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Innovative functional foods based on sweet lupin protein for cardiovascular prevention

Final Report Summary - LUPICARP (Innovative functional foods based on sweet lupin protein for cardiovascular prevention)

Executive Summary:
OBJECTIVES. The lupin seed has the potential to be a useful source of protein ingredients to be exploited in replacement for milk, egg, or soybean proteins, since it contains 30-40% protein. In this context, the main objectives of the LUPICARP project were: a) the development of new nutritive and palatable food products based on lupin; b) the assessment of the health benefits of lupin foods in dyslipidaemia; c) the investigation of the mechanism of action at molecular and functional levels; and d) the concept creation and business development for the SMEs.
DEVELOPMENT OF NEW NUTRITIVE AND PALATABLE FOOD PRODUCTS BASED ON LUPIN. Numerous prototype food products containing lupin protein were designed and optimized for texture and palatability. They represent a variegate portfolio of products that may be consumed in different moments of the day and fulfil the tastes of different kind of consumers, vegan, vegetarians, flexitarians, or simply consumers interested to follow a healthier and more sustainable diet. The comparison of the protein profiles of the raw lupin seeds and these foods demonstrates that the proteins are scarcely modified by the processed applied and thus they maintain their nutritional properties. Some of developed food products have been already commercialised, increasing the annual turnovers of the involved SMEs. The project has thus represented a big stimulus for their business.
ASSESSMENT OF THE HEALTH BENEFITS OF LUPIN FOODS IN DYSLIPIDAEMIA. A clinical study was carried out in two hospitals, NIGUARDA (IT) and HUS (FI). It consisted in a 12 weeks treatment period preceded by a 4 weeks run-in period. Patients were assigned to two groups: the first group (control) consumed a low lipid diet containing both vegetal and animal foods (dairy and meat products); the second group (treatment) consumed 30 g lupin protein per day in replacement of the same amount of animal proteins. The lupin treatment produced small reductions of plasma lipids vs. the control group. In particular, the decrease of LDL-cholesterol, primary end-point of the study, was -5 mg/dL. Greater decreases were observed in the case of total cholesterol (-10.7 mg/dL) and triglycerides (-16.1 mg/dL). Literature clearly indicates that the positive effects of soy protein depend on the baseline cholesterol values of the enrolled subjects. Also in the case of lupin, there was a significant correlation between cholesterol reduction and baseline cholesterol levels (R= -0.6767 P=0.001) whereas no correlation was observed in the control group (P=0.206). This indicates that consuming lupin foods may provide useful health benefits in hypercholesterolemic subjects. These patients are often treated with statins, which can in some cases produce side effects. The available body of data collected so far seems to envisage the possibility of reducing these undesirable side effects combining lower statin doses with a lupin based diet.
INVESTIGATION OF THE MECHANISM OF ACTION AT MOLECULAR AND FUNCTIONAL LEVELS. An investigation at HepG2 cells was aimed at providing information on the mechanism through which lupin peptides may be responsible of the hypocholesterolemic activity. The main findings were the following: i) peptides obtained by hydrolysis with trypsin (T) are able to directly interfere with the HMGCoAR activity, whereas peptides obtained by hydrolysis with pepsin (P) are much less effective; ii) However, both P and T peptides modulate the cholesterol metabolism at HepG2 cell line, through the up-regulation of the pathway involving the LDLR; iii) Both P and T peptide treatments increase the LDL-uptake.
CONCEPT CREATION AND BUSINESS DEVELOPMENT FOR THE SMES. Using innovative techniques, such as Fuzzy Front End, PESTEL analysis, Porter 5-forces analysis, ATAR analysis, and SWOT analysis, RUG developed numerous business cases for each company. Based on the results achieved in this area, SMEs identified an important approach to increase their business: the creation of “common brand “LupaVit” for selling their products. The LupaVit concept exploits the health and environmental benefits of lupin products and has the major advantage that these products will be easily recognized by consumers. Indeed, this common brand has the potential to become a major strong brand in the European market of healthy foods.

