Valorization of the Antioxidant Effect of Mantua PGI Pear By-Product Extracts: Preparation, Analysis and Biological Investigation.

For improving the management of the production chain of PGI Mantua pears (which comprises many varieties, including Abate Fetel), applying the cardinal principles of circular economy and sustainability, the fruits with diseases or defects were recovered for producing dried rounds of pears from the Abate Fetel cultivar, a new product with high nutritional value that extends the remaining life. This process led to the production of secondary and residual by-products, which are mainly composed of the highest and lowest part of the fruits, comprising seeds, pulps, peels and petioles. Hence, this study was focused on the valorization of these secondary by-products of Abate Fetel pears through the production of pear extracts using traditional and "green" extraction methods that involve the use of supercritical CO2 fluid extraction. The produced extracts, together with a reference solvent-derived extract, were analyzed by HPLC-ESI-MS, and in parallel, their direct and cellular antioxidant activity were assessed. Evidence has indicated that all the tested extracts reduced the H2O2-induced reactive oxygen species (ROS), lipid peroxidation and nitric oxide (NO) levels, respectively, in human intestinal Caco-2 cells. Hence, this study clearly suggests that extracts obtained from Mantuan PGI pear by-products may be used as valuable sources of bioactive upcycled phytocomplex for the development of dietary supplements and/or functional foods.

Hempseed (Cannabis sativa) Peptide H3 (IGFLIIWV) Exerts Cholesterol-Lowering Effects in Human Hepatic Cell Line.

Hempseed (Cannabis sativa) protein is an important source of bioactive peptides. H3 (IGFLIIWV), a transepithelial transported intestinal peptide obtained from the hydrolysis of hempseed protein with pepsin, carries out antioxidant and anti-inflammatory activities in HepG2 cells. In this study, the main aim was to assess its hypocholesterolemic effects at a cellular level and the mechanisms behind this health-promoting activity. The results showed that peptide H3 inhibited the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in vitro in a dose-dependent manner with an IC50 value of 59 µM. Furthermore, the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, followed by the increase of low-density lipoprotein (LDL) receptor (LDLR) protein levels, was observed in human hepatic HepG2 cells treated with peptide H3 at 25 µM. Meanwhile, peptide H3 regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Consequently, the augmentation of the LDLR localized on the cellular membranes led to the improved ability of HepG2 cells to uptake extracellular LDL with a positive effect on cholesterol levels. Unlike the complete hempseed hydrolysate (HP), peptide H3 can reduce the proprotein convertase subtilisin/kexin 9 (PCSK9) protein levels and its secretion in the extracellular environment via the decrease of hepatic nuclear factor 1-α (HNF1-α). Considering all these evidences, H3 may represent a new bioactive peptide to be used for the development of dietary supplements and/or peptidomimetics for cardiovascular disease (CVD) prevention.

A heuristic, computer-driven and top-down approach to identify novel bioactive peptides: A proof-of-principle on angiotensin I converting enzyme inhibitory peptides.

Bioactive peptides are short peptides (3-20 amino acid residues in length) endowed of specific biological activities. The identification and characterization of bioactive peptides of food origin are crucial to better understand the physiological consequences of food, as well as to design novel foods, ingredients, supplements, and diets to counteract mild metabolic disorders. For this reason, the identification of bioactive peptides is also relevant from a pharmaceutical standpoint. Nevertheless, the systematic identification of bioactive sequences of food origin is still challenging and relies mainly on the so defined "bottom-up" approaches, which rarely results in the total identification of most active sequences. Conversely, "top-down" approaches aim at identifying bioactive sequences with certain features and may be more suitable for the precise identification of very potent bioactive peptides. In this context, this work presents a top-down, computer-assisted and hypothesis-driven identification of potent angiotensin I converting enzyme inhibitory tripeptides, as a proof of principle. A virtual library of 6840 tripeptides was screened in silico to identify potential highly potent inhibitory peptides. Then, computational results were confirmed experimentally and a very potent novel sequence, LMP was identified. LMP showed an IC50 of 15.8 and 6.8 µM in cell-free and cell-based assays, respectively. In addition, a bioinformatics approach was used to search potential food sources of LMP. Yolk proteins were identified as a possible relevant source to analyze in further experiments. Overall, the method presented may represent a powerful and versatile framework for a systematic, high-throughput and top-down identification of bioactive peptides.

