Antioxidant capacity and polyphenolic content of high-protein lupin products

In order to produce high protein lupin products, α-galactoside extraction from Lupinus angustifolius cv. Troll and cv. Emir and Lupinus albus cv. Multolupa, and protein isolation from L. albus cv. Multolupa were carried out. Trolox equivalent antioxidant capacity (TEAC), DPPH radical-scavenging activity (DPPH-RSA), peroxyl radical-trapping capacity (PRTC), superoxide dismutase-like activity (SOD-like activity), total phenolic compounds (TPC) and total flavonoids were determined in lupin products. In L. angustifolius cv. Troll, L. angustifolius cv. Emir and L. albus cv. Multolupa α-galactoside-low flours, the TEAC and DPPH-RSA decreased (43%, 38%; 73%, 82%; 77%, 38%, respectively). PRTC decreased in L. angustifolius cv. Troll and L. albus cv. Multolupa α-galactoside-low flours (13% and 98%, respectively), while in those of L. angustifolius cv. Emir, PRTC increased (25%). SOD-like activity and TPC were also affected by α-galactoside extraction and reductions of 30–52% and 38–56%, respectively, were observed. The protein isolate of L. albus cv. Multolupa presented lower TEAC (24%), a similar level of PRTC and twice higher level of DPPH-RSA than did α-galactoside-low Multolupa flour. Moreover, the SOD-like activity of lupin protein isolate was sharply reduced but the TPC content was 24% higher. The technological procedures, α-galactoside extraction and protein isolation, provide high protein lupin products but with lower antioxidant capacity and total phenolic compounds than the raw seeds, although the levels of antioxidant capacity of these lupin products resemble those of cereals.     

Development and validation of a sandwich ELISA for the determination of potentially allergenic lupine in food

The paper presents a sandwich enzyme linked immunosorbent assay (ELISA) for the detection of traces of lupine proteins in foods. Anti-lupine antibodies were produced by immunising a rabbit and a hen with a protein extract from white lupine flour. IgY were used as coating and IgG as secondary antibodies. The ELISA detects proteins from white (Lupinus albus) and blue (Lupinus angustifolius) lupine and, with a lower sensitivity, proteins from yellow (Lupinus luteus) lupine. The ELISA does not show any cross-reactivity with 34 plant species potentially used in lupine containing foodstuffs. Accuracy, repeatability, limit of detection (LOD) and limit of quantification (LOQ) were determined by analysing model biscuits and noodles containing from 0 to 10,000 ppm lupine flour. Lupine flour could be detected in the unprocessed doughs as well as in the processed products down to a spiking level of 1 ppm.     

Effects of oligosaccharide removing procedure on the protein profiles of lupin seeds

The effects on the protein pattern of raffinose family oligosaccharides ethanol extraction from intact lupin seeds (Lupinus albus and Lupinus luteus) has not been addressed so far. In this work, 1D and 2D electrophoretic techniques were used to detect changes of the protein profiles upon oligosaccharide removal procedure. Some differences between untreated and treated samples were clearly visible in 1D electrophoresis, where the decrease of some polypeptides was revealed. In addition, a dark zone at the top of each treated sample lane became visible, suggesting the formation of large-sized protein aggregates as a result of the extraction procedure. By using the greater resolution of 2D electrophoresis, the identification of some varying spots was made possible: In particular the bands corresponding to conglutin γ were significantly reduced in the processed samples. The leakage of this protein by ethanol treatment of the seeds was qualitatively and quantitatively confirmed with specific antibodies by Western and dot blotting techniques, respectively. Another polypeptide around 100 kDa undergoing decrease upon treatment was tentatively identified as lupin lipoxygenase, according to literature data and direct lipoxygenase activity measurements on treated and untreated seed extracts. The observed decrease of lipoxygenase activity in the processed seeds was 37%. These results show that the extraction of α-galactosides, while maintaining the overall pattern of lupin storage proteins, led to the reduction of some critical proteins. In particular, conglutin γ and lipoxygenase decrease could be desirable in view of their potential allergenicity and effects on the flour organoleptic characteristics, respectively.     