Project Context and Objectives:
INTRODUCTION
An overwhelming majority (90%) of the population is aware that a correct life style is crucial for maintaining and improving health. Together with a healthier diet, also functional foods and dietary supplements are becoming increasingly favoured by consumers and physicians. A food can be defined as “functional” if it can demonstrate a beneficial efficacy on one or more target physiological functions of the body in a convincing way. Beyond adequate nutritional properties, functional foods should either improve the state of health and well-being and/or reduce disease risk factors [www.ilsi.org].
In this area, a main target is the decrease of cardiovascular risk through cholesterol-lowering foods. In fact, cardiovascular disease and the metabolic syndrome are now the most common cause of death worldwide with emphasis on middle aged and elderly adults. In addition, these disorders result in substantial morbidity and disability and loss of productivity and make a large contribution to the escalating costs of health care, especially with the current ageing of the population. To combat cardiovascular disease, preventive strategies have been planned worldwide, taking into account the main known risk factors. Hypercholesterolemia, hypertension and obesity fulfil all the criteria that are generally used to assess whether an epidemiological association reflects a causal role for a specific risk factor. These risk factors are in part related to dietary habits and can therefore be influenced by a healthy diet and by specific functional foods.
One way of strengthening the competitiveness of SMEs in the health food sector and thus securing existing employment and creating new employment is to enter highly profitable markets with innovative products supported by a unique selling proposition. A great opportunity here is the market of functional foods. Being highly value-added products, the development of functional foods requires, however, great scientific efforts that are well beyond the in-house possibilities of SMEs. They need scientific support in order to assess the bioactivity of ingredients, to integrate these ingredients into their food products and especially to carry out the expensive experimental and clinical studies necessary to demonstrate the health benefits of their products.

PROJECT OBJECTIVES
The overall project objective was to offer a solution to the SMEs of the consortium, by giving them the scientific support, expertise and finances to assess the health benefit of food products based on lupin, a protein-rich grain legume yet insufficiently exploited in human nutrition. The specific objectives of the project were the following:
• To develop and optimise food products based on lupin protein, with optimal nutritional profiles and sensory characteristics that may fulfil the requirements of the consumers and to evaluate the damage induced by processing on protein quality.
• To complete a multicentre 3 months dietary intervention study to assess the hypocholesterolemic activity of lupin foods, having LDL-cholesterol as the main endpoint, but also investigating other metabolic parameters and biomarkers of inflammation.
• To investigate in detail the mechanism of the hypocholesterolemic activity by separating and characterising lupin proteins and assessing their activity using an hepatic cell line.
• To develop new concepts and business scopes for new products and new markets based upon the health benefits of lupin foods.
The consortium of the LUPICARP project ensures that all necessary scientific, technical and managerial competences necessary for the success of the project are covered. It is composed by the following members: 5 lupin food manufacturers (SMEs), i.e. Dominae Trading SRL (DOMINAE), De Korte Weg BV (VEGA), Meatless BV (MEATLESS), Natural Crunch SL (NATURAL CRUNCH), SIRA Foods (SIRA); 1 lupin ingredient producer TERRENA; and 5 RTD performers, i.e. Università degli Studi di Milano (UMIL), Azienda Ospedaliera Ospedale Niguarda Ca' Granda (NIGUARDA), Helsingin Yliopisto (HUS), Rijksuniversiteit Groningen (RUG) and HPF-Nutraceutics SRL (HPF).
Having the objective of entering the market of functional foods with their own ingredients or food products, the SME participants had to solve two main issues: a) they needed to overcome the technological and sensorial problems created by the inclusion of new ingredients; b) they had to complete the experimentation to demonstrate the biological activity of lupin. The latter objective could be accomplished only by out-sourcing it to external RDT performers: indeed LUPICARP has permitted to create a European consortium of excellent RTD partners, having all the specific competencies not available inside the SMEs. In this project, the SME participants took advantage of the different expertise of five RTD performers.
A main research task of the project was a multicenter randomized dietary intervention study with LDL-C as main end-point: the RTD performers NIGUARDA and HUS have performed the intervention study, recruiting the patients following with suitable inclusion criteria. Besides lipid profile, they investigated other inflammatory and metabolic markers, since they are useful indicators of activities related to the prevention of cardiovascular disease and the metabolic syndrome. HUS, finally, analysed the metabolism of non-cholesterol sterols, as a tool for investigating the mechanism at the basis of the hypocholesterolemic activity. Moreover, UMIL and HPF have joined their efforts to perform a detailed investigation of the mechanism of the hypocholesterolemic activity in vitro using a hepatocyte cell line. HPF has also analysed the quinolizidine alkaloids, which may influence negatively the taste of lupin foods. RUG, instead, has worked out a novel method for “New Concept Development” for new products, new services and new markets for enterprises. This method has been applied and further improved in close collaboration with the participating SMEs: the result was a portfolio of qualified business opportunities based upon the new acquired knowledge on the functional properties of lupin foods.

