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The bioaccessibility and bioavailability of dietary fatty acids depend on the lipid to which they are esterified, the organisation of theses lipids in water and their recognition by lipolytic enzymes. In this work, we studied the release of marine long-chain polyunsaturated fatty acids (LC-PUFA), depending on their presentation either in the form of phospholipids (PL) or triacylglycerol (TAG). Two formulations based on marine PL or TAG extracted from salmon heads ( Salmo salar ) were prepared. Lipolysis was first tested in vitro by using individual gastrointestinal lipases and phospholipases to identify the enzymes involved in the digestion. Second, the lipolysis of the prepared formulations by a combination of enzymes was tested under in vitro conditions mimicking the physiological conditions found in the GI tract, both in the stomach and in the upper small intestine, in order to evaluate digestibility of TAG and LC-PUFA-containing liposomes. The in vitro results showed that TAG emulsion was hydrolyzed by porcine pancreatic extracts (PPE) and pure pancreatic lipase (PPL) with its cofactor, colipase, and to a lesser extent by pancreatic-lipase-related protein 2 (PLRP2) and a gastric extract (RGE) containing gastric lipase while no hydrolysis was observed with purified pancreatic phospholipase A2 (PLA2) and carboxyl ester hydrolase (CEH). The PL substrate was found to be hydrolysed by PLA2, PPE and PLRP2. Their phospholipase activities on liposomes formulation was dependent on the presence of bile salts. Using a two-step in vitro digestion model, we measured the kinetics of fatty acid release from TAG and PL during the gastric and intestinal phases of digestion. The highest overall lipolysis level was obtained with liposomes (around 75%) during the intestinal phase while they were preserved during the gastric phase. The overall lipolysis level of TAG emulsion was lower (around 33%), while it started already in the gastric phase. In conclusion, liposomes appear as a better delivery system for intestinal absorption of LC-PUFA than TAG. In addition, their resistance to lipolysis under gastric condition can protect LC-PUFA and provide a gastric stable delivery system for other molecules.

The use of Nuclear Magnetic Resonance spectroscopy for studying lipid digestion in vitro most often consists of quantifying lipolysis products after they have been extracted from the reaction medium using organic solvents. However, the current sensitivity level of NMR spectrometers makes possible to avoid the extraction step and continuously quantify the lipids directly in the reaction medium. We used real-time 1 H NMR spectroscopy and guinea pig pancreatic lipase-related protein 2 (GPLRP2) as biocatalyst to monitor in situ the lipolysis of monogalactosyl diacylglycerol (MGDG) in the form of mixed micelles with the bile salt sodium taurodeoxycholate (NaTDC). Residual substrate and lipolysis products (monogalactosyl monoacylglycerol (MGMG); monogalactosylglycerol (MGG) and octanoic acid (OA) were simultaneously quantified throughout the reaction thanks to specific proton resonances. Lipolysis was complete with the release of all MGDG fatty acids. These results were confirmed by thin layer chromatography (TLC) and densitometry after lipid extraction at different reaction times. Using diffusion-ordered NMR spectroscopy (DOSY), we could also estimate the diffusion coefficients of all the reaction compounds and deduce the hydrodynamic radius of the lipid aggregates in which they were present. It was shown that MGDG-NaTDC mixed micelles with an initial hydrodynamic radius r H of 7.3 ± 0.5 nm were changed into smaller micelles of NaTDC-MGDG-MGMG of 2.3 ± 0.5 nm in the course of the lipolysis reaction, and finally into NaTDC-OA mixed micelles (r H of 2.9 ± 0.5 nm) and water soluble MGG. These results provide a better understanding of the digestion of galactolipids by PLRP2, a process that leads to the complete micellar solubilisation of their fatty acids and renders their intestinal absorption possible., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pancreatic lipase related-protein 2 (PLRP2) exhibits remarkable galactolipase and phospholipase A1 activities, which depend greatly on the supramolecular organization of the substrates and the presence of surfactant molecules such as bile salts. The objective of the study was to understand the modulation of the adsorption mechanisms and enzymatic activity of Guinea pig PLRP2 (gPLRP2), by the physical environment of the enzyme and the physical state of its substrate. Langmuir monolayers were used to reproduce homogeneous and heterogeneous photosynthetic model membranes containing galactolipids (GL), and/or phospholipids (PL), and/or phytosterols (pS), presenting uncharged or charged interfaces. The same lipid mixtures were also used to form micrometric liposomes, and their gPLRP2 catalyzed digestion kinetics were investigated in presence or in absence of bile salts (NaTDC) during static in vitro, so called "bulk", digestion. The enzymatic activity of gPLRP2 onto the galactolipid-based monolayers was characterized with an optimum activity at 15 mN/m, in the absence of bile salts. gPLRP2 showed enhanced adsorption onto biomimetic model monolayer containing negatively charged lipids. However, the compositional complexity in the heterogeneous uncharged model systems induced a lag phase before the initiation of lipolysis. In bulk, no enzymatic activity could be demonstrated on GL-based liposomes in the absence of bile salts, probably due to the high lateral pressure of the lipid bilayers. In the presence of NaTDC (4 mM), however, gPLRP2 showed both high galactolipase and moderate phospholipase A1 activities on liposomes, probably due to a decrease in packing and lateral pressure upon NaTDC adsorption, and subsequent disruption of liposomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Galactolipids are the main lipids from plant photosynthetic membranes and they can be digested by pancreatic lipase related protein 2 (PLRP2), an enzyme found in the pancreatic secretion in many animal species. Here, we used transmission Fourier-transform infrared spectroscopy (FTIR) to monitor continuously the hydrolysis of galactolipids by PLRP2, in situ and in real time. The method was first developed with a model substrate, a synthetic monogalactosyl diacylglycerol with 8-carbon acyl chains (C8-MGDG), in the form of mixed micelles with a bile salt, sodium taurodeoxycholate (NaTDC). The concentrations of the residual substrate and reaction products (monogalactosylmonoglyceride, MGMG; monogalactosylglycerol, MGG; octanoic acid) were estimated from the carbonyl and carboxylate vibration bands after calibration with reference standards. The results were confirmed by thin layer chromatography analysis (TLC) and specific staining of galactosylated compounds with thymol and sulfuric acid. The method was then applied to the lipolysis of more complex substrates, a natural extract of MGDG with long acyl chains, micellized with NaTDC, and intact chloroplasts isolated from spinach leaves. After a calibration performed with α-linolenic acid, the main fatty acid (FA) found in plant galactolipids, FTIR allowed quantitative measurement of chloroplast lipolysis by PLRP2. A full release of FA from membrane galactolipids was observed, that was not dependent on the presence of bile salts. Nevertheless, the evolution of amide vibration band in FTIR spectra suggested the interaction of membrane proteins with NaTDC and lipolysis products., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Most lipids in our diet come under the form of triacylglycerols that are often redispersed and stabilized by surfactants in processed foods. In plant however, lipid assemblies constitute interesting sources of natural bioactive and functional ingredients. In most photosynthetic sources, polar lipids rich in ω3 fatty acids are concentrated. The objective of this review is to summarize all the knowledge about the physico-chemical composition, digestive behavior and oxidative stability of plant polar lipid assemblies to emphasize their potential as functional ingredients in human diet and their potentialities to substitute artificial surfactants/antioxidants. The specific composition of plant membrane assemblies is detailed, including plasma membranes, oil bodies, and chloroplast; emphasizing its concentration in phospholipids, galactolipids, peculiar proteins, and phenolic compounds. These molecular species are hydrolyzed by specific digestive enzymes in the human gastrointestinal tract and reduced the hydrolysis of triacylglycerols and their subsequent absorption. Galactolipids specifically can activate ileal break and intrinsically present an antioxidant (AO) activity and metal chelating activity. In addition, their natural association with phenolic compounds and their physical state (Lα state of digalactosyldiacylglycerols) in membrane assemblies can enhance their stability to oxidation. All these elements make plant membrane molecules and assemblies very promising components with a wide range of potential applications to vectorize ω3 polyunsaturated fatty acids, and equilibrate human diet.

