Effects of sn-2 Palmitic Triacylglycerols and the Ratio of OPL to OPO in Human Milk Fat Substitute on Metabolic Regulation in Sprague-Dawley Rats.

In this study, the influence of total sn-2 palmitic triacylglycerols (TAGs) and ratio of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) to 1,3-dioleoyl-2-palmitoylglycerol (OPO) in human milk fat substitute (HMFS) on the metabolic changes were investigated in Sprague-Dawley rats. Metabolomics and lipidomics profiling analysis indicated that increasing the total sn-2 palmitic TAGs and OPL to OPO ratio in HMFS could significantly influence glycine, serine and threonine metabolism, glycerophospholipid metabolism, glycerolipid metabolism, sphingolipid metabolism, bile acid biosynthesis, and taurine and hypotaurine metabolism pathways in rats after 4 weeks of feeding, which were mainly related to lipid, bile acid and energy metabolism. Meanwhile, the up-regulation of taurine, L-tryptophan, and L-cysteine, and down-regulations of lysoPC (18:0) and hypoxanthine would contribute to the reduction in inflammatory response and oxidative stress, and improvement of immunity function in rats. In addition, analysis of targeted biochemical factors also revealed that HMFS-fed rats had significantly increased levels of anti-inflammatory factor (IL-4), immunoglobulin A (IgA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px), and decreased levels of pro-inflammatory factors (IL-6 and TNF-α) and malondialdehyde (MDA), compared with those of the control fat-fed rats. Collectively, these observations present new in vivo nutritional evidence for the metabolic regulatory effects of the TAG structure and composition of human milk fat substitutes on the host.

Novel Human Milk Fat Substitutes Based on Medium- and Long-Chain Triacylglycerol Regulate Thermogenesis, Lipid Metabolism, and Gut Microbiota Diversity in C57BL/6J Mice.

Human milk is naturally rich in medium- and long-chain triacylglycerols (MLCT), accounting for approximately 30% of the total fat. However, infant formula fat is prepared using a physical blend of vegetable oils, which rarely contains MLCT, similar to human milk. The differences in MLCT between human milk and infant formulas may cause different lipid metabolisms and physiological effects on infants, which are unknown. This study aimed to analyze the metabolic characteristics of formula lipid containing novel human milk fat substitutes based on MLCT (FL-MLCT) and compare their effects with those of the physical blend of vegetable oils (FL-PB) on lipid metabolism and gut microbiota in mice. Compared with the FL-PB group, the FL-MLCT group showed increased energy expenditure, decreased serum triacylglycerol level, and significantly lower aspartate aminotransferase level, epididymal and perirenal fat weight, and adipocyte size. Moreover, the abundances of Firmicutes/Bacteroidota, Actinobacteriota, and Desulfovibrionaceae were significantly decreased in the FL-MLCT group. Novel human milk fat substitutes MLCT could inhibit visceral fat accumulation, improve liver function, and modulate the mice gut microbiota composition, which may contribute to controlling obesity.

Total Sn-2 Palmitic Triacylglycerols and the Ratio of OPL to OPO in Human Milk Fat Substitute Modulated Bile Acid Metabolism and Intestinal Microbiota Composition in Rats.

In this study, the impact of sn-2 palmitic triacyclglycerols (TAGs) in combination with their ratio of two major TAGs (1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) to 1,3-dioleoyl-2-palmitoylglycerol (OPO)) in human milk fat substitute (HMFS) on bile acid (BA) metabolism and intestinal microbiota composition was investigated in newly-weaned Sprague-Dawley rats after four weeks of high-fat feeding. Compared to those of control group rats, HMFS-fed rats had significantly increased contents of six hepatic primary BAs (CDCA, αMCA, ßMCA, TCDCA, TαMCA and TßMCA), four ileal primary BAs (UDCA, TCA, TCDCA and TUDCA) and three secondary BAs (DCA, LCA and ωMCA), especially for the HMFS with the highest sn-2 palmitic acid TAGs of 57.9% and OPL to OPO ratio of 1.4. Meanwhile, the inhibition of ileal FXR-FGF15 and activation of TGR5-GLP-1 signaling pathways in HMFS-fed rats were accompanied by the increased levels of enzymes involved in BA synthesis (CYP7A1, CYP27A1 and CYP7B1) in the liver and two key thermogenic proteins (PGC1α and UCP1) in perirenal adipose tissue, respectively. Moreover, increasing sn-2 palmitic TAGs and OPL to OPO ratio in HMFS also altered the microbiota composition both on the phylum and genus level in rats, predominantly microbes associated with bile-salt hydrolase activity, short-chain fatty acid production and reduced obesity risk, which suggested a beneficial effect on host microbial ecosystem. These observations provided important nutritional evidence for developing new HMFS products for infants.

