bo Lönnerdal a, Carolyn M. Slupsky a, b,⁎ a Department of Nutrition, Davis, One Shields Ave, Davis, CA 95616, USA b Department of Food Science and Technology, University of California, Davis, One Shields Ave, Davis, CA 95616, USA c Department of Clinical Sciences, Pediatrics, Umeå University, SE 901 85 Umeå, Sweden

ARTICLE INFO ABSTRACT

Article history: Lipid is critical for infant development, and yet, the interconnection between lipid Received 17 March 2020 digestion and the microbiota is largely understudied. This review focuses on digestion of the Revised 2 August 2020 human milk fat globule and summarizes the current understanding of the mechanisms Accepted 7 August 2020 underlying this process in infants. We first discuss the partial of milk fat in the stomach, which leads to rearrangement of lipid droplets, creating a lipid-water interface Keywords: necessary for duodenal lipolysis. In the first few months of life, secretion of pancreatic Infant , A2, and bile salts is immature. The dominant aiding fat Neonate digestion in the newborn small intestine are therefore pancreatic lipase-related protein 2 and Lipid digestion bile salt–stimulated lipase from both the exocrine pancreas and milk. We summarize the Milk fat globule interaction between ionic fatty acids and cations to form insoluble soaps and how it is Gut microbiota influenced by various factors, including cation availability, pH, and bile salt concentration, as well as saturation and chain length of fatty acids.Wefurtherarguethattheformationofthe soap complex does not contribute to lipid bioavailability. Next, the possible roles that the gut microbiota plays in lipid digestion and absorption are discussed. Finally, we provide a perspective on how the manufacturing process of infant formula and dairy products may alter the physical properties and structure of lipid droplets, thereby altering the rate of lipolysis. © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Article Outline

1. Introduction ...... 16 2. Partial hydrolysis of MFG in the stomach...... 16 2.1. Release of MFG from the protein clot ...... 16 2.2. The action of lipase in an oil-in-water emulsion ...... 17 2.3. Factors influencing gastric lipid digestion ...... 19 3. Completion of milk fat digestion in the small intestine ...... 19

Abbreviations: Angptl4, angiopoietin-like protein 4; BSSL, bile salt–stimulated lipase; LCPUFA, long-chain polyunsaturated fatty acid; MFGs, milk fat globules; PLRP2, pancreatic lipase-related protein 2; PTL, pancreatic triglyceride lipase. ⁎ Corresponding author at: Department of Nutrition, University of California, Davis. Tel.: +1 530 752 6804. E-mail address: [email protected] (C.M. Slupsky). https://doi.org/10.1016/j.nutres.2020.08.002 0271-5317/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 16 NUTRITION RESEARCH 83 (2020) 15– 29

3.1. Low duodenal bile salts and limited pancreatic triglyceride lipase during early infancy ...... 19 3.2. Major for duodenal lipolysis ...... 20 3.3. Emulsification and digestion in small intestine ...... 20 4. Formation of calcium–fatty acid soap complexes ...... 22 4.1. Factors influencing fatty acid soap formation ...... 22 4.2. The impact of fatty acid–calcium soap on absorption...... 22 5. Bidirectional interaction between gut microbiota and infant lipid digestion...... 22 6. Perspectives on infant formula and dairy products ...... 24 7. Conclusions and future directions ...... 24 Acknowledgment ...... 24 References...... 24