Characterization of the Trans-Epithelial Transport of Green Tea (C. sinensis) Catechin Extracts with In Vitro Inhibitory Effect against the SARS-CoV-2 Papain-like Protease Activity.

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC50 of 0.125 µg mL-1). The untargeted characterization revealed that (-)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC50 value equal to 11.62 ± 0.47 µg mL-1) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.

Impact of Soy ß-Conglycinin Peptides on PCSK9 Protein Expression in HepG2 Cells.

BACKGROUND: Dyslipidaemias, particularly elevated plasma low-density lipoprotein cholesterol (LDL-C) levels, are major risk factors for cardiovascular disease (CVD). Besides pharmacological approaches, a nutritional strategy for CVD prevention has gained increasing attention. Among functional foods, the hypocholesterolemic properties of soy are driven by a stimulation of LDL-receptor (LDL-R) activity. AIM: To characterize the effect of two soy peptides, namely, ß-conglycinin-derived YVVNPDNDEN and YVVNPDNNEN on the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key-regulators of the LDL-R. METHODS: PCSK9 promoter activity (luciferase assay), PCSK9 protein expression (WB) and secretion (ELISA), PCSK9 interaction with LDL-R (binding assay) and human HepG2 cells were the objects of this investigation. RESULTS: Treatment with YVVNPDNNEN peptide has led to a rise in PCSK9 gene expression (90.8%) and transcriptional activity (86.4%), and to a decrement in PCSK9 intracellular and secreted protein (-42.9%) levels. YVVNPDNNEN peptide reduced the protein expression of transcriptional factor HNF1α. Most changes driven by YVVNPDNDEN peptide were not statistically significant. Neither peptide inhibited the PCSK9-LDLR interaction. CONCLUSIONS: Although sharing a common effect on LDL-R levels through the inhibition of 3-hydroxy-3-methylglutaryl CoA reductase activity, only the YVVNPDNNEN peptide has an additional mechanism via the downregulation of PCSK9 protein levels.

Food-derived antioxidants and COVID-19.

SARS-CoV-2 (previously 2019-nCoV), the pathogenic agent of COVID-19 disease, started to expand from Wuhan, China, on December 2019 and in 2 months, it spread worldwide giving origin to a pandemic. COVID-19 has a stronger transmission capacity by inhalation of infectious aerosols and after an incubation time of 3-14 days, it may be responsible for diseases ranging from the asymptomatic to fatal consequences. COVID-19 has emerged as a multifaceted, multisystem, multi-organ disorder, which produces its pathogenic effects through a quite ubiquitous target at the level of multiple organs and in which oxidative stress and inflammatory process play relevant roles. Thus, besides the development of a pharmacological therapy, in the field of alternative and coadjutant therapeutic, the use of dietary supplements or nutraceuticals for the prevention or treatment of SARS-CoV-2 infection may be a useful strategy. Herein, we specifically comment on some literature evidences, which link the food-derived antioxidants and metal-chelating agents with treatment and prevention of oxidative stress and inflammation that play a key role in the progression of COVID-19. PRACTICAL APPLICATIONS: Oxidative stress and inflammation are key factors increasing COVID-19 severity especially in the presence of chronic diseases associated with the antioxidant system fragility. These evidences support the recommendation of antioxidants supplementation as useful strategies against COVID-19. In light with these observations, herein, a comment which describes the major antioxidants and metal-chelating agents from food sources that might be useful for the treatment and prevention of oxidative stress and inflammation during COVID-19.

Extra Virgin Olive Oil Phenol Extracts Exert Hypocholesterolemic Effects through the Modulation of the LDLR Pathway: In Vitro and Cellular Mechanism of Action Elucidation.