Project Results:
1. DEVELOPMENT OF NEW NUTRITIVE AND PALATABLE FOOD PRODUCTS BASED ON LUPIN

1A. DEVELOPMENT OF NEW FOOD PRODUCTS
This is a main achievement of the project. Numerous prototype food products containing lupin protein were designed and optimized by all SMEs. They represent a variegate portfolio of products that may be consumed in different moments of the day and fulfil the tastes of different kind of consumers, vegan, vegetarians, flexitarians, or simply consumers interested to follow a healthier and more sustainable diet. The comparison of the protein profiles of the raw lupin seeds and the food prototypes demonstrate that the proteins are scarcely modified by the processed applied for food preparation and thus maintain their nutritional properties. Some of developed food products have been already commercialised, increasing the annual turnovers of the involved SMEs. The project has thus represented a big stimulus for the business of the five participating SMEs.
This a short description of the most important food products optimized by the SMEs involved in the project.
MEATLESS. MEATLESS is trendsetter in the use of hydrated vegetable fibres and proteins in meat-, fish-, and meat-free products. They renew food products and make them healthier and more sustainable. The emphasis is on flavour and texture, as food should taste good. They do not use any artificial additives, but organic raw materials, such as wheat, rice, lupin and caramel. They are a team of expert professionals, most of whom have decades of experience with food production. Originating out of a meat processing factory, MEATLESS has since 2006 developed into an innovative company using unique techniques to mix vegetable with animal-based products like meat, cheese and fish. MEATLESS has wide experience in the industrial processing of meat, meat-free, fish and cheese products. Using Meatless, food can be made more sustainable and healthier without losing quality or taste. MEATLESS is a business to business (B2B) company, thereby producing a semi-finished product meant for further processing. MEATLESS has an IP protected process for producing vegetable based meat analogues. The lupin version of the product contains whole narrow-leaf lupin, seaweed extract and water only. Therefore, the product is clean label, natural and gluten-free and contains no artificial colorants, aromas or binders. These meat analogues can be based upon wheat, rice or lupin ingredients. Meatless has developed an improved recipe with elevated lupin protein levels for the LUPICARP project. Using lupin concentrate or even isolate, the protein content can be boosted up to 15% (whereas the usual protein content is 8%).
 Meatless is a 100% vegetable fat free fibre, made from lupine, wheat or rice.
 Meatless contains natural raw materials only, no artificial binders, colorizers or taste enhancers.
 Meatless comes ready-to-use in IQF-packaging.
 Meatless is useful to reduce fat content in meat and cheese products without influencing taste.
 Meatless is excellent to improve texture and juiciness in vegetarian products.
 Meatless is produced in a sustainable way and is also available in a hypo-allergenic version.
 Meatless is easy to use, clean label, cost efficient and of unbeatable quality.
