Livrables
Lupin would be a potential raw material to produce vegetable drinks thanks to its high protein content and the suitable functional properties of protein ingredients isolated thereof. Two basic methods for drink production were investigated: from Lupin Protein Isolate(Fraunhofer IVV &A.U.Th)& from Lupin Flour(Terrena)
These new vegetable drinks could be a high quality alternative to milk as they have a similar protein content but with an improved composition of lipids.
Fraunhofer IVV:
Lupin protein isolate was used in concentrations of 2.7 - 3.5% and oil contents of 2 -2.5% in order to produce a lupin drink similar to commercial vegetable drinks (e.g.soy). The drink was prepared by high pressure homogenisation, with a pressure of 200 bars. Viscosity curves of all samples showed Newtonian behaviour with low viscosity. The most stable drink with lower droplet mean diameter than the reference and comparable viscosity was obtained at a lupin protein concentration of 2.75%.
This drink was evaluated in a sensory panel, using a simple descriptive assay. Deviations from the reference sample (commercial soy drink) were evaluated by using a standardized scale with intensities from 0 (no deviation) to 4 (strong difference). The test showed, that the sensory properties were comparable or even better than commercial soy beverages. The lupin drink deviated significantly from the soy reference in most of the characteristics. The most positive deviation was achieved concerning the smell and the taste.
AUTh: Lupin drinks were prepared from lupin protein isolates and concentrates by emulsifying lupin protein solutions and homogenized with an APV laboratory homogenizer. Emulsion mean droplet size, stability against creaming and rheological behavior were evaluated. LPI-E and LPI-F were chosen for the preparation of model lupin drinks for organoleptic evaluation. They were prepared with protein and fat content adjusted to the respective content of cow milk. They were flavoured with vanilla/cocoa. The final recipes (%w/v) are: 3,5% LPI-E, 2% olive oil, 7% sucrose, 0,2% vanilla ; 3,5% LPI-E, 2% olive oil, 10% sucrose, 1,5% cocoa, 0,02% caragennan.
Lupin drinks with vanilla and cocoa were quite satisfying almost as the commercial soy drinks.
TERRENA developed a process for produce a lupin-based drink from lupin flour and produced several batches. The vegetable milk is produced by extraction of lupin protein at basic pH for a best protein solubilization. After settling, the supernatant, containing the soluble lupin proteins, is ultrafiltered to eliminate small molecules. Finally the drink is sterilised by UHT to increase the shelf-life without impairing the taste, colour or nutritional value.
This process allows obtaining a healthy white base-drink with a slight touch of yellow. The new drink presents a high-quality nutritional value: for example rich in protein, source of 3-Omega, source of calcium, iron and magnesium. The product is lactose free and gluten free.
Sensory tests gave good results: three vanilla flavoured beverages were prepared from the basic, unflavoured drink and evaluated by 73 persons: 100%lupin, 100%soya and 50%lupin/50%soya.
Appearance, smell and taste were evaluated in an hedonistic test by the panel. Samples were made anonymous before testing. The sensory test showed that the lupin drink offered a better colour and smelling compared to a standard vanilla flavoured soya drink. Concerning taste, there is not big difference between the answers of the panel, the results are almost identical: 32% of lupin, 33% for soy and 35% for the mixed drink. From this liquid base it is possible to produce different flavoured and high-nutritional vegetable drinks and other products as vegetable yoghurt or vegetable cream.
Potential application: Dietary products sector, functional foods sector
Potential users of results: Food industry, especially SME´s.
Innovative features/benefits: 100% vegetable drink, free of cholsterol, high protein content possible, similar protein content as cow milk but improved lipid composition, product is suitable for diets to prevent cardiovascular disease (cholesterol lowering)
Market potential:Lupin drinks have the potential to be placed on the functional food market which is experiencing strong growth over the next years. Sales of functional products rose by 43% in Europe between 1999 and 2004 reaching a total value of 4 billion - (source: Datamonitor). By far the largest sector was that of beverages, which accounted for 59% of functional product sales in 2003. The Lupin drinks may participate at this market as a (non-GM) alternative to soy based products or products based on animal proteins. The total soy food market in Europe is expected to grow to a market volume of 2 billion in 2007.
Potential barriers: Products only tested in laboratory scale, no market study available, lupin based products are a niche market at the moment and no market data are available.