The specific activities of gastric and pancreatic lipases were measured using triacylglycerols (TAG) from rapeseed oil, purified 1,3-sn-DAG and 1,2(2,3)-sn-DAG produced from this oil, as well as a rapeseed oil enriched with 40% w/w DAG (DAGOIL). Gastric lipase was more active on 1,3-sn-DAG than on 1,2(2,3)-sn-DAG and TAG, whereas pancreatic lipase displayed a reverse selectivity with a higher activity on TAG than on DAG taken as initial substrates. However, in both cases, the highest activities were displayed on DAGOIL. These findings show that DAG mixed with TAG, such as in the course of digestion, is a better substrate for lipases than TAG. The same rapeseed oil acylglycerols were used to investigate intestinal fat absorption in rats with mesenteric lymph duct cannulation. The levels of TAG synthesized in the intestine and total fatty acid concentration in lymph were not different when the rats were fed identical amounts of rapeseed oil TAG, 1,2(2,3)-sn-DAG, 1,3-sn-DAG or DAGOIL. Since the lipolysis of 1,3-sn-DAG by digestive lipases leads to glycerol and not 2-sn-monoacylglycerol (2-sn-MAG) like TAG lipolysis, these results suggest that the re-synthesis of TAG in the enterocytes can entirely occur through the "glycerol-3-phosphate (G3P)" pathway, with the same efficiency as the 2-sn-MAG pathway predominantly involved in the intestinal fat absorption. These findings shed new light on the role played by DAG as intermediate lipolysis products. Depending on their structure, 1,2(2,3)-sn-DAG versus 1,3-sn-DAG, DAG may control the pathway (2-sn-MAG or G3P) by which TAG are re-synthesized in the enterocytes., Competing Interests: Declaration of competing interest We have no conflict of interest to declare., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The rapid and preferential adsorption of a gastric lipase recombinant dog gastric lipase (rDGL) in heterogeneous films of phospholipids and triacylglycerols has previously been unveiled using Langmuir films analyzed by tensiometry, ellipsometry and Langmuir-Blodgett transfer coupled to atomic force microscopy. Here we invest the adsorption behavior of rDGL in heterogeneous galactolipid and mixed galactolipid-phospholipid or galactolipid-phospholipid-phytosterol films representative of plant membrane. Again rDGL, preferentially got adsorbed at the expanded lipid phases of the films underlining the genericity of such adsorption behavior. The addition of phytosterols to these mixtures resulted in the creation of defects, favoring the adsorption of rDGL at the fluid phases, but also improving the adsorption capacities of the lipase at the phase boundaries and towards the defects in the condensed phase. rDGL, like all gastric lipases, does not show any activity on galactolipids and phospholipids but its adsorption impacts their lateral organization and may change the adsorption and activity of other lipolytic enzymes in the course of digestion., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The structural behavior of model assemblies composed of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), the two main galactolipids found in plants, was investigated at the air/water interface and in aqueous dispersion. To approach the composition of the natural photosynthetic membranes, tunable Langmuir model membrane of galactolipids (GL) were used, and were complexified to form either heterogenous binary or ternary assemblies of GL, phospholipids (PL), and phytosterols (pS). The impact of pS, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or both on the structural properties of GL membrane was studied. The nature of the interactions between the different molecules was investigated using biophysical characterizations (ellipsometry, tensiometry, atomic force microscopy). In addition, the phase behavior was determined by SAXS analysis on the model assemblies in aqueous dispersions. Results revealed the good interfacial stability of these specific plant membrane lipids. The morphology of the GL film was characteristic of a fluid phase, with an interfacial roughness induced by the intercalation of monogalactosyl and digalactosyl polar heads of MGDG and DGDG, respectively. A phase heterogeneity in the monolayer was induced by the addition of DPPC and/or pS, which resulted in the modification of galactolipid organization and headgroup interactions. These structural changes were confirmed by SAXS analysis, showing more favorable interactions between MGDG and DPPC than between DGDG and DPPC in aqueous dispersion. This phenomenon was exacerbated in the presence of pS., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO 2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have., Competing Interests: The authors declare no conflict of interest.

An in vitro gastrointestinal human digestion model, with and without additional rapeseed oil, was used to measure the bioaccessibility of the major lipophilic nutrients enriched in chloroplasts: β-carotene; lutein; α-tocopherol; and α-linolenic acid. Chloroplast-rich fraction (CRF) material for this work was prepared from post-harvest pea vine field residue (pea vine haulm, or PVH), an abundant source of freely available, underutilised green biomass. PVH was either steam sterilised (100 °C for 4 min) and then juiced (heat-treated PVH, or HPVH), or was juiced fresh and the juice heated (90 °C for 3 min) (heat-treated juice, or HJ); the CRF from all biomass treatments was recovered from the juice by centrifugation. The impact of postharvest heat treatment of the biomass (HPVH), or of heat treatment of the juice (HJ) derived from the biomass, on the retention and bioaccessibility of the target nutrients was determined. The results showed that both heat treatments increased the apparent retention of β-carotene, lutein, α-tocopherol, and α-linolenic acid in the CRF material during digestion. The presence of edible oil during digestion did not dramatically affect the retention of these nutrients, but it did increase the bioaccessibility of β-carotene, lutein, and α-tocopherol from CRF material derived from heated biomass or juice. The presence of oil also increased the bioaccessibility of β-carotene, but not of lutein, α-tocopherol, or α-linolenic acid, from fresh CRF material.