1. Introduction and pH during the suckling period and (b) describe lipid digestion in detail, emphasizing a few critical enzymes. The Infants are not “small adults.” An infant's physiology un- progressive maturation of the infant digestive system also dergoes several dynamic changes due to the rapid alteration influences the gut microbiota and its actions. We discuss in cellular structure and functional maturation of organs. current views of how the gut microbiota influences lipid During infancy, the brain accounts for a large fraction of digestion and absorption and, finally, provide a perspective energy expenditure [1], much of which is used for brain cell on other dairy products. proliferation, neurogenesis, and myelination [2]. An ever- increasing body of evidence indicates that early life is a unique developmental window that is characterized by 2. Partial hydrolysis of MFG in the stomach considerable plasticity, which can be altered by environmen- tal stresses and diet. Nutrition during this critical period of 2.1. Release of MFG from the protein clot development is of utmost importance, as it may have profound and long-lasting effects on intelligence [3,4] and It has been shown in freshly expressed bovine milk that small the risk of developing chronic diseases [5,6] later in life. casein micelles (average diameter <50 nm) adhere to the MFG Human milk is the preferred form of feeding to support membrane with lipids, as well as other proteins and glyco- optimal growth and development of healthy infants. Milk can proteins, through relatively weak hydrophobic and electro- be characterized as an emulsion of milk fat globules (MFGs) in static interactions [16]. As milk reaches the stomach, the pH an aqueous liquid. The core of these MFGs is predominantly drops and the surface of casein becomes less charged, thereby in the form of triacylglycerols that provide approximately half enhancing hydrophobic interactions between the MFG and of the energy content of milk [7,8]. Beyond energy, milk fat casein [17]. As the ratio of protein to fat in milk increases, the supplies essential nutrients such as n-3 and n-6 long-chain diameter of the casein micelle adsorbed on the surface of the polyunsaturated fatty acids (LCPUFAs), phospholipids, and MFG membrane also increases [16]. cholesterol, which are important for the development of Fig. 1 highlights major differences between infant and brain and cognitive functions during the first year of life [9-11]. adult lipid digestion that will be discussed throughout the The amount of fat in human milk is highly variable, as it following sections. As casein micelles are proteolyzed in the fluctuates throughout the day and with nursing frequency, and stomach by gastric pepsin (the concentrations and efficiency is further influenced by several maternal factors, including of which are relatively low at birth, increasing over time body mass index, age, diet, parity number, and smoking status but still below the adult level [18]), MFGs become entrapped [12-14]. Typical human milk consists of 39% monounsaturated in a “protein clot,” with whey proteins remaining in the serum fatty acid, 35% saturated fatty acid, and 18% LCPUFA [15]. phase (Fig. 2). Over time, gastric pepsin activity gradually Palmitic acid (16:0), oleic acid (18:0 n-9), and linoleic acid (18:2 n-6) reduces the size of protein clot. It has been demonstrated in are the most dominant fatty acids in human milk and remain vitro using bovine milk that addition of protease stable over time [15]. The energetic demands of the developing reduces the particle radius of the κ-casein–coated MFG brain and digestive system, as well as the immaturity of exocrine complex by 10-20 nm at pH 5.5 and 6.0 [17]. MFG membrane pancreatic function during infancy, highlight the importance of components, such as polar lipids and proteins, are essential the complex physical and biochemical processes of milk fat to the physical stability of the MFG, and MFG structural digestion in infancy. stability is pH dependent. As the pH drops below 5.5, the MFG The study of milk fat digestion in infants has received membrane structure visualized becomes less stable and tends considerable attention over the years. Although by no means to favor aggregation and coalescence of lipid particles [19]. complete, there is a growing body of information on lipid Furthermore, protease hydrolyzes transmembrane proteins digestion in the stomach and small intestine during infancy. on the MFG membrane, which further alters the MFG In this review, we (a) summarize the current state of structure and enhances aggregation and coalescence of fat knowledge of the action of lipases in an oil-in-water emulsion globules within the protein matrix. Both in vitro [20-22] and in and their interaction with proteases, stabilizers, emulsifiers, vivo [23] studies of human and bovine MFG have shown NUTRITION RESEARCH 83 (2020) 15– 29 17

Fig. 1 – Summary of major differences in lipid digestion of infants relative to adults.

partial gastric hydrolysis of native MFG membrane proteins pepsin penetration, protein breakdown, and release of resulting in the formation of small peptides (<20 kDa) that entrapped fat from the clot matrix [26]. remain attached to the surface of fat globules [21]. The extent of this protein hydrolysis activity varies by gastric pH and is 2.2. The action of lipase in an oil-in-water emulsion influenced by factors such as age, time after meal, and meal volume [24]. As demonstrated using bovine milk, the break- Fat digestion begins in the stomach by down of the membrane protein coating by gastric protease (encoded by the LIPF gene, EC 3.1.1.3), which is secreted by promotes the release of trapped fat globules from the clot chief (zymogen) cells of the gastric mucosa [27]. It localizes matrix into the digesta [25,26], enhancing the process of primarily in the fundus and body of the stomach [28,29] coalescence. It is important to recognize that different and hydrolyzes the sn-3 position of a triglyceride [30]. structures characteristic of the clot matrix, as demonstrated This hydrolysis occurs at a higher rate for medium-chain in heated and homogenized bovine milk, influence the rate of compared with long-chain triglycerides in