Comparison and enrichment of sn-2 palmitoyl triacylglycerols (OPO/OPL) in fish oil for its potential application as human milk fat substitutes.

Triacylglycerols (TAG) are differences in fatty acid distributions between infant formula and human milk. In this study, fish oil (Tilapia, Golden pompano, Tiger grouper, and Basa) showed the potential as the source of human milk fat substitutes by comparing TAG profiles with infant formula and human milk. The total lipids and TAG of fish were concentrated in the by-products of fish (head and viscera) and contained high levels of palmitic acid, oleic acid, and linoleic acid. Compared with infant formula, fish oil was closer to human milk in sn-2 fatty acid distribution, and sn-2 palmitic acid level in fish oil exceeded 52 % of total palmitic acid, Golden pompano head was the highest (64.46 %). Further research showed that the content of sn-2 palmitoyl TAG (OPO and OPL dominated) increased from 157.16 mg/g TAG to 305.18 mg/g TAG by isopropanol enrichment (solid-liquid ratio: 1:4, temperature: -12 °C, time: 4 h).

Progress and perspectives of enzymatic preparation of human milk fat substitutes.

Human milk fat substitutes (HMFS) with triacylglycerol profiles highly similar to those of human milk fat (HMF) play a crucial role in ensuring the supply in infant nutrition. The synthesis of HMFS as the source of lipids in infant formula has been drawing increasing interest in recent years, since the rate of breastfeeding is getting lower. Due to the mild reaction conditions and the exceptionally high selectivity of enzymes, lipase-mediated HMFS preparation is preferred over chemical catalysis especially for the production of lipids with desired nutritional and functional properties. In this article, recent researches regarding enzymatic production of HMFS are reviewed and specific attention is paid to different enzymatic synthetic route, such as one-step strategy, two-step catalysis and multi-step processes. The key factors influencing enzymatic preparation of HMFS including the specificities of lipase, acyl migration as well as solvent and water activity are presented. This review also highlights the challenges and opportunities for further development of HMFS through enzyme-mediated acylation reactions.

Chemical characterization and solvent fractionation of tilapia oil for its potential application as human milk fat substitute.

The natural source of human milk fat substitute (HMFS) is a field worth exploring. In this study, tilapia oil was extracted and analyzed. In the triacylglycerol fraction, the contents of sn-2 palmitic acid and total sn-1,3 oleic acid and linoleic acid were 48.01% and 66.62%, respectively. The optimal solvent fractionation conditions were determined to be a tilapia oil-to-acetone ratio of 1:8 (w/v), crystallization temperature of -30°C, and crystallization duration of 16 h, giving a solid fraction yield of 64.20%. In fractionated tilapia oil, the total content of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) and 1,3-dioleoyl-2-palmitoylglycerol (OPO) increased by 20.38%, as determined by reversed-phase liquid chromatography. Ultra-high-performance combined-phase chromatography combined with quadrupole time-of-flight mass spectrometry analysis showed that OPL (17.45%) was the most abundant triacylglycerol in fractionated tilapia oil, followed by OPO (13.90%). Fractionated tilapia oil is thus an excellent source of OPL and has great potential for incorporation in HMFS. PRACTICAL APPLICATION: Human milk fat substitutes are an important component of infant formulas. This work provides an excellent natural source of oil rich in OPL, which has great potential in the field of preparing human milk fat substitutes highly similar to human milk fat.

Synthesis of human milk fat substitutes based on enzymatic preparation of low erucic acid acyl-donors from rapeseed oil.