Conventional degreasing of skins and hides in the leather industry requires high amounts of organic solvents and detergents that cause environmental issues. In this study, the LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was shown to be effective in degreasing sheepskins, thus reducing the amount of harmful chemicals. Using 6 mg of lipase/kg of raw skin, successful degreasing was achieved in only 15 min at pH 8 and 30°C. ToF-SIMS mass spectra of chemically and enzymatically treated sheepskins are consistent with a selective elimination process for the enzymatic treatment. Comparative SEM microscopy, ATR-FTIR spectroscopy and physicochemical analyses showed better properties of the enzymatically treated leather than those of the chemical treatment. Effluent physicochemical parameters showed that the enzymatic treatment is a cleaner degreasing operation. Altogether, this work opens new horizons to use the YLLIP2 lipase as a more efficient alternative in the leather industry., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

Fatty acid photodecarboxylase (FAP), one of the few natural photoenzymes characterized so far, is a promising biocatalyst for lipid-to-hydrocarbon conversion using light. However, the optimum supramolecular organization under which the fatty acid (FA) substrate should be presented to FAP has not been addressed. Using palmitic acid embedded in phospholipid liposomes, phospholipid-stabilized microemulsions, and mixed micelles, we show that FAP displays a preference for FAs present in liposomes and at the surface of microemulsions. The kinetics of adsorption onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with a higher affinity in the presence of FAs. The FAP structure reveals a potential interfacial recognition site with clusters of hydrophobic and basic residues surrounding the active site entrance. The resulting dipolar moment suggests the orientation of FAP at negatively charged interfaces. These findings provide important clues about the mode of action of FAP and the development of FAP-based bioconversion processes.

In this study, we evaluated vitamin D and mineral (iron, zinc, magnesium) transfer to the bolus aqueous phase during the digestion of meals with/without pulses. We performed in vitro digestions using test meals made either of i) beef and/or semolina and/or chickpeas, or of ii) potatoes supplemented or not with fibers, phytates, tannins and saponins. Chickpea presence led to a decrease in vitamin D bioaccessibility (-56%, p ≤ 0.05) and mineral solubility (-28% for iron, p ≤ 0.05) compared with meals with beef and/or semolina only. This effect was largely compensated for vitamin D by the fact that this vitamin was more stable during digestion of meals based on plant foods only than of meals with beef. Tannins were the most deleterious compounds for iron solubility, while phytates and tannins decreased vitamin D bioaccessibility. Agronomical or technical solutions to selectively decrease the amount in pulses of compounds that affect micronutrient bioavailability should be further explored., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Galactolipids are the most abundant lipids on earth where they are mainly found in photosynthetic membranes of plant, algae, and cyanobacteria. Pancreatic lipase-related protein 2 (PLRP2) is an enzyme with galactolipase activity allowing mammals, especially herbivores, to digest this important source of fatty acids. We present a method for the quantitative analysis of galactolipids and galactosylated products resulting from their digestion by guinea pig PLRP2 (GPLRP2), using thin-layer-chromatography (TLC), thymol-sulfuric acid as derivatization reagent and scanning densitometry for detection. Thymol-sulfuric acid reagent has been used for the colorimetric detection of carbohydrates. It is shown here that the derivatization of galactosyl group from galactolipids by this reagent is not affected by the bound acyl glycerol, acyl chains length and number of galactose residues in the polar head. This allowed quantifying simultaneously the initial substrate and all galactosylated products generated upon the hydrolysis of monogalactosyl di-octanoylglycerol (C8-MGDG) by GPLRP2 using a single calibration with C8-MGDG as reference standard. The reaction products, monogalactosyl monooctanoyl glycerol (C8-MGMG) and monogalactosyl glycerol (MGG), were identified and quantified, MGG being recovered from the aqueous phase and analyzed by a separate TLC analysis. This method is therefore suitable to quantify the products resulting from the release of both fatty acids present in MGDG and thereby shows that PLRP2 can contribute to the complete digestion of galactolipids and further intestinal absorption of their fatty acids., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The measurement of pancreatic lipase is important for the diagnosis of feline and canine pancreatitis. Recent studies have claimed that lipase assays using the 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) as a substrate are more specific for measuring pancreatic lipase than traditional lipase assays. However, the analytical specificity of this assay for pancreatic lipase has not been demonstrated., Objectives: We aimed to determine whether hepatic and/or lipoprotein lipases can interfere with the DGGR-based assay results in cats and dogs. We, therefore, compared plasma lipase activities measured using DGGR-based and pancreatic lipase immunoreactivity (PLI) assays before and after administering heparin, known to cause the release of hepatic and lipoprotein lipases, in cats and dogs., Methods: Heparin was administered in six cats and six dogs. Blood was collected at baseline and 10, 20, 30, 60, and 120 minutes after heparin administration. Lipase activity was measured using a DGGR-based assay, and PLI concentrations were measured using the Spec fPL and cPL assays for cats and dogs, respectively., Results: Plasma lipase activities, as measured using the DGGR-based assay, increased significantly 10 minutes after heparin administration in both cats (P = .003) and dogs (P = .006) and returned to baseline by 120 minutes. In contrast, PLI concentrations showed no significant changes after heparin administration., Conclusions: DGGR is not only hydrolyzed by pancreatic lipase but also by hepatic lipase, lipoprotein lipase, or both, in cats and dogs. Since these extrapancreatic lipases are also naturally present in cats and dogs, they could contribute to the lack of analytical specificity for the DGGR-based assays., (© 2020 American Society for Veterinary Clinical Pathology.)