Effects of bread making methods,lupin variety and gluten powder on the quality of bread enriched with high percentage of lupin flour

Lupin is an economical source of protein, fibre and bioactive compounds, and to obtain these health and nutritional benefits lupin flour has been used in bread production. However, addition of more than 10% lupin flour markedly reduces bread quality mainly due to gluten dilution. The main aim of this research was to retain lupin bread quality enriched with higher percentages of lupin flour (20%) by addition of vital gluten powder (0%, 2%, 3.5% and 5%), investigating the effects of lupin variety (Lupinus albus and L. angustifolius) and two baking systems (rapid and sponge & dough). Impact on bread staling qualities was also determined through texture analysis of samples over a 72-h storage period. Compared to lupin bread with nil gluten addition, significant improvements in loaf volume and crumb texture were observed with addition of gluten powder especially at 5% which increased loaf volume by an average of 20% across lupin sources and baking methods, and crumb softness by 30–50%. Differences were observed between the lupin flour sources. L. angustifolius had a reduced weakening effect when blended with the base flour compared with L. albus. The Sponge & Dough process was found to be more suitable to the inclusion of lupin flour than the rapid process.     

Chemical composition of dehulled seeds of selected lupin cultivars in comparison to pea and soya bean

With the objective of promoting the cultivation, usage, and consumption of lupin, in the present study, the chemical and fibre composition of dehulled seeds of seven cultivars of different lupin species (Lupinus angustifolius, Lupinus albus and Lupinus luteus) was determined in comparison to pea (Pisum sativum) and soya bean (Glycine max).     

Microstructural changes in Lupinus campestris seed in response to three thermal debittering treatments

BACKGROUND: Lupinus campestris seed has a high protein (350–450 g kg^?1) and oil (180–200 g kg^?1) content, but its use is limited by toxic substances, such as quinolizidine alkaloids (QAs), that can be reduced by debittering thermal treatments (DTTs) which cause biochemical changes in seed compounds such as carbohydrates and proteins and could induce changes in the seed microstructure. This work aims to correlate biochemical and microstructural changes with nutritional composition in lupin seeds in response to DTT. RESULTS: Three DTTs, aqueous (AqT), acid (AcT) and alkaline (AlT), were performed with L. campestris and the effects on nutritional value were evaluated. Scanning electron microscopy was used to evaluate microstructural changes of raw and debittered seeds using image and fractal analysis tools. DTT caused a decrease of QAs, carbohydrate content and protein increase. These effects were more pronounced with A1T. Analysis of microstructural changes indicated decreased rugosity and smoother texture of seed surface subjected to DTT. Smoother surfaces exhibited oval and polyhedrical structures corresponding to globular protein aggregation. CONCLUSIONS: DTT had effects on the seed surface, which showed a smoother texture associated with oval and polyhedrical structures that may be caused by aggregation of globular proteins. These findings allowed associate debittering treatments to microstructural features. Copyright © 2009 Society of Chemical Industry     

Development and Validation of Two Competitive ELISAs for the Detection of Potentially Allergenic Lupine (Lupinus Species) in Food

The paper presents two competitive enzyme linked immunosorbent assays (ELISAs) for the detection of lupine proteins in foods. The ELISAs are based on anti-lupine antibodies produced in a rabbit (IgG) and in a chicken (IgY), respectively. Cross-reactivity tests with 33 foods of plant origin revealed that both ELISAs show slight cross-reactivity (≤1.5 %) with pecan nut. They do, however, not cross-react with 32 species potentially used in lupine containing foodstuffs. Both ELISAs detect proteins from white (Lupinus albus) and blue (Lupinus angustifolius) lupine and, with a lower sensitivity, proteins from yellow (Lupinus luteus) lupine. Matrix effects could be eliminated by diluting the sample extracts 1:10 before loading them onto the microtiter plate. The IgG ELISA allows the detection of 50 ppm white lupine flour in bread, vegetarian patties and rusk. With the IgY ELISA, lupine can be detected in vegetarian patties and bread at the 50 ppm spiking level and in rusk at the 100 ppm spike level.     