Rapeseed oil has a similar oleic acid/linoleic acid ratio to human milk fat (HMF). However, it can hardly be used for human milk fat substitute (HMFS) synthesis due to high erucic acid content. In this study, Candida cylindracea lipase (CCL) was found to strongly discriminate against erucic acid. Free fatty acids containing low erucic acid and high oleic acid and linoleic acid were prepared from rapeseed oil hydrolysis catalyzed by CCL. The erucic acid content was only 1.58% (initial 8.70%), when the degree of hydrolysis reached 79.58%. The free fatty acids were used as acyl-donors in the acidolysis catalyzed by Novozym 40086. Considering acyl incorporation and migration, the optimum conditions were 1:8 (tripalmitin to acyl-donors), 40 °C and 2 h. The erucic acid content dropped to 0.97% in the HMFS. According to the Q-TOF-MS analysis, the HMFS was rich in 1,3-dioleoyl-2-palmitoyl-glycerol (18.20%) and 1-oleoyl-2-palmitoyl-3-linoleoyl-glycerol (17.96%), which was similar to HMF.

The Enzymatic Preparation of Human Milk Fat Substitute Intermediate Rich in Palmitic Acid at sn-2 Position and Low-Unsaturated Fatty Acids at sn-1(3) Positions from Palm Oil Substrate.

The lipid products that consist of structured lipids rich in palmitic acid (16:0) at the sn-2 position of triacylglycerol (TAG) and rich in low-unsaturated fatty acids (FAs) (LUFAs), such as oleic acid; 18:1 and linoleic acid; 18:2 at the sn-1(3) positions, are useful intermediates for manufacturing human milk fat substitute (HMFS), which contains functional lipid components. In this study, the HMFS intermediate (HMFS-IM) was enzymatically prepared from palm oil without using other oil sources. First, the amount of 16:0 at the sn-2 position of TAG substrate was enhanced from 18.9% to more 34.5% via a random esterification reaction using a non-stereospecific lipase, Novozym® 435, to produce a random-palm substrate. Consequently, 2-monoacylglycerol (2-MAG) rich in 16:0 at the sn-2 position over 88%, together with the FA ethyl ester substrates rich in LUFAs, such as 18:1-Et and 18:2-Et above 93.5% was prepared through ethanolysis reaction using the same lipase from the random-palm substrate and by purification with urea complexation, respectively. As the preferred modified method, a continuous use of the same lipase to these reactions were achieved while reducing the usage of enzyme to half. Finally, an HMFS-IM rich in 16:0 at the sn-2 position more than 60% and LUFA at sn-1(3) positions was prepared using these palm oil-based products, including random-palm, palm-Et, and 2-MAG, via the interesterification reaction using a 1,3-stereospecific lipase, Lipozyme® RM-IM. Thus, HMFS-IM was successfully prepared by palm oil materials with a 65 wt% usage ratio. The concept described in this study will be useful for HMFS manufacturing from a single natural oil substrate, which is not initially rich in 16:0 at the sn-2 position.

Enzymatic Synthesis of Human Milk Fat Substitute - A Review on Technological Approaches.

Human milk fat substitute (HMFS) is a structured lipid designed to resemble human milk fat. It contains 60-70% palmitic acid at the sn-2 position and unsaturated fatty acids at the sn-1,3 positions in triacylglycerol structures. HMFS is synthesized by the enzymatic interesterification of vegetable oils, animal fats or a blend of oils. The efficiency of HMFS synthesis can be enhanced through the selection of appropriate substrates, enzymes and reaction methods. This review focuses on the synthesis of HMFS by lipase-catalyzed interesterification and provides a detailed overview of biocatalysts, substrates, synthesis methods, factors influencing the synthesis and purification process for HMFS production. Major challenges and future research in the synthesis of HMFS are also discussed. This review can be used as an information for developing future strategies in producing HMFS.

Enzymatic Preparation and Oxidative Stability of Human Milk Fat Substitute Containing Polyunsaturated Fatty Acid Located at sn-2 Position.