Meatless vision has developed in LUPICARP two portfolios of product applications. The first portfolio is completely meat free, the second portfolio is based upon hybrid applications, i.e. mixtures of meatless product and (minced) meat. Meatless has developed these products in co-creation with customers applications.
Applications with meat free meatless products.
Stir-fry application. Meatless has developed a Thai-inspired stir-fry application based upon Meatless ingredients.
Sauce bolognese application. Meatless has developed a lupin Meatless recipe dedicated for pasta sauces. This Meatless recipe is adapted to the processing conditions for pasta sauces (sauce Bolognese), pasteurization at 80 °C and acid environment.
Oriental application (shoarma, gyros). MEATLESS has developed an oriental mix for eg. shoarma or gyros applications. VEGA has the shoarma mix further developed and adapted to specific taste conditions.
Meatless sate/ teriyaki product style. Meatless products can also be processed into convenient products.
Meatless tuna fish and meatless meat free sausages. Fish applications were developed with proper taste melanges as well as meat-free vegetarian sausages.
Hybrid meat-meatless products. The transition to a less meat consuming society is gradual. Hybrid products, mixtures from animal and vegetable proteins, represent a transition step. Meatless has developed recipes for hybrid products, which have low fat content as unique selling point. Hybrid meat formulations with up to 50% meatless inside were developed. These products are no different in texture and taste from regular 100% meat products, but are far more sustainable and in general have lower fat and lower saturated fatty acids contents. Compared to beef, the use of meatless means a factor 40 improvement on land use, energy use and greenhouse gas output.
VEGA is specialized in the production, marketing and sales of vegetarian products made from vegetable proteins. VEGA distinguishes itself with a whole new generation of meat- and fish substitutes, which chefs and culinary journalists find similar to traditional animal based food items. VEGA developed the following lupin products within the framework of LUPICARP that are already available in the market: www.devegetarischeslager.nl/shop/vegetarische-slager-lijn
Lupain: lupin bread. VEGA has developed a traditional bread Lupain, which contains 45% lupin ingredients (flour, bits and grits). One bread loaf weights 600 gram and is 100% vegetable based. Lupain is prebaked and frozen, and should be stored at -20 °C. Before consumption, it should be baked for 10 min in oven at 200 °C and consumed within 3 days. The ingredients are: water, natural leaven, wheat flour, whole wheat flour, lupin bit, lupin flour, lupin grits, wheat gluten, oat flakes, salt, yeast, barley flakes, flaxseed, rye flakes, millet, sunflower seed, soy grits, wheat flakes, sesame seed, buckwheat groats, potassium iodide, barley malt extract, wheat malt flour. The nutritional value for 100 g is: energy 167 kcal/ 697 kJ, protein 6.91 g, carbohydrates 18.3 g, sugars 0.6 g, fat 1.93 g, fibre 9.45 g, salt (NaCl) 0.01 g, iron 7.0 mg, moisture 4.47%. Other information may be found at: www.devegetarischeslager.nl/shop/vegetarische-slager-lijn/lupain-brood#sthash.77qvQyCa.dpuf.