Germination as a process to improve the antioxidant capacity of Lupinus angustifolius L. var. Zapaton

Antioxidant capacity, measured by glutathione (GSH), superoxide dismutase-like activity (SOD-like activity), peroxyl radical-trapping capacity (PRTC), trolox equivalent antioxidant capacity (TEAC) and inhibition of lipid peroxidation in unilamellar liposomes of egg yolk phosphatidylcholine (PC) has been evaluated in raw and germinated lupin seeds (Lupinus angustifolius L. var. Zapaton) for 2, 3, 4, 5, 6 and 9 days. The content of antioxidant vitamins E and C has been also studied. The tripeptide GSH kept invariable for the first 5 days of germination and suffered a decrease of 20 and 78% after 6 and 9 days, respectively. During lupin germination, SOD-like activity increased slightly whilst PRTC doubled the amount after 9 days. TEAC values changed slightly up to 5 days of germination but after 6 and 9 days a significant increase (25 and 28%, respectively) was found. The oxidation of PC was inhibited by germinated lupin extracts and 9-day germination seeds provided the highest inhibition. Furthermore, germinated lupins provided more vitamin C, vitamin E activity and polyphenols than raw seeds, and the largest amounts of these bioactive compounds were found after 6 days of germination. Therefore, germination of lupin seeds (Lupinus angustifolius L. var. Zapaton) seems to be a good process to enhance their antioxidant capacity.     

The artificial intelligence-based chemometrical characterisation of genotype/chemotype of Lupinus albus and Lupinus angustifolius permits their identification and potentially their traceability

A chemotyping and genotyping comprehensive approach may be useful for the analytical traceability of food ingredients. The interest for lupin (Lupinus spp.) is developing owing to the high protein percentage as well as the positive technological and nutraceutical properties. The objective was the development of innovative models for discerning between Lupinus albus and Lupinus angustifolius, the most used in human nutrition, by applying multivariate statistical analysis (Principal Component Analysis, PCA) and artificial intelligence (Self Organising Maps, SOMs) onto chemical parameters (proximate composition, alkaloids, tocopherols) or Random Polymorphic DNA fingerprints. The application of PCA to either chemical or genetic data permitted the effective discrimination between the two species, whereas the application of the SOM approach to both data-sets enabled clustering some cultivars. The possibility of discriminating L. albus from L. angustifolius is relevant for lupin traceability: the foreseen fields of application are refined flours or ingredients, where morphological analysis is not applicable.     

Some functional properties of lupin proteins modified by lactic fermentation and extrusion

The aim of this work was to determine modifying effects of lactic fermentation and extrusion processes on functionality of lupin proteins. Protein content, surface hydrophobicity, water absorption capacity (WAC), water solubility index (WSI) and emulsifying properties (EAI, ESI) of protein preparations obtained from lupin seeds (Lupinus luteus, Lupinus albus, Lupinus angustifolius), with various contents of hull, were analyzed. Changes of protein properties were affected by lupin cultivar, hull content and applied processing method. An increase of soluble protein content after controlled lactic fermentation of lupin seeds, and changes of surface protein hydrophobicity, WAC and WSI values after each treatment and significant worsening of protein emulsifying properties were observed. Correlations were found between parameters examined in this study.     