Fig. 2 – Interaction between milk fat globule (MFG) and milk proteins in the infant stomach. The degree of this interaction depends on protein concentration and hydrophobicity as well as pH. 18 NUTRITION RESEARCH 83 (2020) 15– 29 vitro [31-33]. Excretion of gastric lipase is well developed contents, but it gradually increases with time and stimula- in infants, and its activity is similar to levels observed in tion of gastric secretions in both infants and adults [43,44]. adults [29]. Gastric pH also increases to around 6-6.5 but decreases over It is important to emphasize that the lipolytic activity of time together with a reduction in gastric volume to create gastric lipases is essential for infant fat digestion [34,35]. optimal conditions for lipid hydrolysis [33,45-47]. The initial Compared with adults, the secretion of colipase-dependent neutral pH environment is likely due to milk, as the pH of pancreatic triglyceride lipase (PTL) and bile acids is immature human milk is between 6.8 and 7.4 [48,49]. Within 180 mi- in the first months of life [36]. PTL alone cannot efficiently nutes, the postprandial pH of the stomach gradually returns hydrolyze an intralipid emulsion or native MFG unless these back to its original value of ~2 [43,46]. During the postprandial emulsions are partially hydrolyzed by other lipases. This has period, the microstructure of the native MFG changes by been shown in vitro using bovine milk fat droplets [37], soy coalescing and forming larger droplets. The size of bovine lipid emulsified with lecithin [38], as well as human MFG [31]. MFG has been shown to increase by 138% after incubating Predigestion of human milk in the stomach may therefore with gastric juice at pH 4 for 1 hour in vitro [50]. This dynamic increase the ability of pancreatic lipase-related protein 2 process has been shown in other in vitro digestion studies [51- (PLRP2) and bile salt–stimulated lipase (BSSL), the latter 53]. Thus, during initial digestion, larger fat globules secreted from both the mammary gland into milk and the are formed because of aggregation and flocculation, which exocrine pancreas to further digest lipids from milk in the is likely due to the simultaneous actions of the acidic pH; duodenum [39-42]. high ionic strength; the action of pepsin on casein, adsorbed Following a meal, the gastric lipase concentration in the whey, and the MFG membrane proteins; as well as lipase stomach initially drops because of the dilution of the gastric binding.

Fig. 3 – Lipolysis of milk fat globule (MFGs) in the stomach. NUTRITION RESEARCH 83 (2020) 15– 29 19

Lipolysis begins by lipase anchoring to the lipid-water glycerophospholipids and glycoproteins (reviewed in [59]). The interface of MFG particles, which in turn, increases the presence of the sphingomyelin- and cholesterol-rich lipid- proportion of endogenously generated lipolytic products ordered domain is important to facilitate the interaction between (free fatty acids, sn-1,2 diacylglycerols) that appear at the lipase and the phospholipid-surface in the stomach. In addition, surface of the fat globule (Fig. 3). Short- and medium-chain glycoproteins from the lipid-disordered phase slow down the rate fatty acids have a high dispersibility in the aqueous environ- of proteolysis by pepsin; thus, to a degree, these residual ment and are therefore more likely to escape the fat globule. membrane-bound glycoproteins and peptides can stabilize the At low pH, a greater proportion of these lipolytic products at globules throughout digestion and may prevent coalescence of the surface of the fat globule are ionized, creating more the globules [20,21]. negative charges on the surface. With more negative charges The mixing action of the stomach further contributes to on the globule surface, the previously aggregated fat globules digestion. Although a portion of the gastric contents moves dissociate, and the resulting flocculated globules have a toward the antrum, the enlarged lipid droplets are subjected different surface composition than what was present in the to a more neutral pH [60] as well as stronger shear and native MFG. grinding forces in comparison to the upper part of the As gastric lipolysis continues, natural emulsifiers gener- stomach. In an adult, the mechanical destructive force in ated from breast milk, primarily free fatty acids and some the antrum can further homogenize and reduce the size of the monoacylglycerols, as well as surface-active phospholipids lipid droplet. Neonatal gastric contractile activity is not from the gastric mucosal barrier [54,55], compete with completely mature compared