The anti-malarial drug Chloroquine (CQ) and its derivative hydroxychloroquine have shown antiviral activities in vitro against many viruses, including coronaviruses, dengue virus and the biosafety level 4 Nipah and Hendra paramyxoviruses. The in vivo efficacy of CQ in the treatment of COVID-19 is currently a matter of debate. CQ is a lysosomotrophic compound that accumulates in lysosomes, as well as in food vacuoles of Plasmodium falciparum. In the treatment of malaria, CQ impairs the digestion and growth of the parasite by increasing the pH of the food vacuole. Similarly, it is assumed that the antiviral effects of CQ results from the increase of lysosome pH and the inhibition of acidic proteases involved in the maturation of virus fusion protein. CQ has however other effects, among which phospholipidosis, characterized by the accumulation of multivesicular bodies within the cell. The increase in phospholipid species particularly concerns bis(monoacylglycero)phosphate (BMP), a specific lipid of late endosomes involved in vesicular trafficking and pH-dependent vesicle budding. It was shown previously that drugs like progesterone, the cationic amphiphile U18666A and the phospholipase inhibitor methyl arachidonyl fluoro phosphonate (MAFP) induce the accumulation of BMP in THP-1 cells and decrease cell infection by human immunodeficiency virus. HIV viral particles were found to be retained into large endosomal-type vesicles, preventing virus spreading. Since BMP was also reported to favour virus entry through hijacking of the endocytic pathway, we propose here that BMP could play a dual role in viral infection, with its antiviral effects triggered by lysosomotropic drugs like CQ., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Caatinga is a Brazilian semi-arid ecosystem that stands out for presenting unique environmental characteristics with a dry, spiny and deciduous shrub/forest vegetation with several species that can be renewable oil sources with potential applicability in oleochemical and nutrition. Caatinga oilseeds have a high content of unsaturated fatty acids, phytosterols and sterols, and this composition is related to its nutritional potential. The present review summarizes the knowledge on the oil contents and fatty acid profiles of seeds from six representatives caatinga species. It was observed that plants species like Caju (Anacardium occidentale L.), Favela (Cnidoscolus quercifolius Pohl), Licuri (Syagrus coronata (Mart.) Becc.), Pinhão-bravo (Jatropha mollissima Pohl Baill), Pequi (Caryocar brasiliense Camb) and Oiticica (Licania rígida Benth) contains approximately 33.1, 33.5, 49.2, 18.3, 70.16 and 57.0% w/w of oil, respectively, on a dry weight basis. Their fatty acid profiles are mostly saturated for Licuri oil, with a high content of lauric acid (up to 40%) and unsaturated for Favela, Pinhão-bravo, Cashew nut, Pequi and Oiticica oils. Oiticica oil shows a high concentration of unusual conjugated polyunsaturated fatty acids, like α-Eleostearic and Licanic acid with 16.90 and 43.20% w/w, respectively., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Galactolipids, mainly monogalactosyl diglycerides and digalactosyl diglycerides are the main lipids found in the membranes of plants, algae and photosynthetic microorganisms like microalgae and cyanobacteria. As such, they are the main lipids present at the surface of earth. They may represent up to 80% of the fatty acid stocks, including a large proportion of polyunsaturated fatty acids mainly α-linolenic acid (ALA). Nevertheless, the interest in these lipids for nutrition and other applications remains overlooked, probably because they are dispersed in the biomass and are not as easy to extract as vegetable oils from oleaginous fruit and oil seeds. Another reason is that galactolipids only represent a small fraction of the acylglycerolipids present in modern human diet. In herbivores such as horses, fish and folivorous insects, galactolipids may however represent the main source of dietary fatty acids due to their dietary habits and digestion physiology. The development of galactolipase assays has led to the identification and characterization of the enzymes involved in the digestion of galactolipids in the gastrointestinal tract, as well as by microorganisms. Pancreatic lipase-related protein 2 (PLRP2) has been identified as an important factor of galactolipid digestion in humans, together with pancreatic carboxyl ester hydrolase (CEH). The levels of PLRP2 are particularly high in monogastric herbivores thus highlighting the peculiar role of PLRP2 in the digestion of plant lipids. Similarly, pancreatic lipase homologs are found to be expressed in the midgut of folivorous insects, in which a high galactolipase activity can be measured. In fish, however, CEH is the main galactolipase involved. This review discusses the origins and fatty acid composition of galactolipids and the physiological contribution of galactolipid digestion in various species. This overlooked aspect of lipid digestion ensures not only the intake of ALA from its main natural source, but also the main lipid source of energy for growth of some herbivorous species.

The link between food and human health is increasingly a topic of interest. One avenue of study has been to assess food disintegration and interactions within the gastrointestinal tract. In vitro digestion models have been widely used to overcome the constrictions associated with in vivo methodology. The COST Action INFOGEST developed an international, harmonised protocol for static simulation of digestion in the upper gastrointestinal tract of adults. This protocol is widely used; however, it is restricted to providing end-point assessment without considering the possible structural changes. On the other hand, there are dynamic models that provide more physiologically relevant data but are expensive and difficult to access. There is a gap between these models. The method outlined in this article provides an intermediate model; it builds upon the harmonised static model and now includes crucial kinetic aspects associated with the gastric phase of digestion, including gradual acidification, fluid and enzyme secretion and emptying. This paper provides guidance and standardised recommendations of a physiologically relevant semi-dynamic in vitro simulation of upper gastrointestinal tract digestion, with particular focus on the gastric phase. Adaptations of this model have already been used to provide kinetic data on nutrient digestion and structural changes during the gastric phase that impact on nutrient absorption. Moreover, it provides a simple tool that can be used in a wide range of laboratories.

Milk fat globule membrane conditions the reactivity and enzymatic susceptibility of milk lipids. The use of bovine membrane extracts to make infant formulas more biomimetic of human milk has been suggested recently. A comparison of the physico-chemical behavior of human and bovine milk membrane extracts and their interaction with gastric lipase is here undertaken using biophysical tools. Milk membrane extracts (70% of polar lipids) were obtained either pooling of mature human milk (n = 5) or bovine buttermilk. Human extract contained more anionic glycerophospholipids, less phosphatidylethanolamine and more unsaturated fatty acids (57% versus 46%) than bovine extract. Human extract presented a higher compressibility, with slower increase of surface pressure, than bovine extract. Micronic liquid condensed (LC) domains were evidenced in both extracts at 10 mN/m, but the evolution differs upon compression. Upon gastric lipase addition, an adsorption preference for liquid expanded phase (LE) was observed for both extracts. However, insertion was more homogeneous in terms of height level in human extract and impacted less its lipid lateral organization than in bovine extract. Both membrane extracts share close physico-chemical properties, however human membrane higher compressibility may favour gastric lipase insertion and higher interfacial reactivity in gastric conditions., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ., (Copyright © 2019. Published by Elsevier B.V.)