Industrial orange juice debittering: Impact on bioactive compounds and nutritional value

The impact of an industrial debittering process (DP) on nutritional and bioactive compounds in orange juice (OJ) was studied. The DP was aimed at removing bitter components in OJ by physical adsorption in a resin. The levels of bioactive compounds (carotenoids, ascorbic acid and phenolics), total antioxidant activity and the colour in the fresh orange juices (non-debittered) and in the debittered counterparts were measured. The results demonstrated that the carotenoid contents were not significantly affected by the treatment. However, the debittered orange juices showed a reduction (p < 0.001) of 26% in ascorbic acid, 32% in hydroxycinnamic acids, 28% of flavones and 41% of flavanones in comparison with the non-treated juices. The antioxidant activity of the hydrophilic fraction (HF) was significantly higher (p < 0.05) in untreated juice than in debittered juices. Some colour parameters (L^∗, a^∗ and h_ab) were also affected. Discriminant analysis revealed that the canonical function related to the levels of HF compounds allowed a 100% correct classifications of the different types of juices.     

Cross-reactivity between peanut and lupin proteins

Peanut-allergic individuals may also react to lupin, which, for this reason, has been included in the EU list of food allergens. Since there is not yet any general consensus on the major allergen/s in lupin, the objective of this investigation was to compare the reactivity of the main lupin proteins towards the IgE of the sera of allergic patients. Both Lupinus albus and Lupinus angustifolius were investigated. ELISA’s, Western blotting and mass spectrometry, including also de novo sequencing of the unknown lupin proteins, were used for identifying the IgE-binding proteins. Significant differences in the protein reactivities were observed. In particular, there was a direct relationship between the level of peanut-specific IgE’s and the cross-reactivity to lupin proteins; also the reactivity of each serum appeared to be unique. Although numerous lupin proteins bind IgE’s, our data suggest a predominant contribution of α-conglutin in the reactivity of both L. albus and L. angustifolius.     

Raffinose family oligosaccharides and sucrose contents in 13 Spanish lupin cultivars

The contents of raffinose family oligosaccharides (RFOs) and sucrose in 13 Spanish cultivars of lupin (Lupinus albus, Lupinus luteus and Lupinus angustifolius) were studied, with the aim of selecting those with highest amounts of these oligosaccharides in order to obtain pure RFOs for use as ingredients in functional foods. The levels of sucrose and RFOs (raffinose, stachyose and verbascose) were determined using high perfornnance liquid chromatography (HPLC). There were large variations in the levels of individual RFOs between lupin cultivars. L. albus seeds were characterised by the lowest verbascose content (∼0.4%), L. luteus seeds by the highest contents of stachyose (∼7.4%) and verbascose (∼3.1%) and the lowest of sucrose (∼1.2%),and L. angustifolius seeds by the highest sucrose (∼3.4%) and the lowest stachyose (∼4.6%) contents. Furthermore, there was a wide variation in total α-galactosides between species, with a remarkably high content found in L. luteus (9.5–12.3%) which was about twice that of other lupin cultivars. For this reason, L. luteus seeds are the best choice for obtaining pure RFOs for use as prebiotics in functional foods.     

Highly automated and fast determination of raffinose family oligosaccharides in Lupinus seeds using pressurized liquid extraction and high‐performance anion‐exchange chromatography with pulsed amperometric detection

BACKGROUND: Taking into account several requirements for the determination of raffinose family oligosaccharides (RFOs) from Lupinus seeds—e.g., conducting plant breeding projects or food product development—a reasonable combination of efficient automated sample preparation and reliable analysis need to be developed and validated. RESULTS: In this regard pressurized liquid extraction was applied to extract the RFOs from ground and defatted lupin flour. Compared to many other publications, no further pretreatment, such as protein precipitation, was necessary to obtain satisfactory results applying ion chromatography with pulsed amperometric detection. The oligosaccharide content for the examined Lupinus albus samples were in the range 5.19–9.25 g kg⁻¹ and for Lupinus angustifolius RFOs 3.49–4.75 g kg⁻¹. Stachyose has always been the main component followed by raffinose and verbascose. CONCLUSION: The developed sample preparation and analytical method is suited to quantify raffinose, stachyose, verbascose and the disaccharide sucrose and, owing to a high degree of automation for sample preparation and relatively short analysis times by pretty peak separation, particularly high sample numbers can be accomplished. Copyright © 2008 Society of Chemical Industry     