Bio Shoarma. The second food products produced by VEGA is Bio-Shoarma. Again, it is already on the market: www.devegetarischeslager.nl/shop/vegetarische-slager-lijn/de-vegetarische-slager-bio-shoarma-van-lupine#sthash.S6tNJsxK.dpuf. The special marinated slices are made from biological lupin and other ingredients. They have an authentic oriental taste. Lupin ingredients are from Dutch biological lupin crops. The ingredients are: 86% lupin flour, vegetable oils (rapeseed and sunflower), herbs and spices, sodium alginate, salt, maltodextrin, wheat-protein, coriander, cumin, sweet pepper, clove, turmeric, pepper, ginger, mace, fenugreek, yeast extract, palm fat. The nutritional value for 100 g are: energy 151 kcal/ 631 kJ, protein 8.7 g, fat 7.9 g, carbohydrates 6.7 g, fibre 9.1 g, salt 1.7 g.

SIRA is specialized in new product and service development for the agro-food and lifestyle domains. It has developed several prototypes of protein-rich products, which may have beneficial health effects.
Lupin drinks: milk and smoothie. The drinks family of products consists of the base recipe of lupin milk with about 3-4% protein content. Fortified protein drinks up to 10-15% are possible as well, but fit within the domain of sports drinks and shakes. Lupin milk was the basis of the smoothie products, where especially “super fruits” and “super foods” were included: in particular, Acai berry, Goiji berry, or Chia seed were formulated with the lupin milk base.
Lupin desserts: lupin yoghurt, tofu and ice cream. Lupin protein was fermented and/or coagulated with special bacteria populations and/or coagulants thereby creating yoghurt or (smooth-silken) tofu type of desserts. Lupin milk was the base for lupin ice cream. Lupin yoghurts and lupin tofu’s have protein contents of 8-10%.
Lupin dough products: bread, pasta and rösti. Lupin sourdough bread was the result of natural (wild) fermentation with lactic acid bacteria. Sourdough bread, with lupin protein content up to 20%, was formulated. Microbial solid state fermentation cultures were adapted to the composition of the raw material (lupin flour/oat flour/wheat flour) after the first spontaneous wild fermentations. Starter cultures were established after three-four fermentation cycles. The prototypes of lupin pasta were prepared mixing durum wheat flour and lupin flour (up to 25% lupin flour resulting in 10% lupin protein). The spaghetti type of pasta was produced. Lupin rösti was inspired by a traditional Austrian/German recipe of fried grated potato. Lupin rösti is a 50% mixture of grated lupin dough and grated potato, which is fried in a frying pan or deep-fried for industrial applications. Lupin rösti has a lupin protein content of 20-25%.
Lupin spread. Lupin spreads were inspired upon the Turkish hummus dishes. The original hummus is soaked and grinded chicken peas, but the same procedure can be applied to lupin beans as well. Hummus can be mixed with spices and herbs to create different tastes like garlic, cumin, or green herbs Mediterranean style. The protein content of lupin hummus can be up to 30%.
Lupin lifestyle: lupin protein bars, lupin powder protein shakes. Lupin protein bars were produced with rolled oats, lupin bits and dried fruits from super-berries and others. The lupin protein content was increased by using lupin protein concentrates. Lupin shakes are powders, which needs to be dissolved in blenders by the customer. Lupin shake powders were produced with spray-dried lupin protein concentrate with natural flavours. The lupin protein content can be up to 30%.
NATURAL-CRUNCH has developed and optimized new food products containing lupin protein in order to provide a wide range of products to put on the market. The aim was to develop a complete range of lupin protein based food items, covering all daily moments from breakfast, to lunch and dinner including also midnight snack. These foods are: crunchy texturized lupin protein flakes mixtured with other texturized fruits and vegetables or cereals and crunchy texturized lupin flakes covered with milk-free chocolate or joney, and energetic or dietetic bars made with them. The ingredients were lupin flakes mixed with crunchy fruits powders and pieces of crunchy fruits. The food quality and shelf-life were tested and the formulations optimized to overcome any appearing issue. They were tested for their acceptability with some panel tests based on consumers. Several prototypes for each of these products were developed. All these products are additive-free.
Ingredients used:
- Crunchy dried apples slices + crunchy apples powder + crunchy texturized lupin flakes.
- Crunchy dried banana coins + crunchy banana powder + crunchy texturized lupin flakes.
- Crunchy dried pineapple triangle + crunchy pineapple powder + crunchy texturized lupin flakes.
- Crunchy dried strawberries slices + crunchy strawberries powder + crunchy texturized lupin flakes.
- Crunchy dried mango sticks + crunchy mango powder + crunchy texturized lupin flakes.
- Crunchy tomato slices + crunchy pineapple powder + crunchy texturized lupin flakes.
- Crunchy paprika strips+ crunchy pineapple powder + crunchy texturized lupin flakes.
- Crunchy pumpkin strips + crunchy pineapple powder + crunchy texturized lupin flakes
- Crunchy onion scales + crunchy pineapple powder + crunchy texturized lupin flakes.
- Crunchy beetroot slices + crunchy pineapple powder + crunchy texturized lupin flakes.
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The sensory evaluation gave these descriptions:
- Sweet and crunchy
- Good colors and appearance
- Good powder mixtures
- Good consumer acceptance
- Mixtures with fruits are very successful for mixing with yogurts, deserts or ice creams.
- Mixtures with vegetables are very suitable for topping on salads, sauces and soups, garnishes, canapes or starters.