Accumulation of reserve compounds (i.e., lipids and chrysolaminarin) in diatoms depends on the environmental conditions, and is often triggered by stress conditions, such as nutrient limitation. Manipulation of CO 2 supply can also be used to improve both lipids and carbohydrates accumulation. Given the high diversity among diatoms, we studied the two marine model diatoms- Thalassiosira pseudonana and Phaeodactylum tricornutum , a freshwater diatom, Asterionella formosa , and Navicula pelliculosa -found in fresh- and sea-water environments. We measured the accumulation of reserve compounds and the activity of enzymes involved in carbon metabolism in these diatoms grown at high and atmospheric CO 2 . We observed that biomass and lipid accumulation in cells grown at high CO 2 differ among the diatoms. Lipid accumulation increased only in P. tricornutum and N. pelliculosa grown in seawater in response to elevated CO 2 . Moreover, accumulation of lipids was also accompanied by an increased activity of the enzymes tested. However, lipid accumulation and enzyme activity decreased in N. pelliculosa cultured in fresh water. Chrysolaminarin accumulation was also affected by CO 2 concentration; however, there was no clear relation with lipids accumulation. Our results are relevant to understand better the ecological role of the environment in the diatom adaptation to CO 2 and the mechanisms underpinning the production of storage compounds considering diatom diversity.

The removal of intact chloroplasts from their cell wall confinement offers a novel way to obtain lipophilic nutrients from green biomass, especially carotenoids and galactolipids. These latter are the main membrane lipids in plants and they represent a major source of the essential α-linolenic acid (18:3; ALA). Nevertheless, knowledge on their digestion is still limited. We have developed a physical method of recovering a chloroplast-rich fraction (CRF) from green biomass and tested its digestibility in vitro under simulated gastrointestinal conditions. Using a two-step static model, CRF from both spinach leaves and postharvest, pea vine field residue (haulm) were first exposed to enzymes from rabbit gastric extracts and then either to pancreatic enzymes from human pancreatic juice (HPJ) or to porcine pancreatic extracts (PPE). The lipolysis of monogalactosyldiacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) was monitored by thin layer chromatography and gas chromatography of fatty acid methyl esters. For both CRF preparations, MGDG and DGDG were converted to monogalactosylmonoacylglycerol (MGMG) and digalactosylmonoacylglycerol (DGMG), respectively, during the intestinal phase and ALA was the main fatty acid released. Galactolipids were more effectively hydrolysed by HPJ than by PPE, and PPE showed a higher activity on MGDG than on DGDG. These findings may be explained by the higher levels of galactolipase activity in HPJ compared to PPE, which mainly results from pancreatic lipase-related protein 2. Thus, we showed that CRF galactolipids are well digested by pancreatic enzymes and represent an interesting vehicle for ALA supplementation in human diet.

Intrinsically disordered proteins (IDPs) are proteins that provide many functional advantages in a large number of metabolic and signalling pathways. Because of their high flexibility that endows them with pressure-, heat- and acid-resistance, IDPs are valuable metabolic regulators that help algae to cope with extreme conditions of pH, temperature, pressure and light. They have, however, been overlooked in these organisms. In this review, we present some well-known algal IDPs, including the conditionally disordered CP12, a protein involved in the regulation of CO 2 assimilation, as probably the best known example, whose disorder content is strongly dependent on the redox conditions, and the essential pyrenoid component 1 that serves as a scaffold for ribulose-1, 5-bisphosphate carboxylase/oxygenase. We also describe how some enzymes are regulated by protein regions, called intrinsically disordered regions (IDRs), such as ribulose-1, 5-bisphosphate carboxylase/oxygenase activase, the A 2 B 2 form of glyceraldehyde-3-phosphate dehydrogenase and the adenylate kinase. Several molecular chaperones, which are crucial for cell proteostasis, also display significant disorder propensities such as the algal heat shock proteins HSP33, HSP70 and HSP90. This review confirms the wide distribution of IDPs in algae but highlights that further studies are needed to uncover their full role in orchestrating algal metabolism., (Copyright © 2019. Published by Elsevier Inc.)

The inhibition of recombinant CpLIP2 lipase/acyltransferase from Candida parapsiolosis was considered a key model for novel antifungal drug discovery and a potential therapeutic target for candidiasis. Lipases have identified recently as potent virulence factors in C. parapsilosis and some other yeasts. The inhibition effects of orlistat and four flavonols (galangin, kaempferol, quercetin and myricetin) characterized by an increasing degree of hydroxylation in B-ring, were investigated using ethyl oleate hydrolysis as the model reaction. Orlistat and kaempferol (14 µM) strongly inhibited CpLIP2 catalytic activity within 1 min of pre-incubation, by 90% and 80%, respectively. The relative potency of flavonols as inhibitors was: kaempferol > quercetin > myricetin > galangin. The results suggested that orlistat bound to the catalytic site while kaempferol interacted with W294 on the protein lid. A static mechanism of interactions between flavonols and CpLIP2 lipase was confirmed by fluorescence quenching analyses, indicating that the interactions were mainly driven by hydrophobic bonds and electrostatic forces. From the Lehrer equation, fractions of tryptophan accessibility to the quencher were evaluated, and a relationship with the calculated number of binding sites was suggested., Competing Interests: The authors declare no conflict of interest

Correction for 'Free fatty acid release from vegetable and bovine milk fat-based infant formulas and human milk during two-phase in vitro digestion' by Jeske H. J. Hageman et al., Food Funct., 2019, 10, 2102-2113.

Background: Bovine milk fat is increasingly used in infant formula (IF). The triacylglycerol (TAG) structure of bovine milk fat might be beneficial for digestion and absorption. We investigated the release of fatty acids (FAs) of IF containing different fat blends and compared this to human milk., Methods: Fresh human milk was sampled and two IFs were produced; one containing 100% vegetable fat (IF1) and one with 67% bovine milk fat and 33% vegetable fat (IF2). Using a static in vitro infant digestion model, consisting of a gastric and duodenal phase, the time dependent release of individual free fatty acids (FFA) was studied and analysed using GC-MS, and residual TAG levels were determined by GC-FID., Results: Human milk and the IFs showed comparable total FA release. In the gastric phase, 4-11% of lipolysis occurred, and mainly short (SCFA)- and medium chain fatty acids (MCFA) were released. In the duodenal phase, lipolysis proceeded with release of C4:0 but was marked by a fast release of long-chain fatty acids (LCFA). The digestion of the IFs resulted in different FFA profiles during and at the end of digestion. IF2 gave more release of C4:0-C11:0, which reflects the FA composition of bovine milk., Conclusion: The addition of bovine milk fat to IF resulted in a total FA release comparable to an IF with only vegetable fat and human milk. However, it did lead to a different time-dependent release of individual FAs, which might result in differences in absorption and other health effects in vivo.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.