This study was aimed at investigating the hypocholesterolemic effects of extra virgin olive oil (EVOO) phenols and the mechanisms behind the effect. Two phenolic extracts were prepared from EVOO of different cultivars and analyzed using the International Olive Council (IOC) official method for total phenols, a recently validated hydrolytic procedure for total hydroxytyrosol and tyrosol, and 1H-NMR analysis in order to assess their secoiridoid profiles. Both of the extracts inhibited in vitro the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in a dose-dependent manner. After the treatment of human hepatic HepG2 cells (25 µg/mL), they increased the low-density lipoprotein (LDL) receptor protein levels through the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, leading to a better ability of HepG2 cells to uptake extracellular LDL molecules with a final hypocholesterolemic effect. Moreover, both of the extracts regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Unlike pravastatin, they did not produce any unfavorable effect on proprotein convertase subtilisin/kexin 9 (PCSK9) protein level. Finally, the fact that extracts with different secoiridoid profiles induce practically the same biological effects suggests that the hydroxytyrosol and tyrosol derivatives may have similar roles in hypocholesterolemic activity.

Assessment of the Multifunctional Behavior of Lupin Peptide P7 and Its Metabolite Using an Integrated Strategy.

LTFPGSAED (P7) is a multifunctional hypocholesterolemic and hypoglycemic lupin peptide. While assessing its angiotensin-converting enzyme (ACE) inhibitory activity, it was more effective in intestinal Caco-2 cells (IC50 of 13.7 µM) than in renal HK-2 cells (IC50 of 79.6 µM). This discrepancy was explained by the metabolic transformation mediated by intestinal peptidases, which produced two main detected peptides, TFPGSAED and LTFPG. Indeed LTFPG, dynamically generated by intestinal dipeptidyl peptidase IV as well as its parent peptide P7 were linearly absorbed by mature Caco-2 cells. An in silico study demonstrated that the metabolite was a better ligand of the ACE enzyme than P7. These results are in agreement with an in vivo study, previously performed by Aluko et al., which has shown that LTFPG is an effective hypotensive peptide. Our work highlights the dynamic nature of bioactive food peptides that may be modulated by the metabolic activity of intestinal cells.

"Bottom-Up" Strategy for the Identification of Novel Soybean Peptides with Angiotensin-Converting Enzyme Inhibitory Activity.

IAVPTGVA (Soy1) and LPYP are two soybean peptides, which display a multifunctional behavior, showing in vitro hypocholesterolemic and hypoglycemic activities. A preliminary screening of their structures using BIOPEP suggested that they might be potential angiotensin-converting enzyme (ACE) inhibitors. Therefore, a bottom-up-aided approach was developed in order to clarify the in vitro hypotensive activity. Soy1 and LPYP dropped the intestinal and renal ACE enzyme activity with IC50 values equal to 14.7 ± 0.28 and 5.0 ± 0.28 µM (Caco-2 cells), and 6.0 ± 0.35 and 6.8 ± 0.20 µM (HK-2 cells), respectively. In parallel, a molecular modeling study suggested their capability to act as competitive inhibitors of this enzyme. Finally, in order to increase both their stability and hypotensive properties, a suitable strategy for the harmless control of their release from a nanomaterial was developed through their encapsulation into the RADA16-assembling peptide.

Soybean Peptides Exert Multifunctional Bioactivity Modulating 3-Hydroxy-3-Methylglutaryl-CoA Reductase and Dipeptidyl Peptidase-IV Targets in Vitro.

This study was aimed at evaluating the cellular mechanism through which peptic (P) and tryptic (T) soybean hydrolysates modulate the targets involved in hypocholesterolemic pathways in HepG2 and antidiabetic pathways in Caco-2 cells. Both hydrolysates (tested in the concentration range of 0.5-2.5 mg/mL) inhibited the 3-hydroxy-3-methylglutaryl-CoA reductase activity in HepG2 cells. In addition, Soybean P increased LDLR protein levels on HepG2 membranes by 51.5 ± 11.6% and 63.0 ± 6.9% (0.5-1.0 mg/mL) whereas Soybean T increased them by 55.2 ± 9.7% and 85.8 ± 21.5% (0.5-1.0 mg/mL) vs the control, with a final improved HepG2 capacity in the uptake of extracellular LDL. Soybean P reduced in vitro the dipeptidyl peptidase-IV activity by 16.3 ± 3.0% and 31.4 ± 0.12% (1.0 and 2.5 mg/mL), whereas Soybean T reduced it by 15.3 ± 11.0% and 11.0 ± 0.30% (1.0 and 2.5 mg/mL) vs the control. Finally, both hydrolysates inhibited dipeptidyl peptidase-IV activity in situ in human intestinal Caco-2 cells. This investigation may help to explain the activities observed in experimental and clinical studies.