In the present study, we compared the effects of lupin and soy proteins on blood pressure and vascular function in two experimental animal models: one for type II diabetes (GK), and the other for salt-sensitive hypertension (DSS). In both studies the development of hypertension was accelerated by sodium loading. We showed for the first time, that in the diabetic animals both protein treatments lowered the elevated blood pressure to the level of normotensive controls and both of the protein treatments also normalised the impaired vasocontraction. However, only the lupin treatment improved the severely diminished vascular relaxation. These beneficial cardiovascular effects were hypothesised to be due to the high arginine content of lupin protein and the hypothesis was studied further in DSS rats. The lupin and soy protein treatments did not, however, show any beneficial effects in DSS rats, even though a comparable amount of L-arginine in the drinking water was able to produce favourable effects (data not shown).
The results of the studies have been presented in the Healthy ProFood meetings in Helsinki (8.-10.7.2004), Copenhagen (15.-16.4.2005) and Milan (9.-10.11.2005). An abstract presenting the results of both of the studies has been published in the book of proceedings of the final conference. A manuscript of the first study has been submitted to the Journal of Physiology and Pharmacology.
The possible beneficial cardiovascular effects of lupin protein in other animal models as well as in humans, and the mechanisms involved, should be studied further before the expected benefits of these results can be evaluated.
The influence of lupin protein on the cardiovascular disease risk markers and oxidative stress related to smoking and environmental hypercholesterolemia was assessed and clinical studies on influence of lupin protein on lipids, lipoproteins, markers of inflammation and oxidative stress were performed: The studied group consisted of 55 subjects. All subjects were heavy smokers. All study participants during 3 months (90 days) taken 2 glasses (2X 250 ml) of the lupin protein rich drink per day The lupin protein drink was prepared by TERRENA (France). The 2 glasses of lupin drink contained 36 g of lupin protein. This amount of lupin protein is similar to the amounts of soy protein for which significant metabolic effects were reported. During our nutritional experiments no adverse effects of the diet enriched in lupin protein were observed during clinical examination and no one of study participant have complained. Therefore, the consumption of lupin protein can be recognized as safety.
The studied group consisted of normolipemic and mildly hyperlipemic subjects. A significant drop in total cholesterol levels was noted and in more than 60% of studied subjects the mean serum cholesterol decrease was higher than 10% of initial value. Significant drop was also noted in LDL cholesterol concentrations. HDL cholesterol did not change significantly on lupin protein rich diet.In subjects with serum triglyceride levels >150mg/dl a significant drop in TG concentrations was noted and more than 90% of studied subjects were recognised as hyperresponders.
Significant changes in systolic and diastolic blood pressure was observed on diet rich in lupin protein. More than 50 % of studied subjects were recognized as hyerresponders. On the lupin protein rich diet significant decrease in glucose levels (p<0.001) was observed. On lupin rich diet significant decrease in urinary F2-isoprostane (the accepted marker of in vivo oxidation) and in ex vivo ROS (reactive oxygen species) production by different blood leukocyte subpopulations were observed. These changes suggest that lupin protein possesses antioxidant properties and decrease the possibility of development of inflammatory process, which plays an important role in development of atherosclerosis and cardiovascular diseases.
Based on the results of performed studies lupin protein can be recognized as a factor, which significantly influences metabolic processes related to cardiovascular disease development. Lupin protein significantly decreases serum cholesterol, LDL cholesterol and triglyceride levels, and blood pressure. All these parameters are well-accepted markers of enhanced CHD risk. Lupin protein consumption also significantly decreases serum glucose levels. Lupin protein can be recognized as factor, which significantly influences processes associated with metabolic syndrome and diabetes development.
It can be concluded that food containing lupin protein helps to normalize lipid profile, glucose levels and blood pressure. Therefore, lupin protein might be recognized as functional food and might play a significant role in the prevention of cardiovascular diseases.
The objective of this research was to show the potential of lupin protein isolate (LPI) and lupin flour for the production of bakery products. LPI was used as a substitute for egg and milk proteins and lupin flour was used as a partial replacement of wheat flour. Two types of model foods have been successfully developed: lupin muffins and lupin biscuits.