The lipids and some surfactants present in oral lipid-based drug delivery systems are potential substrates for the various lipases involved in gastrointestinal (GI) lipolysis. The levels of these enzymes, together with pH and biliairy secretion, are important parameters that condition the fate of lipid-based formulations (LBF) and the dispersion, solubilization and absorption of lipophilic drugs in the GI tract. Since in vitro methods of digestion are now combined with dissolution assays for a better assessment of LBF performance, it is essential to have a basic knowledge on lipase, pH and bile acid (BA) levels in vivo to develop relevant in vitro models. While these parameters and their variations in healthy subjects are today well documented, in vivo data on specific populations (age groups, patients with various diseases, patients with treatment affecting GI tract parameters, …) are scarce and obtaining them from clinical studies is sometimes difficult due to ethical limitations. Here we collected some in vivo data already available on the levels of digestive lipases, gastric and intestinal pH, and BAs at various ages and in patients with exocrine pancreatic insufficiency, a pathological situation that leads to drastic changes in GI tract parameters and impacts pharmacological treatments., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.

Rabbit gastric extract (RGE) is a source of gastric enzymes for in vitro digestion studies. While its gastric lipase activity has been characterized and compared to other lipases, its pepsin activity has not been studied. We measured pepsin activity in RGE using both hemoglobin and azocoll as substrates, and identified the protein separated by SDS-PAGE as a type II-4 mature pepsin of 328 amino acid residues using Edman sequencing, LC-MS/MS analysis and intact mass measurement. As a proof-of-concept that RGE was suitable for in vitro digestion of both proteins and lipids, it was used for studying the proteolysis of β-casein under conditions mimicking the early stages of intragastric digestion. β-Casein was displayed either in solution or at the surface of a β-casein-stabilized rapeseed oil emulsion to investigate the impact of lipids and lipolysis on proteolysis. Proteolysis of β-casein was quantified based on the kinetics of β-casein disappearance, the identification of various peptides generated upon digestion and their variation with time. The results obtained with RGE were highly similar to those obtained with equivalent amounts of porcine pepsin used as a reference standard. Digestion of β-casein was slower when it was displayed at the oil-water interface and some degradation peptides were transiently observed at higher levels and for a longer time than with β-casein in solution, or accumulated upon digestion. N-terminal sequencing of the main isolated peptides revealed a sequential action of pepsin starting from the hydrophobic C-terminal end of β-casein, which was impaired by the interaction of β-casein with lipids.

Bile acid (BA) secretion and circulation in chronic pancreatitis (CP) patients with exocrine pancreatic insufficiency (EPI) were investigated by simultaneously measuring postprandial levels of individual BAs in duodenal contents and blood plasma using LC-MS/MS. CP patients and healthy volunteers (HVs) were intubated with gastric and duodenal tubes prior to the administration of a test meal and continuous aspiration of duodenal contents. Pancreatic lipase outputs in CP patients were very low (0.7 ± 0.2 mg) versus HVs (116.7 ± 68.1 mg; P < 0.005), thus confirming the severity of EPI. Duodenal BA outputs were reduced in CP patients (1.00 ± 0.89 mmol; 0.47 ± 0.42 g) versus HVs (5.52 ± 4.53 mmol; 2.62 ± 2.14 g; P < 0.15). Primary to secondary BA ratio was considerably higher in CP patients (38.09 ± 48.1) than HVs (4.15 ± 2.37; P < 0.15), indicating an impaired transformation of BAs by gut microbiota. BA concentrations were found below the critical micellar concentration in CP patients, while a high BA concentration peak corresponding to gallbladder emptying was evidenced in HVs. Conversely, BA plasma concentration was increased in CP patients versus HVs suggesting a cholangiohepatic shunt of BA secretion. Alterations of BA circulation and levels may result from the main biliary duct stenosis observed in these CP patients and may aggravate the consequences of EPI on lipid malabsorption., (Copyright © 2018 Humbert et al.)

Talaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10 ng of enzymes, against 100 ng to 10 μg with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Heterodimeric phospholipase A 2 from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity., Methods: Molecular cloning of Sm-PLGV and heterologous expression in Escherichia coli of three recombinant forms was used to determine the role of the short chain on enzymatic activity. Infrared spectroscopy assisted 3D model building of the three recombinant constructs (phospholipases with and without the penta-peptide and Long chain only) allowed us to propose an explanation of the differences in specific activities and their interaction with various phospholipids., Results: Nucleotide sequence of Sm-PLGV encodes 129 residues corresponding to the Long chain, the penta-peptide and the short chain. Although recombinant phospholipases without and with the penta-peptide have different specific activities, they display a similar substrate specificity on various phospholipid monolayers and similar bell-shaped activity profiles with maxima at high surface pressure. The absence of the short chain reduces significantly enzymatic and hemolytic activities. The 3D models pointed to an interaction of the short chain with the catalytic residues, what might explain the difference in activities of our constructs., Conclusion: Infrared spectroscopy data and 3D modeling confirm the experimental findings that highlight the importance of the short chain for the Sm-PLGV activity., General Significance: New informations are given to further establish the structure-function relationships of the Sm-PLGV., (Copyright © 2018. Published by Elsevier B.V.)

