Nutrient value and stability of chloroplast-rich fraction (CRF) material from pea vine haulm, and the role of galactolipolytic enzymes during digestion

Underutilised green plant material is a rich source of chloroplasts, organelles in plants and other photosynthetic organisms, which are responsible for the conversion of light energy into chemical energy. The removal of intact chloroplasts from their cell wall confinement offers a novel way to obtain lipophilic nutrients and galactolipids from green biomass, especially carotenoids, vitamin E, and galactolipids (the main membrane lipids in plants and they represent a major source of the essential alpha-linolenic acid (18:3, ALA)). We have developed a physical method of recovering a chloroplast-rich fraction (CRF) from postharvest, pea vine field residue (haulm) by using slow-screw twin-gear juicer without added water. The CRF from pea vine haulm (PVH), like CRF from spinach leaves, is a good source of lipophilic nutrients, including beta-carotene (provitamin A), lutein, alpha-tocopherol (vitamin E), phylloquinone (vitamin K), and essential fatty acids. Steam sterilisation of pea vine haulm prior to generating CRF was used to inactivate enzymes after harvesting and extend the shelf life of nutrients. The stability of selected nutrients (beta-carotene, lutein, and alpha-tocopherol) in the freeze-dried CRF material was measured over 84 days; the impact of storage temperature (-20°C, 4°C, 25°C and 40°C), light and air on nutrient stability was established. All three nutrients were stable at -20°C and 4°C in the presence or absence of air; this stability was lost at higher temperatures in the presence of air. The extent and rate of nutrient breakdown significantly increased when the CRF samples were exposed to light. beta-Carotene appeared to be more susceptible to degradation than lutein and tocopherol at 40°C in the presence of air, but when CRF was exposed to light all three nutrients measured were significantly broken-down during storage at 25°C or 40°C, whether exposed to air or not. The bioaccessibility (% nutrient available for uptake) of some essential lipophilic nutrients, contained in a chloroplast-rich fraction (CRF) recovered from post-harvest pea vine field residue (haulm) (PVH), was measured using an in vitro gastrointestinal human digestion model with and without additional rapeseed oil. The impact on nutrient stability during digestion (in-digesta) and bioaccessibility of postharvest heat treatment of the biomass (HPVH), or the juice (HJ) derived from the biomass, was determined. The results show that both heat treatments stabilised beta-carotene, lutein, alpha-tocopherol and alpha-linolenic acid in the CRF material in-digesta. The presence of oil during digestion appears to improve retention of beta-carotene, and the bioaccessibility of beta-carotene in CRF from HPVH and HJ, except in the case of lutein and alpha-tocopherol. Conversely, the combination of heat treatment both HPVH and HJ and the presence of oil in PVH CRF enhanced the retention and bioaccessibility of beta-carotene, lutein, and alpha-tocopherol. The release of nutrients that are embedded into the chloroplast structure is likely to be affected by the extent to which thylakoid membranes are hydrolysed during digestion. The PVH CRF and spinach CRF were therefore measured in vitro under simulated gastrointestinal conditions. Using a two-step static model, CRF from both spinach leaves and postharvest pea vine field residue (haulm) was 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 digalactosyldiacylglycerol (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 mono galactosylmonoacylglycerol (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, is probably due to presence of pancreatic lipase-related protein 2 (PLRP2) in HPJ. Thus, we showed that CRF galactolipids are well digested by pancreatic enzymes and represent an interesting vehicle for ALA supplementation in human diet

Bioaccessibility of carotenoids (β-carotene and lutein) from intact and disrupted microalgae (Chlamydomonas reinhardtii)

Chlamydomonas reinhardtii (C. reinhardtii) has a potential as a novel source for food/feed because it contains several constituents including bioactive compounds. However, its multilayer cell wall (hydroxyproline-rich glycoprotein [HRPGs]) may restrict the bioaccessibility of its nutrients. Therefore, using disruption techniques such as hydrodynamic cavitation (HDC) can be useful for assessing single cell compounds. This work aims to evaluate the impact of HDC on the bioaccessibility of carotenoids (β-carotene and lutein) from C. reinhardtii. Our results illustrated that digestive enzymes cannot fully break down the cell walls beside HDC process generates their significant change. The intact C. reinhardtii (ICR) and disrupted C. reinhardtii (DCR) have a comparable lutein bioaccessibility, in contrast, DCR decreased the biocessibility of β-carotene. HDC decreased the biocessibility of β-carotene in the small intestine although 37% of total carotenoids from DRC were absorbed

Chloroplast/thylakoid-rich material: A possible alternative to the chemically synthesised flow enhancer polyglycerol polyricinoleate in oil-based systems