to adults [61]; hence, a lower surface-active molecules already present on the surface of shear rate and weaker mixing occurs. Lipid emulsions are the fat globules and further incorporate into the lipid-water formed and stabilized by amphiphiles such as milk proteins, interface to stabilize the surface tension of the fat globule. As phospholipids, and lipolysis products. a result, there is a change in the type of surface molecules Foods are highly complex systems, and the efficiency of that appear at the lipid-water interface. Over time, more fat gastric digestion of lipids can be further influenced by the globules with larger sizes arise from coalescence, although available food matrix. Thickening and emulsifying agents, as some flocculation may still be observed [51,53,56,57]. The well as common dietary viscous fibers such as guar gum, pectin, action of coalescence likely occurs due to the low surface and xanthan gum, are additives in weaning foods and some viscosity and elasticity of fatty acids and monoacylglycerols infant formulas. When introduced during breastfeeding, the at the new globule surface. differing food matrix is expected to influence the lipid-water MFGs are digested at different rates according to the size of interface, thereby reducing the efficiency of the digestive lipase theirnativeformandtheintermediateformduringpartial activity. Interestingly, consumption of breast milk showed faster hydrolysis. Larger lipid droplets with greater surface area are gastric emptying than formula milk, and a trend toward faster less efficiently hydrolyzed than smaller droplets, as shown gastric emptying in whey-predominant formula compared in vitro using both large-sized bovine MFG [50] and lipid droplets to casein-predominant formula has been suggested (reviewed [52]. This effect can be partially explained by the accumulation of in [62]). Together, the packing property of food microstructures lipolytic products at the lipid globule surface, as these products and the extent of lipid emulsification are key factors in lipid form specific particles that entrap gastric lipase and reduce its bioavailability and digestion. activity, especially when the concentration of these hydrolysis products is high [52]. Additionally, several small droplets would provide a greater total surface area for lipase to act upon relative to the same volume of fats in larger droplets. 3. Completion of milk fat digestion in the small intestine 2.3. Factors influencing gastric lipid digestion 3.1. Low duodenal bile salts and limited pancreatic The structure, composition, and domain morphology of the MFG triglyceride lipase during early infancy membrane layer can also play a unique role in gastric digestion of lipids. Gastric lipase activity is substantially higher in bovine Both humans [63-65] and experimental animals [66,67]exhibit milk phospholipid emulsified lipid droplets than in similarly low secretion of bile from the gallbladder into the duodenum, sized soy lecithin emulsified lipid droplets [58]. One possible resulting in low intraluminal bile salt concentration at birth that explanation for the observed differences in lipase activity is increases over time. Analysis of duodenal aspirates from infants the differing phospholipid profiles of the two droplets. Bovine has indicated that the bile acid profile predominantly consists milk phospholipids predominantly consist of sphingomyelin of primary bile acids (cholic acid and chenodeoxycholic acid) and phosphatidylcholine, whereas soy lecithin is primarily with a greater proportion of cholic acid and its conjugates than phosphatidylcholine and phosphatidylinositol. Increasing the chenodeoxycholic acid and its conjugates [65,68,69]. Although concentration of sphingomyelin in the milk phospholipid interindividual variation in bile acid profiles is noted in adults, membrane significantly increases the hydrolysis rate of gastric median concentrations of colic acid and its conjugates are lower lipase in vitro, but interestingly, the hydrolysis rate is unaffected than those of chenodeoxycholic acid and its conjugates [70]. A by addition of sphingomyelin to soy lecithin [58]. The MFG high proportion of taurine-to-glycine conjugated bile salts has membrane presents a very unique architecture with the coexis- been observed in early infancy [65,71]. This proportion decreases tence of a lipid-ordered phase, rich in sphingomyelin and with age, with a greater proportion of glycine conjugates being cholesterol, and a lipid-disordered phase (fluid matrix), rich in observed in adults [68,70,72]. 20 NUTRITION RESEARCH 83 (2020) 15– 29