The development of human milk fat substitutes (HMFSs), rich in palmitic acid (16:0) at the sn-2 position of triacylglycerol (TAG) and rich in unsaturated fatty acids (FAs) (oleic acid, 18:1 and linoleic acid, 18:2) at the sn-1(3) positions, has gained popularity. In this study, HMFSs containing polyunsaturated fatty acids (PUFAs) predominantly at the sn-2 position were prepared, and their oxidation stabilities were compared. First, a non-PUFA-containing HMFS (NP-HMFS) was produced by enzymatic reactions using Novozyme® 435 and Lipozyme® RM-IM as the enzymes and lard as the raw material. Second, HMFSs, containing 10 % PUFA at the sn-2 or sn-1(3) position, were individually prepared by enzymatic reactions using lard and fish oil as raw materials. Here, sn-2-PUFA-monoacylglycerol (MAG) was extracted from the reaction solution using a mixture of hexane and ethanol/water (70:30, v/v) to produce high-purity sn-2-PUFA-MAG with 78.1 % yield. For the PUFA-containing HMFS substrates, comparable oxidation stability was confirmed by an auto-oxidation test. Finally, HMFSs containing 10 % or 2 % sn-1,3-18:1-sn-2-PUFA-TAG species were prepared by enzymatic reactions and subsequent physical blending. The oxidative stability of sn-1,3-18:1-sn-2-PUFA-HMFS was two-fold higher than that of 1/2/3-PUFA-HMFS in which each PUFA was located without stereospecific limitations in TAG. The removal of PUFA-TAG molecular species with higher concentrations of unsaturated units had a significant effect. In addition, the oxidation stability increased with the addition of tocopherol as an antioxidant. Thus, the combined use of two strategies, that is, the removal of PUFA-TAG molecular species with high concentrations of unsaturated units and the addition of antioxidants, would provide a PUFA-containing HMFS substrate with high oxidative stability.

Facile and Green Production of Human Milk Fat Substitute through Rhodococcus opacus Fermentation.

Human milk fat substitute (HMFS) is a class of structured lipids widely used in infant formulas. Herein, HMFS was prepared by Rhodococcus opacus fermentation. The substrate oils suitable for HMFS production were coconut oil (66.1-57.5%), soybean oil (17.5-26.5%), high oleic acid sunflower oil (5.4-4.5%), Antarctic krill oil (9-9.5%), and fungal oil (2%). Six HMFSs were prepared, among which HMFS V and VI were similar to human milk fat from Chinese in terms of fatty acid composition and triacylglycerol species. The sn-2 position of HMFS was occupied by palmitic acid (49.31 and 43.48% in HMFS V and VI, respectively). The major triacylglycerols were OPL, OPO, and LPL, accounting for 15.90, 9.49, and 6.84 and 17.52, 8.44, and 8.55% in HMFS V and VI, respectively. This study is the first to prepare structured lipids intended for infant formula through fermentation, providing a novel strategy for the edible oil industry.

Carbon source modify lipids composition of Rhodococcus opacus intended for infant formula.

Human milk fat substitutes (HMFSs) are the structured lipids intended for infant formula. It provides energy and essential fatty acid for infant. HMFSs are mainly prepared by enzymatic method. In this study, we aim to explore the potential for producing HMFSs by fermentation using R. opacus. The results indicated that different compounds with chain length from 12 to 18, used as carbon source, could be incorporated into triacylglycerols directly. Polyunsaturated fatty acids in term of ARA, EPA, DHA could enter the kennedy pathway directly and involved in the biosynthesis of triacylglycerols. GC, UPLC-MS and 13C-NMR analysis demonstrated that typical structured lipids ß-OPL (40.09%) was synthesized in R. opacus. Transcriptome analysis revealed that ß-oxidation, fatty acid elongation and kennedy pathways existed in R. opacus. It was concluded that fatty acid supplied as carbon source could enter the kennedy pathways directly or via the de novo fatty acid biosynthesis pathway depending on the chain length, thus, affect the triacylglycerol species formed in the Rhodococcus opacus.

Optimization of Microencapsulation of Human Milk Fat Substitute by Response Surface Methodology.