The interaction of pancreatic lipase-related protein 2 (PLRP2) with various micelles containing phospholipids was investigated using pHstat enzyme activity measurements, differential light scattering, size exclusion chromatography (SEC) and transmission IR spectroscopy. Various micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and lysophosphatidylcholine were prepared with either bile salts (sodium taurodeoxycholate or glycodeoxycholate) or Triton X-100, which are substrate-dispersing agents commonly used for measuring phospholipase activities. PLRP2 displayed a high activity on all phospholipid-bile salt micelles, but was totally inactive on phospholipid-Triton X-100 micelles. These findings clearly differentiate PLRP2 from secreted pancreatic phospholipase A2 which is highly active on both types of micelles. Using an inactive variant of PLRP2, SEC experiments allowed identifying two populations of PLRP2-DPPC-bile salt complexes corresponding to a high molecular weight 1:1 PLRP2-micelle association and to a low molecular weight association of PLRP2 with few monomers of DPPC/bile salts. IR spectroscopy analysis showed how DPPC-bile salt micelles differ from DPPC-Triton X-100 micelles by a higher fluidity of acyl chains and higher hydration/H-bonding of the interfacial carbonyl region. The presence of bile salts allowed observing changes in the IR spectrum of DPPC upon addition of PLRP2 (higher rigidity of acyl chains, dehydration of the interfacial carbonyl region), while no change was observed with Triton X-100. The differences between these surfactants and their impact on substrate recognition by PLRP2 are discussed, as well as the mechanism by which high and low molecular weight PLRP2-DPPC-bile salt complexes may be involved in the overall process of DPPC hydrolysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Usual methods for the continuous assay of lipolytic enzyme activities are mainly based on the titration of free fatty acids, surface pressure monitoring or spectrophotometry using substrates labeled with specific probes. These approaches only give a partial information on the chemistry of the lipolysis reaction and additional end-point analyses are often required to quantify both residual substrate and lipolysis products. We used transmission infrared (IR) spectroscopy to monitor simultaneously the hydrolysis of phospholipids by guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the release of lipolysis products. The substrate (DPPC, 1,2-Dipalmitoyl phosphatidylcholine) was mixed with sodium taurodeoxycholate (NaTDC) to form mixed micelles in D 2 O buffer at pD 6 and 8. After hydrogen/deuterium exchange, DPPC hydrolysis by GPLRP2 (100nM) was monitored at 35°C in a liquid cell by recording IR spectra and time-course variations in the CO stretching region. These changes were correlated to variations in the concentrations of DPPC, lysophospholipids (lysoPC) and palmitic acid (Pam) using calibration curves established with these compounds individually mixed with NaTDC. We were thus able to quantify each compound and its time-course variations during the phospholipolysis reaction and to estimate the enzyme activity. To validate the IR analysis, variations in residual DPPC, lysoPC and Pam were also quantified by thin-layer chromatography coupled to densitometry and similar hydrolysis profiles were obtained using both methods. IR spectroscopy can therefore be used to monitor the enzymatic hydrolysis of phospholipids and obtain simultaneously chemical and physicochemical information on substrate and all reaction products (H-bonding, hydration, acyl chain mobility)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Guinea pig pancreatic lipase-related protein 2 (GPLRP2) is an interesting model enzyme that can hydrolyze a large set of acylglycerols in vitro but displays however some selectivity depending on the supramolecular structure of substrate and the presence of surfactants like bile salts. We showed that GPLRP2 hydrolyzes 1,2-dipalmitoyl phosphatidylcholine (DPPC) present in mixed micelles with sodium taurodeoxycholate (NaTDC) but not in multilamellar (MLV) and large unilamellar (LUV) vesicles of DPPC. After characterization of these lipid aggregates by dynamic light scattering (DLS), the discriminative recognition of DPPC in DPPC/NaTDC micelles versus MLV and LUV by an inactive variant (S152G) of GPLRP2 to avoid the effect of substrate hydrolysis was investigated using Fourier transform infrared spectroscopy (FTIR). IR spectra were recorded after hydrogen/deuterium exchange, at pD 6 and various temperatures to study phase transitions. We analyzed the methylene asymmetric stretching (ν(CH2) as ), the carbonyl stretching (ν(CO)) and the composite polar head-group vibration bands, first to characterized differences in DPPC micelles and vesicles, and second to estimate the degree of interaction of GPLRP2 S152G with phospholipid. Our results indicate that a significant interaction between GPLRP2 S152G and DPPC is only observed when NaTDC is added to the system to form micelles and this can be explained by the different organization of DPPC in mixed micelles compared to lamellar vesicles (higher hydration of polar head, higher mobility of alkyl chains) that favors GPLRP2 penetration into the phospholipid layer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Food effects on oral drug bioavailability can have significant impact on the provision of safe and reliable oral pharmacotherapy. A mechanistic understanding of the events that contribute to the occurrence of food effects is therefore critical. An increased oral bioavailability is often seen for poorly water-soluble drugs after co-administration with lipids, including lipids in food, and is commonly explained by the ability of lipids to enhance drug solubility in intestinal luminal fluids. In contrast, the impact of lipids on drug solubilisation in the stomach has received less attention. This is in spite of the fact that lipid digestion is initiated in the stomach by human gastric lipase and that gastric events also initiate emulsification of lipids in the gastrointestinal tract. The stomach therefore acts to 'pre-process' lipids for subsequent events in the intestine and may significantly affect downstream events at intestinal drug absorption sites. In this article, the mechanisms by which lipids are processed in the stomach are reviewed and the potential impact of these processes on drug absorption discussed. Attention is also focused on in vitro methods that are used to assess gastric processing of lipids and their application to better understand food effects on drug release and absorption.

Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure under physiological conditions in the absence of their biological partners and thus exist as dynamic ensembles of interconverting conformers, often highly soluble in water. However, in some cases, IDPs such as the ones involved in neurodegenerative diseases can form protein aggregates and their aggregation process may be triggered by the interaction with membranes. Although the interfacial behavior of globular proteins has been extensively studied, experimental data on IDPs at the air/water (A/W) and water/lipid interfaces are scarce. We studied here the intrinsically disordered C-terminal domain of the Hendra virus nucleoprotein (N TAIL ) and compared its interfacial properties to those of lysozyme that is taken as a model globular protein of similar molecular mass. Adsorption of N TAIL at the A/W interface was studied in the absence and presence of phospholipids using Langmuir films, polarization modulated-infrared reflection-absorption spectroscopy, and an automated drop tensiometer for interfacial tension and elastic modulus determination with oscillating bubbles. N TAIL showed a significant surface activity, with a higher adsorption capacity at the A/W interface and penetration into egg phosphatidylcholine monolayer compared to lysozyme. Whereas lysozyme remains folded upon compression of the protein layer at the A/W interface and shows a quasi-pure elastic behavior, N TAIL shows a much higher molecular area and forms a highly viscoelastic film with a high dilational modulus. To our knowledge, a new disorder-to-order transition is thus observed for the N TAIL protein that folds into an antiparallel β-sheet at the A/W interface and presents strong intermolecular interactions., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The cDNA encoding human gastric lipase (HGL) was integrated into the genome of Pichia pastoris using the pGAPZα A transfer vector. The HGL signal peptide was replaced by the yeast α-factor to achieve an efficient secretion. Active rHGL was produced by the transformed yeast but its levels and stability were dependent on the pH. The highest activity was obtained upon buffering the culture medium at pH5, a condition that allowed preserving enzyme activity over time. A large fraction (72±2%) of secreted rHGL remained however bound to the yeast cells, and was released by washing the cell pellet with an acid glycine-HCl buffer (pH2.2). This procedure allowed establishing a first step of purification that was completed by size exclusion chromatography. N-terminal sequencing and MALDI-ToF mass spectrometry revealed that rHGL was produced in its mature form, with a global mass of 50,837±32Da corresponding to a N-glycosylated form of HGL polypeptide (43,193Da). rHGL activity was characterized as a function of pH, various substrates and in the presence of bile salts and pepsin, and was found similar to native HGL, except for slight changes in pH optima. We then studied by site-directed mutagenesis the role of three key residues (K4, E225, R229) involved in salt bridges stabilizing the lid domain that controls the access to the active site and is part of the interfacial recognition site. Their substitution has an impact on the pH-dependent activity of rHGL and its relative activities on medium and long chain triglycerides., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background & Aims: It has been suggested that homogenization of Holder-pasteurized human milk (PHM) could improve fat absorption and weight gain in preterm infants, but the impact on the PHM digestive kinetics has never been studied. Our objective was to determine the impact of PHM homogenization on gastric digestion in preterm infants., Methods: In a randomized controlled trial, eight hospitalized tube-fed preterm infants were their own control to compare the gastric digestion of PHM and of homogenized PHM (PHHM). PHM was obtained from donors and, for half of it, was homogenized by ultrasonication. Over a six-day sequence, gastric aspirates were collected twice a day, before and 35, 60 or 90 min after the start of PHM or PHHM ingestion. The impact of homogenization on PHM digestive kinetics and disintegration was tested using a general linear mixed model. Results were expressed as means ± SD., Results: Homogenization leaded to a six-fold increase in the specific surface (P < 0.01) of lipid droplets. The types of aggregates formed during digestion were different between PHM and PHHM, but the lipid fraction kept its initial structure all over the gastric digestion (native globules in PHM vs. blend of droplets in PHHM). Homogenization increased the gastric lipolysis level (P < 0.01), particularly at 35 and 60 min (22 and 24% higher for PHHM, respectively). Homogenization enhanced the proteolysis of serum albumin (P < 0.05) and reduced the meal emptying rate (P < 0.001, half-time estimated at 30 min for PHM and 38 min for PHHM). The postprandial gastric pH was not affected (4.7 ± 0.9 at 90 min)., Conclusions: Homogenization of PHM increased the gastric lipolysis level. This could be a potential strategy to improve fat absorption, and thus growth and development in infants fed with PHM; however, its gastrointestinal tolerance needs to be investigated further. This trial was registered at clinicaltrials.gov as NCT02112331., (Copyright © 2017 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl- sn -glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology., Competing Interests: The authors declare there are no competing interests.