Characterization of Lupine Proteins

Proteins of eight cultivars of two lupine species (Lupinus albus and L. angustifolius) were examined by several procedures. Differences in protein solubility between the species and among varieties within a species were observed. Defatting of the lupine flour altered protein solubility. Amino acid compositions of total protein and protein fractions showed only minor differences between the species and among varieties within a species (except for cv. Kali). Sodium dodecylsulfate polyacrylamide gel electrophoresis patterns of protein fractions revealed both qualitative and quantitative differences between species and among varieties.     

Evaluation of debittered and germinated fenugreek (Trigonella foenum graecum L.) seed flour on the chemical characteristics,biological activities,and sensory profile of fortified bread

Germination of fenugreek seed showed better nutraceutical profile as evaluated by in vivo (oral glucose tolerance and acute antihypertriglyceridemic tests) on rats and in vitro DPPH and enzyme inhibition assays (α‐glucosidase, lipase, and lipoxygenase). Attempts were made to develop debittered and germinated fenugreek seed flour (DGFSF) by soaking in acesulfame (0.25%, 8 hr), curd (1:1 in water, 8 hr) and sucrose (5%, 8 hr) solution at 1:4 (wt/vol) ratio for debittering and then germinated, of which curd showed the maximum extent of debittering. The effect of substitution of wheat flour with 5, 10, 15, and 20% (wt/wt) of DGFSF on chemical characteristics and sensory properties was investigated; 10% fortified bread was organoleptically acceptable and had enhanced protein and fiber contents. Fortification increased the bulk density and crumb firmness, decreased the specific loaf volume, and darkened the crumb color. Glycemic index of the bread decreased with 10% DGFSF, which is indicative of desirable health benefits.

Practical applications

Fenugreek seeds are major constituent of Indian spices, bitter to taste and possess various medicinal properties. These seeds are an excellent source of dietary fiber and protein. Although incorporated into traditional foods, its bitterness limits its wide usage. In India, soaking and germination of seeds in water or curd was practiced traditionally since ancient days. Germination and debittering improved antioxidant activity, and various in vitro and in vivo bioactivities. Incorporation of debittered flour improved the polyphenolic, protein, and dietary fiber contents in bread which in turn demonstrated reduced rate of in vitro starch digestibility or lower glycemic index     

The Use of Vacuum Impregnation for Debittering Green Olives

The aim of this research was to assess the possibility of shortening the length of the debittering process for green table olive ‘Domat’ cultivar by vacuum impregnation (VI). For this purpose, debittering was carried out with NaCl (3 %), NaOH (1.5 %) and NaOH (1.5 %) + NaCl (3 %) solutions at atmospheric conditions and under vacuum (68 kPa). The effects of these applications on some physicochemical properties (total dry matter, total ash, titratable acidity, salt, protein, oil, oleuropein, total phenolics, antioxidant activity and colour) of the processed samples were determined. Total dry matter, titratable acidity, salt, protein and oil contents of the samples changed between 24.23 and 27.90, 0.22 and 0.45, 2.27 and 2.58, 0.50 and 1.26 and 6.79 and 9.42 % (w/w), respectively. Colour parameters (lightness (L*); redness (a*); yellowness (b*)) of the processed olives were measured as between 41.72 and 51.29, 15.09 and 13.30 and 22.79 and 34.84, respectively. Hue angles of the processed samples were changed between 59.78 and 68.52. VI was a useful tool for the shortening of the period of debittering process. Use of NaOH combined with NaCl in VI process caused the debittering period to decrease to 6 h. However, reduction of the amount of total phenolic compounds was the highest (21.43 %) in this condition. Use of alkali for debittering caused more dry matter diffusion from olive to the solution. There was no statistically significant difference between the oleuropein and total ash contents of the processed samples (p?<?0.01). Alkali treatment also caused more oil loss from the olives. Antioxidant activity of the samples reduced up to 59.89 %. This research was the first using VI for debittering of olives; further studies are necessary to optimise process conditions of debittering for limiting loss of natural antioxidants and other components and to ensure maximum benefits to the consumer.     