For infants at 2 weeks of age, the fasting duodenal bile salt tri-, di-, and monoacylglycerols; phospholipids; and ceramide concentration is approximately 3.5 mmol/L. However, the bile (reviewed in [91,92]). The origin of BSSL in the infant is from salt concentration drops sharply after consuming evaporated both the milk (as it is expressed in the mammary gland) and milk or modified formula and 0.5-1.5 hours after a meal the pancreas [41,93]. Either pancreatic secretion or milk itreaches its lowest concentration of approximately 1 mmol/L, delivery is required for complete fat digestion in infants. It which is below the critical micelle concentration of 2 mmol/L for was shown that Cel knockout mouse pups (who therefore lack bile salts under physiological conditions [73]. After 3 hours, the pancreatic excretion of BSSL) nursed by Cel knockout dams level of bile salts returns to the fasting state [71]. Older children (therefore lacking BSSL in milk) had significantly higher (from 3 to 72 months) also exhibit a similar postprandial decrease glycerolipids (tri-, di-, and free glycerol) in in bile salt concentrations; however, the lowest bile salt their ileum compared with heterozygous pups nursed by a concentration observed after a meal well exceeds that of the heterozygous dam [94]. Moreover, the lack of BSSL from both critical micelle concentration [71]. mother's milk and the pancreas results in a high excretion of Compared to adults, the secretion of colipase-dependent PTL undigested fat in stool [95]. Under low intraluminal bile salt (encoded by the Pnlip gene, EC 3.1.1.3) is much lower in infants, concentrations, BSSL is important for hydrolyzing glycerol especially those with low gestational age, but progressively esters of LCPUFAs, such as docosahexaenoic acid 22:6(n-3) increases over time [74-78]. Duodenal lipase activity is higher and arachidonic acid 20:4(n-6) [96], and it has been shown to during the fasting state, decreases markedly during the first be more active against medium- and long-chain triglycerides postprandial hour because of dilution, and then gradually than PLRP2 [87]. Moreover, in vitro studies have shown that increases again over the subsequent 2 hours [74,75]. In early BSSL and PLRP2 can work synergistically toward a more infancy, PTL in the duodenum is insufficiently low PLRP2, and efficient lipid hydrolysis of human milk (but not formula milk) BSSL from pancreas and milk are the dominant lipases for fat than either alone [39,42]. Although the physical-chemical digestion in the small intestine [79]. explanation behind the synergism is still unknown, this synergistic effect could be important to infants prior to 3.2. Major enzymes for duodenal lipolysis maturation of the exocrine pancreas and the rise of luminal bile salt concentrations. PLRP2 (encoded by the Pnliprp2 gene, EC 3.1.1.3, EC 3.1.1.26) is Maturation of the digestive system is a dynamic and expressed during the neonatal period [78,80-83] and shares continuous process where a different set of key enzymes arises 65% amino acid identity with PTL [84]. Unlike PTL, PLRP2 does with a compensatory role to aid lipolysis. Throughout the first not have the positional specificity and can hydrolyze all sn-1, year of life, exocrine pancreatic function gradually matures and -2 and -3 positions of triglycerides [42]; however, its specific PTL becomes increasingly important for lipid digestion [75,77]. activity is not as high as PTL [85]. In addition, PLRP2 has low Studies conducted using rodents [80,82] and pigs [97]indicatea affinity for colipase and does not absolutely require colipase significant increase of PTL after weaning; however, to date, there to anchor at the bile salt–coated lipid-water interface [85-87]. are no human data available that indicate when the level of PTL PLRP2 has broad substrate specificity that can hydrolyze level reaches an adult level. Nonetheless, with more PTL excreted triglycerides, galactolipids, phospholipids, and retinyl palmi- as infants age, a synergistic effect between PTL and other lipases tate [86,88,89]. PLRP2 has the highest activity on diglycerides, is expected. For example, it was shown that when PTL and PLRP2 followed by galactolipids. Although its phospholipase activity were incubated together with native bovine MFGs, the hydrolysis is weak, it has greater activity toward phosphatidylethanol- rate for both was higher than the sum of hydrolysis rates amine, phosphatidylglycerol, and phosphatidylcholine than measured for each individually [98]. Similar results were phosphatidylserine [86,89]. In rodents, the expression of shown when PTL and BSSL were incubated with human milk [40]. PLRP2 is