DOMINAE is and Italian SME, that was founded after the positive results of the preceding EU project HEALTHY-PROFOOD to develop in Italy the market of lupin foods. They are now developing their business in collaboration with CONBIO, another Italian SME, which has now started selling the developed products with great success. Some of these products, that are meat-less ready-to-use foods, such as cutlets, beefsteak, burger, and fricassee as well as frankfurter and hummus, were especially designed to fulfil the tastes and customs of Italian consumers. These foods are targeted to the specialised vegan and vegetarian markets and have constituted a main revenue of the company in 2013.
1b. ANALYSIS OF QUINOLIZINE ALKALOIDS IN THE FOOD PRODUCTS AND INGREDIENTS.
Quinolizidine alkaloids, which should never overcome the limit of 200 mg/kg, represents the most important contamination problem in lupin ingredients and foods. They are toxic secondary metabolites synthesized by plants belonging to the genus Lupinus. They have a bitter taste and may thus impair the sensory characteristic of the foods. HPF has quantified the alkaloids in the lupin ingredients and foods provided by the SMEs, as well as in lupin ingredients available on the market. It was possible to confirm that lupin foods can be very safely added in the diet of European consumers, since all analysed samples respect the limit of 200 mg/kg fixed by the Australia, New Zealand, France and UK Health Authorities.

1c. LUPIN PROTEIN PROFILE AND CHARACTERIZATION OF THE MAIN STORAGE PROTEINS IN LUPIN FOODS
UMIL and HPF have also worked together to characterize the main proteins in the total protein extracts of L. angustifolius and L. albus, since the products of the SMEs are based on these two lupin species. The analyses were performed by 2D-electrophoresis and mass spectrometry. The main storage proteins in lupin seed are b-conglutin, a-conglutin, g-conglutin and d-conglutin, with great differences in the relative abundance of each fraction between the two species. Anion exchange chromatography allowed the partial purification of three major protein fraction: a-conglutin, b-conglutin and g-conglutin. There are relevant differences in the chromatographic profiles of the two species: the main protein in L. angustifolius is a-conglutin, whereas in L. albus it is b-conglutin. This prospects that the health benefits provided by each species may be different.
Much effort was dedicated to evaluating how much the processes applied to the preparation of the food prototypes had modified the protein sequences. The experimentation consisted in the comparison of the profiles of total protein extracts from lupin seeds (untreated samples) with those of the food samples, by using as analytical techniques SDS-PAGE, 2D-electrophoresis and anion exchange chromatography. The main results of this investigation ae the following: a) the ratios between -conglutin and -conglutin were higher in the foods than in the seeds, indicating that the resistance of the former to heat treatments is more satisfactory; b) the γ-conglutin peak was much smaller in all food samples, suggesting that this protein is very susceptible to degradation. However, the processes applied during the preparation of these food prototypes appear to be sufficiently mild, since the protein profiles were in general only marginally modified.

2) ASSESSMENT OF THE HEALTH BENEFITS ON DISLIPIDEMIA PREVENTION OF THE FOOD PRODUCTS BASED ON LUPIN PROTEINS.

2a. EFFECTS ON LIPIDS
Another main objective of the project was the assessment of the health benefits of lupin foods in cardiovascular prevention. A clinical study, with a multicentre, randomized, parallel group design, was carried out in two hospitals, NIGUARDA and HUS, after approval by their Ethical Committees. It consisted in a 4 weeks run-in period followed by a 12 weeks treatment period. The trial involved two groups of 40 patients each. The patients of the first group (control) consumed a low lipid diet containing both vegetal and animal foods (dairy and meat products); in the diet of the patients of the second group (treatment) animal foods were substituted by a combination of lupin foods, which allowed including in their diet 30 g lupin protein in replacement of the same amount of animal proteins. The patients of each group consumed these foods as part of a personalized balanced diet. Each subject was scheduled for 6 visits during the study: 4 weeks before randomization, at baseline, and 4 and 8 weeks after treatment. A final visit at 4-6 weeks after the end of the study was also performed. Blood samples were collected at each visit.

Visit V-1 (-4 weeks from Time 0 of the study)
After obtaining written informed consent, the patients have started the 4 weeks run-in period.
Procedures listed below have been performed:
 Physical examination
 Fasting blood sample for:
- total cholesterol
- triglycerides
- LDL-cholesterol
- HDL-cholesterol

Visit V0 (Time 0 of the study)
The subjects were randomized to one treatment group and started the treatment period. Procedures listed below have been performed:
 Physical examination
 Fasting blood sample for :
- total cholesterol
- triglycerides
- LDL-cholesterol
- HDL-cholesterol
- Fasting glucose and insulin
- cholesterol, squalene and non-cholesterol sterols
- IL-6
- hsCRP
- sICAM-1
- adiponectin

At this visit, the subjects have received the food products for the following 4 weeks.