Hypocholesterolaemic Activity of Lupin Peptides: Investigation on the Crosstalk between Human Enterocytes and Hepatocytes Using a Co-Culture System Including Caco-2 and HepG2 Cells.

Literature indicates that peptic and tryptic peptides derived from the enzymatic hydrolysis of lupin protein are able to modulate cholesterol metabolism in human hepatic HepG2 cells and that part of these peptides are absorbed in a small intestine model based on differentiated human Caco-2 cells. In this paper, a co-culture system, including Caco-2 and HepG2 cells, was investigated with two objectives: (a) to verify whether cholesterol metabolism in HepG2 cells was modified by the peptides absorption through Caco-2 cells; (b) to investigate how lupin peptides influence cholesterol metabolism in Caco-2 cells. The experiments showed that the absorbed peptides, not only maintained their bioactivity on HepG2 cells, but that this activity was improved by the crosstalk of the two cells systems in co-culture. In addition, lupin peptides showed a positive influence on cholesterol metabolism in Caco-2 cells, decreasing the proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion.

Nutraceuticals for blood pressure control.

Significant effects on blood pressure (BP) have been reported from large nutritional interventions, particularly the Dietary Approaches to Stop Hypertension (DASH) and the Mediterranean diet. In more recent years, numerous studies have investigated the possible BP-lowering effect of different nutraceuticals; these range from specific foods to minerals, lipids, whole proteins, peptides, amino acids, probiotics, and vitamins. While a very large body of evidence supports the use of potassium, L-arginine, vitamins C and D, cocoa flavonoids, beetroot juice, some probiotics, coenzyme Q10, controlled-release melatonin, aged garlic extract, and coffee, the use of other nutraceuticals, such as green tea, flaxseed, and resveratrol, has not as yet been supported by adequate evidence. In some cases, e.g. proteins/peptides, the responsible component needs also to be fully uncovered. Finally, while for most of the products only short-term studies are available, with no specific end-points, an ongoing very large prospective study on chocolate flavanols will answer the question whether this may reduce cardiovascular risk. Thus, in addition to data on long-term safety, further clinical research is advisable in order to identify, among active nutraceuticals, those with the best cost-effectiveness and risk-benefit ratio for a wide use in the general population with a raised cardiovascular risk consequent to uncomplicated hypertension.

A simple and high-throughput in-cell Western assay using HepG2 cell line for investigating the potential hypocholesterolemic effects of food components and nutraceutics.

Since saving time and money are critical issues while developing innovative functional foods and nutraceutics, the use of specific and high-throughput assays for the fast screening of potentially bioactive ingredients is crucial. In this context, the aim of the present investigation was the development of an in-cell Western (ICW) assay, a quantitative colorimetric cell-based technique, at the HepG2 cell line for screening and evaluating the effects of potentially bioactive compounds on the low density lipoprotein (LDL) receptor (LDLR). It is known that LDLR plays a pivotal role in the binding and endocytosis of circulating LDL, increasing its plasma clearance. The ICW was optimised and validated using monacolin K, the main hypocholesterolemic component of red yeast rice. This provided a robust and reproducible assay useful for characterising the cholesterol-lowering properties of bioactive food components. To our knowledge, this is the first application of the ICW technique in the field of functional foods and nutraceutics.

Nutraceutical approach to moderate cardiometabolic risk: results of a randomized, double-blind and crossover study with Armolipid Plus.