highest during the suckling period and significantly reduces after weaning [82,83]. PLRP2-deficient mouse pups 3.3. Emulsification and digestion in small intestine showed fat malabsorption with large quantities of undigested fat in the stool [90], suggesting that PLRP2 plays a crucial role Phospholipids and bile salts secreted from the gallbladder are in digestion of dietary fats in infancy. Furthermore, PLRP2 structured as either small micelles (3-6 nm diameter) or vesicles lipase activity can be drastically reduced in vitro by high bile (25-130 nm diameter), depending on the ratio of bile acid to salt concentrations [86,87,89]. Therefore, in comparison to phosphatidylcholine and cholesterol in the vesicle [99]. These adults, the low luminal bile salt concentration in the infant biliary mixed micelles and vesicles become rapidly diluted when duodenum favors activity of PLRP2. It is considered that the reaching the duodenum, mixing with fats and lipolysis products contribution of PLRP2 to triglyceride hydrolysis in normal from the stomach. It is important to recognize the age-related adult conditions is low compared to infancy. The main difference in biliary lipid secretion and its potential implication physiological function of PLRP2 in adults favors the digestion on emulsification of lipids. The levels of bile salts and phospho- of galactolipids, which are the main lipids present in lipids in the gallbladder are lower in infants (from birth to vegetable foods [86,89]. 6 months of age) as compared to older infants and children (from In the small intestine, BSSL (also referred to as carboxyl 6 months to 12 years of age) [100]. ester lipase, carboxyl ester , and bile salt–dependent Bile salts are surface-active molecules that ensure the lipase), encoded by the Cel gene (EC 3.1.1.3, EC 3.1.1.13, EC removal of lipolytic products from the oil-water interface, 3.1.1.6), is activated by primary bile salts (cholate and coordinate micellar solubilization, and stabilize lipid droplets chenodeoxycholate) and has the ability to hydrolyze a broad against aggregation. As higher proportions of phospholipids range of substrates including cholesteryl esters; retinol esters; and lipolytic products (fatty acids, mono- or diacylglycerols) NUTRITION RESEARCH 83 (2020) 15– 29 21 accumulate at the surface of pre-existing emulsified lipid An increasing proportion of lipolytic products become droplets, they become highly unstable because of their emulsified in the duodenum through the weak mechanical solubility in water [101], thus favoring mixed micelle forma- mixing of the duodenal motility (a low shear force) and the tion. The actions of trypsin and chymotrypsin further presence of bile salts (reviewed in [106,107]). As the core of the hydrolyze residual MFG membrane proteins that are still emulsion droplet shrinks, lipid products appear mainly at the attached to the lipid droplet surface, producing peptides and emulsion surface of the lipid droplet, presumably as the polypeptides that are less surface active than the intact surface coat of this multilamellar liquid-crystalline structure. protein. Within a few minutes, low concentrations of bile salts are Phospholipids on the MFG membrane are not digested in sufficient to release these membrane components, including the infant stomach [102,103]. In addition, expression of phospholipids and residual membrane-bound peptides into pancreatic is likely insignificant during the lumen [108]. Short-chain and medium-chain fatty acids early infancy, as demonstrated in neonatal rodents [80]; are readily soluble in an aqueous environment. In compari- therefore, PLRP2 and BSSL are more important for hydrolyzing son, long-chain fatty acids have lower solubility in water and MFG membrane phospholipids in infants. Sphingomyelin, the thus are incorporated into mixed bile salt micelles or vesicles. most abundant polar lipid species in human MFGs [59], can be In the lumen of the small intestine, lipolytic products self- digested to ceramide by biliary secretion of alkaline assemble into various lipid-crystalline structures depending sphingomyelinase (encoded by the Enpp7 gene, EC 3.1.4.12, on their molar ratio relative to bile salts (Fig. 4). Because the reviewed in [104]). The ceramide is then further hydrolyzed by duodenal bile salt level is low immediately after a meal and BSSL to release fatty acids and sphingosine [105]. Digestion of the neonate's duodenal pH is neutral or slightly acidic [46], polar lipids at the globule surface may further destabilize the lamellar or multilamellar liquid-crystalline aggregates are lipid globule. expected to form when the concentration ratio of fatty acids