Visit V1 (Time +4 weeks of the study)
Procedures were performed as detailed for visit V0. At this visit the subjects have received the food products for the following 4 weeks.

Visit V2 (Time +8 weeks of the study)
Procedures were performed as detailed for visit V0. At this visit, the subjects received the food products for the following 4 weeks.

Visit V3 (Time +12 weeks of the study)
The subjects ended the study. Procedures have been performed as detailed for visit V0

Visit V4 (Time +4/6 weeks after the end of the study)
Procedures were performed as detailed for visit V0.

The clinical study enrolled hypercholesterolemic patients according to the indicated inclusion and exclusion criteria.
Inclusion Criteria:
(i) males and postmenopausal females;
(ii) from 18 years to 75 years of age;
(iii) body mass index > 25;
(iv) LDL-cholesterol levels > 130 mg/dl and < 190 mg/dl.
Exclusion Criteria:
• Any past history of cerebro-vascular or coronary events, including unstable angina, myocardial infarction, angioplasty, or coronary artery bypass graft.
• Subjects affected by any kind of food allergy.
• Any concomitant therapy known to alter any of the parameter to assess.
• History of or current alcohol or drug abuse.
• Any clinically significant medical condition that could interfere with the conduct of the study.
• Known or suspected diagnosis of hepatitis or human immunodeficiency virus (HIV) infection
• Unable or unwilling to comply with the protocol requirements, or deemed by the investigator to be unfit for the study.
All patients were provided with full written information about the modalities and objectives of the study and signed an informed consent form. Patients were randomized to the lupin foods (treatment) or the control diet. The 3-months treatment program was completed by:
 patients randomized to lupin protein treatment: 26;
 patients randomized to control (milk protein) treatment: 27.
The lipid parameters, LDL cholesterol (primary endpoint), total and HDL cholesterol, triglycerides, ApoA-I, ApoB, and glucose were assayed by means of an automated system (Roche Diagnostics) by NIGUARDA and by UMIL. Plasma insulin and the other metabolic, inflammatory risk markers (adiponectin, hsCRP, sICAM-1, interleukin-6) were assayed by specific commercial ELISA kits (Mercodia, insulin) and R&D Systems (adiponectin, hsCRP, sICAM-1, interleukin-6). Serum non-cholesterol sterols were assayed by HUS. Data were expressed as mean + SD. All differences were assessed by paired Student’s t test. All tests are 2-sided, and P values < 0.05 are considered as statistically significant. Normality was assessed by the Kolmogorov-Smirnov test. Statistical analysis was performed by using the SAS Software version 20 (SAS Inc, Cary, NC).
Starting from the control group, there were no significant changes in all parameters in the patients who consumed the control diet. This fact strongly supports the reliability of the study. In fact, this occurs in dietary studies only when the run-in period is correctly used to balance the entire diet that should afterward remain constant during all the trial period. In case of long studies, this may be difficult.
The lupin treatment produced small modifications of the main lipid parameters. In particular, the decrease of the end-point of the study, i.e. LDL-cholesterol, was only -5 mg/dL. Greater decreases were observed in the case of total cholesterol (-10.7 mg/dL) and triglycerides (-16.1 mg/dL).
Literature (Anderson et al, 1995; Sirtori et al, 2007) clearly indicates that the positive effects of soy protein depend on the baseline cholesterol values of the subjects submitted to the intervention studies. We have thus prepared charts to evaluate how the cholesterol changes depend on the initial cholesterol values in each group. The lupin chart indicates that there is a clear correlation between the observed changes and the initial values, which is highly significant (P=0.0011) whereas no correlation exists in the control group (P=0.2059). This indicates that consuming lupin foods may provide useful health benefits especially in the subjects that mostly need to control their cholesterol.
The decision to evaluate the hypocholesterolemic activity of lupin foods was based on previous results from UMIL and NIGUARDA and other literature data. Some investigations in a rat model of hypercholesterolemia have demonstrated that diets containing either L. albus protein (Bettzieche et al., 2008b; Sirtori et al., 2004) or L. angustifolius protein (Bettzieche et al., 2008a; Parolini et al., 2012) may significantly reduce both total cholesterol and low-density-lipoprotein cholesterol (LDL-C) levels vs. control diets containing casein. The cholesterol-lowering activity of L. albus has also been confirmed in a hamster model of dislipidemia (Fontanari et al., 2012). Moreover, in a rabbit model of the atheromatous plaque generated at the common carotid arteries (Chiesa et al., 2001), L. albus protein slowed down the plaque formation induced by a hyperlipidemic diet (Marchesi et al., 2008). Finally, an uncontrolled clinical trial on L. albus (Naruszewicz et al., 2006) and two double blind ones on L. angustifolius (Sirtori et al., 2012; Bähr et al, 2014) have demonstrated that consuming foods containing 25-30 g lupin protein has a positive effect on cholesterolemia also in humans. The first study evaluated a lupin beverage (EU project HEALTHY-PROFOOD), the second a dietary bar containing a lupin protein isolate (EU project BIOPROFIBRE), and the third a portfolio of different foods, such bread, roll, sausage, and vegetarian spread.
As in the LUPICARP trial, also in all these published studies, the greater reduction observed in patients with higher baseline cholesterol suggests that a nutritional intervention with lupin protein may provide health benefits especially in the presence of high initial cholesterol. These highly hypercholesterolemic subjects are often treated with statins, which often produce well-known side effects. The available body of data collected so far seems to envisage the possibility of reducing these undesirable side effects combining lower statin doses with a lupin based diet.