Chloroplasts are abundant organelles in a diverse range of plant materials; they are predominantly composed of multicomponent thylakoid membranes which are lipid and protein rich. Intact or unravelled thylakoid membranes should, in principle, have interfacial activity, but little has been published on their activity in oil-in-water systems, and nothing on their performance on an oil continuous system. In this work different physical methods were used to produce a range of chloroplast/thylakoid suspensions with varying degrees of membrane integrity. Transmission electron microscopy revealed that pressure homogenisation led to the greatest extent of membrane and organelle disruption compared to less energy intensive preparation methods The ability of the derived materials to modulate the flow behaviour of a chocolate model system (65% (w/w) sugar/ sunflower oil (natural amphiphiles removed) suspension) was investigated by acquiring rheological parameters. All chloroplast/thylakoid preparations reduced yield stress, apparent viscosity, tangent flow point and cross over point in a concentration-dependent fashion, although not as significantly as polyglycerol polyricinoleate applied at a commercially relevant concentration in the same chocolate model system. Confocal laser scanning microscopy confirmed presence of the alternative flow enhancer material at the sugar surfaces. This research reveals that low-energy processing methods that do not extensively disrupt thylakoid membranes are applicable to generating materials with marked capacity to affect the flow behaviour of a chocolate model system. In conclusion, chloroplast/thylakoid materials hold strong potential as natural alternatives to synthetic rheology modifiers for lipid-based systems such as PGPR

In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts

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

Nutrient value and stability of chloroplast-rich fraction (CRF) material from pea vine haulm, and the role of galactolipolytic enzymes during digestion

Underutilised green plant material is a rich source of chloroplasts, organelles in plants and other photosynthetic organisms, which are responsible for the conversion of light energy into chemical energy. The removal of intact chloroplasts from their cell wall confinement offers a novel way to obtain lipophilic nutrients and galactolipids from green biomass, especially carotenoids, vitamin E, and galactolipids (the main membrane lipids in plants and they represent a major source of the essential alpha-linolenic acid (18:3, ALA)). We have developed a physical method of recovering a chloroplast-rich fraction (CRF) from postharvest, pea vine field residue (haulm) by using slow-screw twin-gear juicer without added water. The CRF from pea vine haulm (PVH), like CRF from spinach leaves, is a good source of lipophilic nutrients, including beta-carotene (provitamin A), lutein, alpha-tocopherol (vitamin E), phylloquinone (vitamin K), and essential fatty acids. Steam sterilisation of pea vine haulm prior to generating CRF was used to inactivate enzymes after harvesting and extend the shelf life of nutrients. The stability of selected nutrients (beta-carotene, lutein, and alpha-tocopherol) in the freeze-dried CRF material was measured over 84 days; the impact of storage temperature (-20°C, 4°C, 25°C and 40°C), light and air on nutrient stability was established. All three nutrients were stable at -20°C and 4°C in the presence or absence of air; this stability was lost at higher temperatures in the presence of air. The extent and rate of nutrient breakdown significantly increased when the CRF samples were exposed to light. beta-Carotene appeared to be more susceptible to degradation than lutein and tocopherol at 40°C in the presence of air, but when CRF was exposed to light all three nutrients measured were significantly broken-down during storage at 25°C or 40°C, whether exposed to air or not. The bioaccessibility (% nutrient available for uptake) of some essential lipophilic nutrients, contained in a chloroplast-rich fraction (CRF) recovered from post-harvest pea vine field residue (haulm) (PVH), was measured using an in vitro gastrointestinal human digestion model with and without additional rapeseed oil. The impact on nutrient stability during digestion (in-digesta) and bioaccessibility of postharvest heat treatment of the biomass (HPVH), or the juice (HJ) derived from the biomass, was determined. The results show that both heat treatments stabilised beta-carotene, lutein, alpha-tocopherol and alpha-linolenic acid in the CRF material in-digesta. The presence of oil during digestion appears to improve retention of beta-carotene, and the bioaccessibility of beta-carotene in CRF from HPVH and HJ, except in the case of lutein and alpha-tocopherol. Conversely, the combination of heat treatment both HPVH and HJ and the presence of oil in PVH CRF enhanced the retention and bioaccessibility of beta-carotene, lutein, and alpha-tocopherol. The release of nutrients that are embedded into the chloroplast structure is likely to be affected by the extent to which thylakoid membranes are hydrolysed during digestion. The PVH CRF and spinach CRF were therefore measured in vitro under simulated gastrointestinal conditions. Using a two-step static model, CRF from both spinach leaves and postharvest pea vine field residue (haulm) was 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 digalactosyldiacylglycerol (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 mono galactosylmonoacylglycerol (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, is probably due to presence of pancreatic lipase-related protein 2 (PLRP2) in HPJ. Thus, we showed that CRF galactolipids are well digested by pancreatic enzymes and represent an interesting vehicle for ALA supplementation in human diet

In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves

International audienceThe 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 -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

Bioaccessibility of essential lipophilic nutrients in a chloroplast-rich fraction (CRF) from agricultural green waste during simulated human gastrointestinal tract digestion

International audienceThe lipophilic nutrients in a chloroplast-rich fraction derived from pea vine postharvest field-residue are released in an in vitro digestion model; the extent of their release (bioaccessibility) is affected by heat-treatment of biomass or juice

The digestion of galactolipids and its ubiquitous function in Nature for the uptake of the essential α-linolenic acid

International audienceGalactolipids, 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 the earth. They may represent up to 80 % of the fatty acid stocks, including a large proportion of polyunsaturated fatty acids mainly -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