Lupin muffins: LPI-E was used in concentrations of 1.5 - 3.7% (w/w) as a substitute for egg and milk proteins in order to get 100% vegetable products low in cholesterol. The egg/milk protein (2.2%) in the reference formulation was substituted in steps of 25% up to a substitution ratio of 175%. The best texture and taste was achieved at a lupin protein concentration of 2.2%, replacing 100% of the milk/egg protein of the original recipe.
These muffins had a better expansion and a slightly bigger firmness than the reference muffin. Substitution ratios of 175% were possible but resulting muffins showed softer crumb and lower expansion. In a sensory test with an expert panel (11 evaluators, descriptive assay with 10 point scale) the lupin muffins with 2.2% LPI were compared to reference products based on egg or soy protein respectively. With regard to the sensory attributes evaluated, no significant differences were found in most of the sensory descriptors, particularly those related to aroma and texture. The lupin muffin showed significantly more bean like taste and the overall acceptability was slightly lower than the egg reference. This demonstrates that LPI is a possible animal protein replacement in bakery products especially for vegetarians and people allergic against egg/milk products.
Lupin biscuits: Several biscuits were tested by Fraunhofer IVV and Terrena in order to develop a healthy biscuit, high in protein, 100% vegetable, with low fat and long shelf life.
The most successful models tested were an almond containing cantucci biscuit (1) and a sesame biscuit (2).
(1): Lupin cantucci could be developed from an egg based reference by totally substituting the egg protein by LPI and partial substitution of wheat flour by lupin flour and LPI. The best texture and taste were achieved at lupin protein concentrations of 6% to 8% (w/w), but acceptable products were possible up to 12% of lupin protein. Generally the hardness of the biscuits increased with the amount of lupin protein added. At additions higher than 13% the biscuits developed a remarkable lupin taste. A consumer acceptance test with 83 untrained persons using a hedonic scale from 1 (like extremely) to 7 (dislike) was performed with lupin biscuits containing 6% and 12% of lupin protein and a reference based on egg protein. The reference sample was the most accepted. The 12% lupin biscuit was the least accepted but the score did not vary significantly from the 6% lupin biscuit. Standard deviations between 1.4 and 1.7 for the overall acceptance of the two lupin biscuits demonstrated that these cookies polarised the testers. Some of them liked or even tolerated the taste whereas an equal group disliked the samples.
(2): Biscuits containing 7.5% and 15% of lupin flour/grits (corresponding to 3% and 6% lupin protein concentration respectively) were prepared. The hardness profile of both biscuits was comparable to the reference product without lupin. A consumer acceptance test with 30 evaluators using a hedonic scale from 1 (dislike) to 9 (like extremely) didn t show significant differences between the samples. The mean scores of 7.0 for the overall appearance, 6.0 for the texture and 5.6 for the overall acceptability indicated a good acceptability. Cookies produced with a similar recipe with the addition of 7.5% and 15% of LPI-E (equal to a lupin protein concentration of 6.8% and 13.4% respectively) increased the hardness of the biscuits. The product with addition of 7.5% of LPI reached a value of 5.3 for the overall acceptability, which was comparable to the lupin flour, based biscuits.
Lupin-based snacks: The purpose was to produce new snacks, richer in proteins and fibres and less energetic than those found on the market. Because lupin flour contains about 40% of protein, 30% of fibres and 10% of lipids, it is used to improve snacks nutritional value. On the other hand lupin flour does not contain starch, so it cannot be extruded alone. Corn semolina and rice flour have been used to bring the minimum necessary quantity of starch. 60% of corn semolina or rice flour + 40% of lupin flour was the base mix to produce flavoured snacks. Lupin snacks produced presented very high nutritional interest: 21% of protein, 15% of fibres and about 5% of lipids against 7% of protein, 2% of fibres and 35% of lipids for standard snacks.