Fig. 4 – Emulsification and lipolysis in small intestine. Bile salts are surface-active molecules that are critical to facilitate emulsification of lipids by adsorbing to the droplet surface. Lipid hydrolysis in the duodenum is carried out through the action of several key lipases including pancreatic lipase‐related protein 2 (PLRP2) and bile salt‐stimulated lipase (BSSL) (also referred to as carboxyl ester lipase (CEL), carboxyl ester hydrolase (CEH), and bile salt–dependent lipase (BSDL)). Mixed micelles and vesicles transport lipolytic products, cholesterol, phospholipids, and lysophospholipids, as well as fat-soluble vitamins, across the unstirred water layer to the intestinal brush boarder prior to absorption. 22 NUTRITION RESEARCH 83 (2020) 15– 29 to monoacylglycerols exceeds their micellar solubility. If more impacts in vivo digestion is currently unknown, especially lipolytic products are produced while bile salt concentrations when the role of pH is considered. are low, unilamellar vesicles grow into large multilamellar In the duodenum, the Brunner glands and the pancreas vesicles, which are multilayer particles with a greater capacity produce alkaline secretions that serve to neutralize the acidic to accommodate lipid digestion products. When bile salts are not chyme from the stomach, so the intestinal pH gradually rises saturated with lipids (the ratio of bile salts to lipolytic products from the proximal to the distal portion of the small intestine. >1.0), the lipolysis products are spontaneously removed from the In adults, the pH of the small intestine ranges from weakly water-oil interface and liquid-crystalline phase and disperse into acidic to neutral (duodenum: 5.0-7.4; jejunum: 5.5-7.7; ileum: smaller mixed micellar products [109]. The micellar phase is 6.6-7.9 [124]), whereas in infants, the pH approaches neutral- heterogeneous in both size and composition. In addition to the ity (duodenum: 6.4; jejunum: 6.6; ileum: 6.9 [46]). The rectal pH mixed micelles, different structures of liquid-crystalline phases of healthy neonates (<28 days) is approximately 6.5 (range are expected to occur in the intestine [110]. These highly 6.3-6.7) and is only slightly more basic in infants (≥28 days dispersed colloids provide an effective transport shuttle for and <1 year) at 6.90 (range 6.68-7.12) [125]. These differ from hydrophobic species across the viscous unstirred water layer the adult rectal pH of approximately 7.9 [126]. Colonic pH is toward the brush border of enterocytes, greatly enhancing their influenced by gut microbial activity. uptake by the enterocytes (reviewed in [106,107]). Absorption of The presence of bicarbonate and higher pH leads to a the products of lipid digestion occurs through two mechanisms: higher proportion of ionized fatty acids, enhancing fatty acid passive diffusion and carrier-mediated transporter system soap formation and a higher degree of lipolysis [127,128]. (reviewed in [111,112]). Because the pKa of a single fatty acid increases with decreasing saturation and increasing chain length [129] and mixtures of long-chain fatty acids have a lower pKa than that of each acid [130], then theoretically, under the condition of – 4. Formation of calcium fatty acid soap pH neutrality in the infant jejunum, the majority of long- complexes chain free fatty acids that are not incorporated into micelles will be protonated, therefore preventing their binding with 4.1. Factors influencing fatty acid soap formation free Ca2+. Importantly, as the gut matures over time and bile salt concentrations increase, the absorption of saturated fatty On one hand, multiple nutritionally relevant cations, including acids will be significantly improved [131] and therefore less Ca2+,Mg2+,Zn2+,Fe2+,andCu2+,havebeenshowninvitroto available for soap formation. interact with ionized free fatty acids to form fatty acid soaps; however, the extent of soap formation is dependent on both type 4.2. The impact of fatty acid–calcium soap on absorption and availability of the cations and fatty acids present [113]. Because the concentration of calcium in both human milk and Upon hydrolysis, fatty acids can be absorbed throughout the milk-based formulas greatly exceeds that of the other cations, small intestine but are predominantly absorbed in the jejunum. calcium is the predominant cation involved in soap formation. In With increasing dietary fat intake, the amount of fat absorbed in human milk, the majority of calcium (~79%) is found in the the jejunum increases and a higher proportion of fat passes into soluble (whey) fraction [114-116], whereas only about half (~58%) the ileum [132,133]. Although the distal intestine is capable of fat is associated with the bovine whey fraction [119]. On the other absorption [133], the expression of fatty acid–binding protein and hand, ~6% and ~41% of calcium are found in casein micelles of fatty acid is higher in the proximal jejunum than in human and bovine milk, respectively [119], which are only the ileum [134,135]. Additionally, calcium is actively absorbed in released upon protein digestion [117]. the duodenum and proximal jejunum and passively absorbed As lipase adsorbs to the oil-water interface and hydrolyzes throughout the length of the colon [136]. In contrast, the majority triglycerides, short- to medium-chain fatty acids are removed of insoluble fatty acid soap formation occurs in the distal small because of their greater water solubility relative to long-chain intestine, with negligible formation occurring in the stomach or fatty acids (shown in vitro)[118]. However, ionized long-chain colon [113,137](Fig. 5). As such, the formation of insoluble fatty fatty acids will accumulate at the interface and can complex with acid–calcium soaps in the intestinal tract likely has a negligible local free Ca2+ in a 1:2 molar ratio to form a layer of crystalline impact on calcium and lipid absorption in healthy breastfed calcium–fattyacidsoapattheoil-waterinterface[119]. When bile infants. salt concentration is low, the accumulation of lipolytic products at the surface of the oil droplet can slow down the activity of lipase [120], thereby limiting further digestion. Bile salts are important for helping to solubilize the liberated fatty acids, but 5. Bidirectional interaction between gut given that bile salts are significantly lower in the neonate, the microbiota and infant lipid digestion formation of calcium–fatty acid soap complexes is favored. Bile salts have been shown to disrupt the formation of a In humans, the microbiota differs throughout the various fatty acid soap network [119]. In vitro studies suggest that the regions of the [138-140], and animal presence of calcium can increase lipolysis through the studies have indicated that the microbial profile of the formation of calcium–fatty acid soaps [119,121-123] but only jejunum appears to be more similar to the ileum [141], when there are insufficient emulsifying reagents such as bile whereas the colon is more similar to feces [142,143]. There- salts [122]. However, the extent to which this interaction fore, observing changes in fecal microbiota under different fat NUTRITION RESEARCH 83 (2020) 15– 29 23