BACKGROUND: Primary cardiovascular prevention may be achieved by lifestyle/nutrition improvements and specific drugs, although a relevant role is now emerging for specific functional foods and nutraceuticals. OBJECTIVES: The aim of this study was to evaluate the usefulness of a nutraceutical multitarget approach in subjects with moderate cardiovascular risk and to compare it with pravastatin treatment. SUBJECTS: Thirty patients with moderate dyslipidemia and metabolic syndrome (according to the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults) were included in an 8-week randomized, double-blind crossover study and took either placebo or a nutraceutical combination that contained red yeast rice extract, berberine, policosanol, astaxanthin, coenzyme Q10, and folic acid (Armolipid Plus). Subsequently, they were subjected to another 8-week treatment with pravastatin 10 mg/d. This dosage was selected on the basis of its expected -20% efficacy in reducing low-density lipoprotein-cholesterol. RESULTS: Treatment with Armolipid Plus led to a significant reduction of total cholesterol (-12.8%) and low-density lipoprotein-cholesterol (-21.1%), similar to pravastatin (-16% and -22.6%, respectively), and an increase of high-density lipoprotein-cholesterol (4.8%). Armolipid Plus improved the leptin-to-adiponectin ratio, whereas adiponectin levels were unchanged. CONCLUSIONS: These results indicate that this nutraceutical approach shows a lipid-lowering activity comparable to pravastatin treatment. Hence, it may be a safe and useful option, especially in conditions of moderate cardiovascular risk, in which a pharmacologic intervention may not be appropriate.

Hypocholesterolaemic effects of lupin protein and pea protein/fibre combinations in moderately hypercholesterolaemic individuals.

The present study was aimed to evaluate the effect of plant proteins (lupin protein or pea protein) and their combinations with soluble fibres (oat fibre or apple pectin) on plasma total and LDL-cholesterol levels. A randomised, double-blind, parallel group design was followed: after a 4-week run-in period, participants were randomised into seven treatment groups, each consisting of twenty-five participants. Each group consumed two bars containing specific protein/fibre combinations: the reference group consumed casein+cellulose; the second and third groups consumed bars containing lupin or pea proteins+cellulose; the fourth and fifth groups consumed bars containing casein and oat fibre or apple pectin; the sixth group and seventh group received bars containing combinations of pea protein and oat fibre or apple pectin, respectively. Bars containing lupin protein+cellulose ( - 116 mg/l, - 4·2%), casein+apple pectin ( - 152 mg/l, - 5·3%), pea protein+oat fibre ( - 135 mg/l, - 4·7%) or pea protein+apple pectin ( - 168 mg/l, - 6·4%) resulted in significant reductions of total cholesterol levels (P<0·05), whereas no cholesterol changes were observed in the subjects consuming the bars containing casein+cellulose, casein+oat fibre or pea protein+cellulose. The present study shows the hypocholesterolaemic activity and potential clinical benefits of consuming lupin protein or combinations of pea protein and a soluble fibre, such as oat fibre or apple pectin.

Functional foods for dyslipidaemia and cardiovascular risk prevention.

A food can be regarded as 'functional' if it can demonstrate a beneficial efficacy on one or more target functions in the body in a convincing way. Beyond adequate nutritional qualities, functional foods should either improve the state of health and wellbeing and/or reduce the risk of disease. Functional foods that are marketed with claims of heart disease reduction focus primarily on the major risk factors, i.e. cholesterol, diabetes and hypertension. Some of the most innovative products are designed to be enriched with 'protective' ingredients, believed to reduce risk. They may contain, for example, soluble fibre (from oat and psyllium), useful both for lowering cholesterol and blood pressure, or fructans, effective in diabetes. Phytosterols and stanols lower LDL-cholesterol in a dose-dependent manner. Soya protein is more hypocholesterolaemic in subjects with very high initial cholesterol and recent data indicate also favourable activities in the metabolic syndrome. n-3 Fatty acids appear to exert significant hypotriacylglycerolaemic effects, possibly partly responsible for their preventive activity. Dark chocolate is gaining much attention for its multifunctional activities, useful both for the prevention of dyslipidaemia as well as hypertension. Finally, consensus opinions about tea and coffee have not emerged yet, and the benefits of vitamin E, garlic, fenugreek and policosanols in the management of dyslipidaemia and prevention of arterial disease are still controversial.

Nutritional and nutraceutical approaches to dyslipidemia and atherosclerosis prevention: Focus on dietary proteins.