The project work focused on the development and evaluation of successful processes to produce functional lupin protein isolates and concentrates with improved sensory and nutritional properties. Two different processes have been developed and implemented:
- Fraunhofer IVV researchers developed an innovative process to separate native protein products from sweet white lupin seeds (L. albus). With this new process, efficient protein enrichment and purification was accomplished. The process starts with dry-milling (cleaning, de-hulling, flaking) of the seeds followed by de-oiling with hexane to get white flakes (50-55% protein, <2% fat) as an input material for the protein extraction. The following mild aqueous extraction and purification delivered two types protein isolates with more than 90% of protein (N*6.25): Lupin protein isolate type E (LPI-E) recovered by isoelectric precipitation, with a good emulsifying capacity, and lupin protein isolate type F (LPI-F), ) isolated by acid extraction and subsequent ultra-/diafiltration showing a high capability of foam formation and stabilization. The protein extraction was accomplished in a new pilot plant facility, with batches of 150 kg to 200 kg of white flakes. Within the project, a sequence of experiments was performed in order to select the best conditions for the separation and drying of the lupin protein isolates and to guarantee the reproducibility of product yields and quality. Low temperature-time exposure and a careful selection of the processing conditions, preserved the native protein properties and permitted to obtain ingredients with low thermal damage. About 29% to 31% of the input dry mass and around 50% to 55% of the initial protein could be recovered as protein isolates, 29% of LPI-E and 2.5% of LPI-F. The reproducibility of the protein extraction process was demonstrated on two different varieties of white lupin.
- Bioraf has developed a process without flour defatting (non-solvent), based on the use of enzymes and subsequent ultrafiltration of the protein extract. This process delivered a native protein concentrate, Protein VI, with a protein concentration of 80-90% and fat content around 7.8%) The process results in four fractions respectively hulls, fibres, low molecular weight compounds and a protein isolate (Protein VI) containing 90 % protein (N x 6,25). The Protein VI isolate contain proteins largely reflected by the profile of the proteins found in the seeds. These proteins differ when the different seed sources are used (L. luteus compared to L. albus) with respect to both protein subunits (determined by SDS-PAGE) and isoelectric points (determined by isoelectric focusing, IEF). Apart from the proteins contained in the protein isolate other compound groups are represented covering lipid, lipid soluble compounds and various other low molecular compounds as well as ash. Other lipid soluble compounds as fat soluble vitamins and phenolics contributing as antioxidants are as well in part retained in the protein product VI. Evaluation of the functionality properties of Protein VI shows that the emulsifying properties are competitive in different applications compared to solvent defatted protein isolates. However, Protein VI do not show foaming properties.
The economical feasibility of both process was investigated by cost models. Based on the assumptions and estimates made in the models, the processing of white and yellow lupins into lupin protein isolates would be an economically feasible investment.
Terrena, the third partner, was in charge of the selection and supplying of lupin seeds for the production of lupin protein concentrates or isolates by the other partners. Three varieties gave good results for the following criteria : stability of the protein, fat and fibre contents, year after year, low level of antinutritional compounds (especially alkaloids), stability of the yield in culture : between 25 and 40 quintals/ha. The three varieties selected are the following: Aster, a winter variety, Energy and Ares, springtime varieties.
Unfortunately, because of the non-availability of two varieties the two last years, only Ares could be sent to the other partners for production of lupin concentrates and isolates. Terrena produced lupin flour and improved its features in order to use it as raw material in concentrate and isolates production.
By using an animal food allergy model based on oral co-administration of cholera toxin and food proteins to mice to assess the potential allergenicity of raw lupin and lupin protein products, we have demonstrated that although the lupin specific Ig response induced by co-administration of cholera toxin and lupin proteins appears to be dose dependent, the IgE response appears to depend merely on some intrinsic properties of the proteins as well as some factors of the protein matrix. We found that the lupin protein g-conglutin gave rise to an IgE response, but only when administered as part of the crude lupin extract as opposed to processed and fractionated protein. Our findings are in accordance with studies on human sera, which likewise identified g-conglutin as the major allergen in lupin, and thus indicate that the cholera toxin mouse model is an appropriate model for assessment of allergenicy of novel foods. Furthermore, the findings indicate that it is possible to fractionate lupin proteins to produce health-promoting lupin protein isolates, which will not pose any risk for consumers that are not already allergic to lupin or other legumes, such as peanut and soy.
When the capability of lupin and peanut proteins to induce an allergenic IgE response was compared, we demonstrated that IgE antibodies against g-conglutin raised by feeding with lupin cross-reacted with the peanut allergen Ara h3 despite no sequence homology between the two proteins. Likewise, IgE from mice fed peanut reacted with both Ara h3 from peanut and g-conglutin from lupin.
These results show for the first time that allergenic proteins from different food sources despite no sequence homology may cross-react and, therefore, substantiate the need for proper animal models for prediction of allergenicity and allergen identification in novel foods.
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