Human milk fat substitutes (HMFS) are rich in polyunsaturated fatty acids which upon microencapsulation, can be used as a source of high quality lipids in infant formula. The response surface methodology (RSM) was employed to optimize the microencapsulation condition of HMFS as a functional product. The microencapsulation efficiency (MEE) of microencapsulated HMFS was investigated with respect to four variables including concentration of soy lecithin (A), ratio of demineralized whey powder to malt dextrin (B), HFMS concentration (C), and homogenizing pressure (D). The optimum conditions for efficient microencapsulation of HMFS by the spray drying technique were determined as follows: the amount of soybean lecithin-0.96%, ratio of desalted whey powder to malt dextrin-2.04:1, oil content-17.37% and homogeneous pressure-0.46MPa. Under these conditions, the MEE was 84.72%, and the basic indices of the microcapsules were good. The structure of the microcapsules, as observed by scanning electron microscopy (SEM), revealed spherical, smooth-surfaced capsules with diameters ranging between 10-50 µm. Compared with HFMS, the peroxide value (POV) and acid value (AV) of the microcapsule were significantly lower during storage indicating that the microencapsulation process increases stability and shelf life. Infrared spectroscopic analyses indicated that HFMS had the same characteristic functional groups as the oil extracted from microcapsules. Simulated in vitro digestion revealed that the microcapsules were digested completely within 2h with maximum lipid absorption rate of 64%. Furthermore, these results advocate the embedding process of HFMS by RSM due to its efficacy.

Lipozyme RM IM-catalyzed acidolysis of Cinnamomum camphora seed oil with oleic acid to produce human milk fat substitutes enriched in medium-chain fatty acids.

In the present study, a human milk fat substitute (HMFS) enriched in medium-chain fatty acids (MCFAs) was synthesized through acidolysis reaction from Cinnamomum camphora seed oil (CCSO) with oleic acid in a solvent-free system. A commercial immobilized lipase, Lipozyme RM IM, from Rhizomucor miehei, was facilitated as a biocatalyst. Effects of different reaction conditions, including substrate molar ratio, enzyme concentration, reaction temperature, and reaction time were investigated using response surface methodology (RSM) to obtain the optimal oleic acid incorporation. After optimization, results showed that the maximal incorporation of oleic acid into HMFS was 59.68%. Compared with CCSO, medium-chain fatty acids at the sn-2 position of HMFS accounted for >70%, whereas oleic acid was occupied predominantly at the sn-1,3 position (78.69%). Meanwhile, triacylglycerol (TAG) components of OCO (23.93%), CCO (14.94%), LaCO (13.58%), OLaO (12.66%), and OOO (11.13%) were determined as the major TAG species in HMFS. The final optimal reaction conditions were carried out as follows: substrate molar ratio (oleic acid/CCSO), 5:1; enzyme concentration, 12.5% (w/w total reactants); reaction temperature, 60 °C; and reaction time, 28 h. The reusability of Lipozyme RM IM in the acidolysis reaction was also evaluated, and it was found that it could be reused up to 9 times without significant loss of activities. Urea inclusion method was used to separate and purify the synthetic product. As the ratio of HMFS/urea increased to 1:2, the acid value lowered to the minimum. In a scale-up experiment, the contents of TAG and total tocopherols in HMFS (modified CCSO) were 77.28% and 12.27 mg/100 g, respectively. All of the physicochemical indices of purified product were within food standards. Therefore, such a MCFA-enriched HMFS produced by using the acidolysis method might have potential application in the infant formula industry.

The effect of fatty acid positioning in dietary triacylglycerols and intake of long-chain n-3 polyunsaturated fatty acids on bone mineral accretion in growing piglets.

Long-chain n-3 PUFA (LCPUFA) and palmitate (16:0) positioning in the triacylglycerol (TAG) of infant formula may affect calcium-uptake which could affect bone health. We investigated if a human milk fat substitute (HMFS) with a modified TAG structure holding 16:0 predominantly in the sn-2-position compared with a control (CONT) and if increasing n-3LCPUFA intake giving fish oil (FO) compared with sunflower oil (SO) would affect bone parameters in piglets in two sets of controlled 14d-interventions (n=12/group). We assessed this by dual-energy x-ray absorptiometry, and ex vivo peripheral quantitative computed tomography and mechanical strength. Bone mineral content (BMC) was higher in the FO compared to the SO-group (p=0.03). Despite similar weight gain in HMFS- and CONT-groups, body fat accumulation was higher with HMFS (p<0.001), and BMC, bone area (BA) and cortical BA in femur were lower (p=0.002, p=0.005, and p=0.02, respectively), indicating importance of both n-3LCPUFA and 16:0 TAG-positioning in infant formulas.