Fig. 5 – pH of intestinal content and fatty acid soap formed throughout the gastrointestinal tract of growing male piglets (n = 8) after receiving a tallow-based diet for 9 days. pH and digesta throughout the gastrointestinal tract were obtained from each piglet immediately following anesthesia and euthanasia. Fecal values were evaluated from a pooled sample consisting of the last 3 days. The tallow-based diet contained a total of 109 g fat/kg, 25.9 g palmitic acid/kg, 26.4 g stearic acid/kg, and 27.9 g oleic acid/kg. Values are extracted from chapter 6 of Natascha Stroebinger's thesis [113] and expressed in means ± SEM.

intake might not be applicable to support any direct influence microbial density and activity. Although data are limited, the on small intestinal function. presence of Streptococcus and Bifidobacterium in the infant In analyzing a reference genome consisting of all available ileum [149] may contribute to acetic acid and lactic acid bacterial genomes associated with the gastrointestinal tract, production [150,151], which would decrease the intraluminal 53% of microbes were found to encode genes to be equally pH, possibly disfavoring the formation of calcium–fatty acid specialized in saccharolytic, proteolytic, and lipolytic activi- soaps. Acetic acid and lactic acid can also act as intracellular ties. It is uncommon to find a group of microbes with signaling molecules that differently regulate enterocyte lipid predominately lipolytic activities, although several absorption, storage, and oxidation [152]. Proteobacteria and Eubacterium spp were noted to have high Gut microbiota can influence lipid digestion by altering lipolytic potential [144]. Metagenomic analyses of microbes host gene expression. It has been shown that germ-free mice from samples obtained from the adult duodenum reveal that have severely impaired lipid digestion and absorption with a smaller proportion of functional genes may be assigned to elevated fecal lipid excretion compared with conventionally (2.38%) in comparison to raised mice [153,154]. In comparison to germ-free mice, mice (7.65%) and amino acid (13.26%) metabolism. Several micro- colonized with Bacteroides thetaiotaomicron showed elevated bial genes encode for enzymes that are associated with gene expression of PLRP-2, colipase, liver-type fatty acid– glycerophospholipid metabolism, as well as fatty acid degra- binding protein, and apolipoprotein A-IV in the ileum [155]. dation and synthesis, including outer membrane phospholi- Intestinal gene expression of angiopoietin-like protein 4 pase A1, acyl-CoA dehydrogenase, acyl-CoA dehydrogenase, (Angptl4) was 2.5-fold higher in pigs fed a milk-based diet and enoyl-[acyl carrier protein] reductase [140]. Similarly, than a control diet [156]. Angptl4 may be part of a negative metagenomics profiles of ileostomy effluent reveal genes feedback mechanism to protect against lipotoxicity [157], as coding for fatty acid/phospholipid biosynthesis enzymes, its activity reduces the activity of PTL and increases fat acyl-coenzyme A synthetases, and outer membrane phos- excretion in stool [158]. Immediately after weaning, conven- pholipase A [141]. tionally raised mice showed lower expression of Angptl4 in Through in vitro culture of human stool and comparison the small intestine compared to their germ-free counterparts [ between germ-free and conventionally raised mice, a few 159,160]. Butyrate and butyrate-producing bacteria can also intestinal bacteria, including a few Bifidobacterium and Lacto- increase expression of Angptl4 in the small intestine in a bacillus species, have demonstrated the capability to metab- PPAR-γ–independent manner [160]. Finally, the microbiota olize linoleic acid to conjugated linoleic acids, which can has been shown to contribute to the diurnal variation in lipid subsequently be converted to vaccenic acid and then stearic absorption, as well as other nutrients, by altering the acid [145-148]. This specific activity might occur more rapidly expression of histone deacetylase 3 [161]. The mechanism by in the colon than in small intestine, as the colon has higher which this may occur in infancy is not completely 24 NUTRITION RESEARCH 83 (2020) 15– 29 understood. Further study is warranted to understand how understanding of how native MFGs from the mammary gland are different microbial taxa and microbial by-products differen- transformed to lipolytic products that are readily absorbed by tially or additively affect host lipid digestion and absorption. enterocytes and used by infants. Understanding the physiology of the digestive process in infants is the foundation to startunravelling the interactions 6. Perspectives on infant formula and dairy between lipid digestion and the early microbiota during this products critical period of development. Studies in swine models have indicated that the primary source of small intestinal coloni- Dairy processing techniques such as homogenization, heat zation was mother's milk [143] and that the colonization treatment, and microfiltration, as well as chemical and pattern differed between sow-fed and formula-fed piglets [ enzymatic reactions from the cheese-making process, can 171]. Germ-free mice have severely impaired lipid digestion alter the size and destroy the native structure of MFGs. As a and absorption with elevated fecal lipid excretion, compared result, new and less stable fat globules are formed, where with conventionally raised mice [153]. When colonized with a thermally denatured and fragmented whey, casein, or MFG high-fat diet–induced jejunal microbiota, mice exhibited membrane proteins are adsorbed onto the lipid droplet increased lipid absorption on both high-fat and low-fat diets interface (reviewed in [162-164]). [153]. This provides several interesting avenues of future To mimic the fat composition of breast milk, fat in infant research, including a determination of which microbial formulas is provided through homogenization of vegetable species are contributing to lipid digestion in infants and the oils with added emulsifying agents, such as whey protein associated mechanisms. Additional research is also needed to isolate, whey protein hydrolysate, maltodextrin, citric acid determine whether any products of infant lipid digestion esters of mono- and di-, or soybean lecithin [165]. direct small intestinal colonization and whether any contri- Spray-drying can cause protein aggregation, breaking, and butions are dependent on fat source (human milk vs formula) coalescence of oil globules. The resultant lipid particle is and any subsequent processing (freezing, pasteurization, etc). much smaller than MFG from human milk and has a different surface composition depending on its formulation and the processing conditions [164-166]. The impact of dairy process- Acknowledgment ing is also expected to influence the interfacial structure and composition of lipid droplets in infant formula. One conse- The authors declare that they have no conflicts of interest quence is the reduced rate of lipolysis, which may be due with the contents of this article. This project was supported to the lower accessibility of gastric lipase by accumulation by the USDA National Institute of Food and Agriculture Hatch of a denatured milk protein barrier on the lipid droplet surface Project 1021411 and funds from the Kinsella Endowed Chair in [167,168], as well as the lack of BSSL present in human milk. Food, Nutrition, and Health awarded to CMS. 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