A correct lifestyle is crucial in the primary and secondary prevention of cardiovascular disease. Innovative nutritional strategies to reduce the main risk factors have been developed including either dietary changes or consumption of specifically targeted functional foods and dietary supplements. These nutraceutical products may also provide an alternative to lipid lowering, antihypertensive, and antidiabetic drugs. Functional foods and beverages have the appearance of normal foods, but contain specific components whose activity on at least one measurable risk factor has been scientifically demonstrated. Dietary supplements, having formulations similar to drugs, allow the delivery of a bioactive ingredient in dosages that exceed those obtainable from food products. Among bioactive components, at present dietary proteins from both vegetable and animal sources are of high interest, because of their specific effects on cholesterolemia and blood pressure. Active peptides have been identified for the latter indication, whereas works is in progress in attempting to identify specific cholesterol lowering peptides.

Parameters for the evaluation of the thermal damage and nutraceutical potential of lupin-based ingredients and food products.

Foods based on sweet lupin proteins are gaining attention from industry and consumers because of their possible role in the prevention of cardiovascular disease. When promoting lupin-based foods for inclusion in a daily diet, the thermal damage suffered during processing is of relevance to the bioactive and nutritional quality of the food product. N-(2-furoylmethyl)-L-lysine (furosine) quantification demonstrates that currently available sweet lupin protein isolates have a thermal damage comparable to or lower than other traditional food ingredients, and are a good source of lysine in non-dairy products. In lupin-based foods claiming to have cholesterol-lowering potential, shotgun proteomics offers itself as a fast and effective screening method for assessing the biological availability of active peptides. Such a method is readily applicable to other legume-enriched food products.

Phytoestrogens: end of a tale?

Phytoestrogens are plant-derived hormone-like diphenolic compounds of dietary origin that are present at high levels in plasma of subjects living in areas with low atherosclerosis and cancer incidence. The term phytoestrogen is commonly applied to the soy isoflavones genistein, daidzein and glycitein. As outlined in a previous review article in this journal by Adlercreutz and Mazur 1, these compounds are weakly estrogenic and appear to influence the cardiovascular system, the production, metabolism and biological activity of sex-hormones, as well as malignant cell proliferation, differentiation and angiogenesis. Recently skepticism has developed concerning the true potential of phytoestrogens to beneficially modify these processes. A critical analysis of the early findings from supplementing the diet with soy protein has failed to confirm phytoestrogens as the responsible agent for beneficial cardiovascular effects, be it by way of lipid reduction, vasodilation or lipoprotein oxidation. Furthermore, contrasting data have been reported on the potential of phytoestrogens to prevent hormone-dependent cancers (e.g. breast and prostate) and to successfully treat post-menopausal complaints, an indication for which they are widely used. These potentially negative findings have led health authorities in several countries to suggest maximum daily intake levels for phytoestrogens. There is now growing interest in the use of soy products containing low levels of phytoestrogens and in research on other phytoestrogen free legumes such as lupin.

Analysis of Lupinus albus storage proteins by two-dimensional electrophoresis and mass spectrometry.

A laboratory-prepared total protein extract (TPE) and a lupin protein isolate (LPI-E) produced in a pilot plant were submitted to a detailed two-dimensional (2DE) proteomic investigation. Recent findings have indicated that in an established rodent model of hyperlipidemia, moderate daily intakes of LPI-Es lead to a reduction of total and low-density lipoprotein cholesterol levels, and the knowledge of the actual composition of the protein sample used in that study is at the basis of further structure/action investigations. The experimental results indicate that the semi-industrial procedure used for the production of LPI-E damages only marginally the proteins. It does, however, cleave some disulfide bridges and induce mild proteolysis, as confirmed by the higher number of resolved protein spots in the low Mr and acidic pI region of the 2DE map. Out of 72 spots submitted to mass spectrometry and compared with available protein databases, 42 correspond to fragments of beta-conglutin, the 7S globulin of lupin, spanning between positions 37 and 495 of the protein sequence. Using the bioinformatic tool BlastP, these peptides were compared to the alpha'-subunit of beta-conglycinin, the 7S globulin of soybean, this being the most active hypocholesterolemic component of soybean protein, as shown by in vitro and in vivo experiments. At least 18 peptides derived from beta-conglutin, having a percentage identity higher than 50% and a similarity percentage higher than 70% vs the alpha'-subunit of beta-conglycinin, are likely candidates to be the biologically active components of lupin protein.