Nutritional and quality aspects of wheat sourdough bread using L. luteus and L. angustifolius flours fermented by Pedioccocus acidilactici

Lupine has the potential to be a new domestic source of vegetable protein due to its comparable quality to the commonly used soy proteins. However, the bioprocessing that take place in the production of wheat bread with non‐conventional flours could play an important role. The wholemeal Lupinus angustifolius and Lupinus luteus flours were fermented by bacteriocin‐producing strain of Pediococcus acidilactici. The effect of lupine flour supplementation on wheat bread quality, sensory and safety criteria was studied. The lupine additives significantly decreased the quality of bread. The fermented L. luteus flour (10% of flour basis) had a slightly higher positive effect on the specific volume and crumb porosity (5.4%) and lowering of crumb hardness (9.5%) than those of L. angustifolius. In contrary, consumers rated higher for bread with L. angustifolius sourdough, which contributed to a stronger taste score. The levels of tyramine, histamine and putrescine (32.6–215.8, 20.8–96.7 and 33.7–195.2 mg kg^?1, respectively) do not present a health risk for consumers due to their relatively low levels in lupine fermented products. Bioprocessing used for wheat bread production with lupine flour additives could improve the nutritional profile of bread without increasing the risk of biogenic amine formation.     

Sorption of citrus flavour compounds on XAD‐7HP resin during the debittering of grapefruit juice

Grapefruit juice made from concentrate was debittered using a laboratory‐scale downflow column packed with XAD‐7HP adsorbent resin. Sorption of the bitter principle naringin was analysed by high‐performance liquid chromatography. In addition, the influence of the debittering process on the flavour of the juice was investigated using headspace gas chromatography for the volatile flavour compounds α‐pinene, β‐myrcene, d‐limonene, α‐terpineol and β‐caryophyllene. Kinetic constants were determined for all substances using the Lagergren adsorption model. The debittered juices showed a selective shift in their reduced flavour profile. Reduction of naringin to 51.28% of its initial value led to a decrease in α‐terpineol to 43.05%, whereas the other flavour compounds ranged from 76.27% (β‐myrcene) to 92.76% (β‐caryophyllene). The column flow rate influenced the adsorption of α‐terpineol and naringin. The highest affinity for adsorption was consistently observed for the off‐flavour α‐terpineol, which indicates a promising option for its selective removal from processed juices during debittering.     

Primary structure of 2S albumin from seeds of Lupinus albus

 The 2S albumin from the seeds of white lupin (Lupinus albus L.) was purified by ammonium sulfate precipitation and ultrafiltration followed by anion-exchange and reversed-phase HPLC. The complete amino acid sequences of the large and the dominating small subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin (a) consists of 37 amino acid residues resulting in a molecular mass M _r of 4407. The large subunit (b) contains 75 amino acid residues (M _r=8827). The two polypeptide chains are linked by two interchain disulfide bonds (Cys^a8-Cys^b24 and Cys^a20-Cys^b13 or, more probably, Cys^a20-Cys^b12). In addition, the large polypeptide contains two intrachain disulfide bridges (Cys^b26-Cys^b68 and Cys^b12-Cys^b60 or, more probably, Cys^b13-Cys^b60) and one free sulfhydryl group (Cys^b40). A high degree of homology (88%) exists between the primary structure of the 2S albumin from L. albus and that of a comparable 2S protein from L. angustifolius, designated conglutin δ. Received: 4 April 1996