The gene coding for a lipase of Fusarium solani, designated as FSL2, shows an open reading frame of 906bp encoding a 301-amino acid polypeptide with a molecular mass of 30kDa. Based on sequence similarity with other fungal lipases, FSL2 contains a catalytic triad, consisting of Ser144, Asp198, and His256. FSL2 cDNA was subcloned into the pGAPZαA vector containing the Saccharomyces cerevisiae α-factor signal sequence and this construct was used to transform Pichia pastoris and achieve a high-level extracellular production of a FSL2 lipase. Maximum lipase activity was observed after 48h. The optimum activity of the purified recombinant enzyme was measured at pH 8.0-9.0 and 37°C. FSL2 is remarkably stable at alkaline pH values up to 12 and at temperatures below 40°C. It has high catalytic efficiency towards triglycerides with short to long chain fatty acids but with a marked preference for medium and long chain fatty acids. FSL2 activity is decreased at sodium taurodeoxycholate concentrations above the Critical Micelle Concentration (CMC) of this anionic detergent. However, lipase activity is enhanced by Ca 2+ and inhibited by EDTA or Cu 2+ and partially by Mg 2+ or K + . In silico docking of medium chain triglycerides, monogalctolipids (MGDG), digalactolipids (DGDG) and long chain phospholipids in the active site of FSL2 reveals structural solutions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human milk feeding is an important recommendation for preterm newborns considering their vulnerability and digestive immaturity. Holder pasteurization (62.5°C, 30min) applied in milk banks modifies its biological quality and its microstructure. We investigated the impact of pasteurization of preterm human milk on its gastrointestinal kinetics of lipolysis, proteolysis and structural disintegration. An in vitro dynamic system was set up to simulate the gastrointestinal digestion of preterm newborns. A pool of preterm human milk was digested as raw or after Holder pasteurization. Pasteurization impacted the microstructure of undigested human milk, its gastrointestinal disintegration and tended to limit the intestinal lipolysis. Furthermore, the gastrointestinal bioaccessibility of some fatty acids was decreased by pasteurization, while the intestinal bioaccessibility of some amino acids was selectively modulated. The impact of pasteurization on the digestion of human milk may have nutritional relevance in vivo and potentially modulates preterm development and growth., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Based on a previous study and in silico molecular docking experiments, we have designed and synthesized a new series of ten 5-Alkoxy-N-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one derivatives (RmPPOX). These molecules were further evaluated as selective and potent inhibitors of mammalian digestive lipases: purified dog gastric lipase (DGL) and guinea pig pancreatic lipase related protein 2 (GPLRP2), as well as porcine (PPL) and human (HPL) pancreatic lipases contained in porcine pancreatic extracts (PPE) and human pancreatic juices (HPJ), respectively. These compounds were found to strongly discriminate classical pancreatic lipases (poorly inhibited) from gastric lipase (fully inhibited). Among them, the 5-(2-(Benzyloxy)ethoxy)-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one (BemPPOX) was identified as the most potent inhibitor of DGL, even more active than the FDA-approved drug Orlistat. BemPPOX and Orlistat were further compared in vitro in the course of test meal digestion, and in vivo with a mesenteric lymph duct cannulated rat model to evaluate their respective impacts on fat absorption. While Orlistat inhibited both gastric and duodenal lipolysis and drastically reduced fat absorption in rats, BemPPOX showed a specific action on gastric lipolysis that slowed down the overall lipolysis process and led to a subsequent reduction of around 55% of the intestinal absorption of fatty acids compared to controls. All these data promote BemPPOX as a potent candidate to efficiently regulate the gastrointestinal lipolysis, and to investigate its link with satiety mechanisms and therefore develop new strategies to "fight against obesity"., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Delayed fat digestion might help to fight obesity. Fat digestion begins in the stomach by adsorption of gastric lipases to oil/water interfaces. In this study we show how biopolymer covered interfaces can act as a physical barrier for recombinant dog gastric lipase (rDGL) adsorption and thus gastric lipolysis. We used β-lactoglobulin (β-lg) and thermosensitive methylated nanocrystalline cellulose (metNCC) as model biopolymers to investigate the role of interfacial fluid dynamics and morphology for interfacial displacement processes by rDGL and polysorbate 20 (P20) under gastric conditions. Moreover, the influence of the combination of the flexible β-lg and the elastic metNCC was studied. The interfaces were investigated combining interfacial techniques, such as pendant drop, interfacial shear and dilatational rheology, and neutron reflectometry. Displacement of biopolymer layers depended mainly on the fluid dynamics and thickness of the layers, both of which were drastically increased by the thermal induced gelation of metNCC at body temperature. Soft, thin β-lg interfaces were almost fully displaced from the interface, whereas the composite β-lg-metNCC layer thermogelled to a thick interfacial layer incorporating β-lg as filler material and therefore resisted higher shear forces than a pure metNCC layer. Hence, with metNCC alone lipolysis by rDGL was inhibited, whereas the layer performance could be increased by the combination with β-lg.