2B) EFFECTS ON CHOLESTEROL METABOLISM
The main components of whole-body cholesterol metabolism are the carefully regulated intestinal absorption efficiency of cholesterol and hepatic de-novo cholesterol synthesis as well as with cholesterol trafficking to and from tissues, and cholesterol excretion from the body. Since the analysis of cholesterol absorption efficiency and cholesterol synthesis is laborious, the analysis of serum non-cholesterol sterols and squalene has been developed and generally accepted as surrogate markers of cholesterol metabolism. The serum cholesterol precursors squalene, cholestenol, desmosterol, and lathosterol reflect cholesterol synthesis, whereas plant sterols campesterol and sitosterol, and cholestanol, a metabolite of cholesterol, reflect cholesterol absorption efficiency (Miettinen et al, Nutr Metab Cardiovasc Dis 2011).
Since the lupin foods contain small amounts of plant sterols, we hypothesised that they may inhibit cholesterol absorption efficiency. The results of this investigation indicated that a) the endogenous cholesterol synthesis was unchanged in both groups; b) both diets increased slightly the cholesterol absorption efficiency as depicted by the increased serum cholestanol levels in both groups; c) the phytosterol levels were higher in the lupin group.
The study population was moderately hypercholesterolemic not having a profile of high cholesterol absorption or high cholesterol synthesis rate. In this regard, the study population was optimal to test the possible changes in cholesterol metabolism. When consuming a natural diet, how much phytosterols are needed to reduce LDL cholesterol? In a literature study, the subjects consumed two controlled diets, phytosterol poor (126 mg/2000 kcal) and phytosterol abundant (449 mg/2000 kcal) ones. Compared to the phytosterol poor diet, phytosterol abundant diet increased plasma phytosterol ratios to cholesterol by 68%, decreased plasma cholestanol ratio to cholesterol by 45%, and decreased cholesterol absorption by 18%, but total and LDL cholesterol were unchanged. The difference between the amount of phytosterols in the diets was 323 mg/2000 kcal; with this amount cholesterol absorption was reduced about one fifth with no effect on LDL cholesterol concentration. In the present study, in the lupin group the increased consumption of plant sterols by 60 mg/day elevated serum plant sterol levels from 22 to 39%, but cholesterol absorption did not decrease. On the contrary, it increased phytosterol absorption. It can only be assumed that the reason for the increased serum cholestanol level (and increased absorption) in both groups is based on some other component in both test products, the protein moiety, which increases cholesterol absorption.

3. INVESTIGATION OF THE MECHANISM OF ACTION AT MOLECULAR AND FUNCTIONAL LEVELS

Numerous data in the literature indicate that the LDL receptor (LDLr) pathway may be implicated in the hypocholesterolemic activity of soy protein via the activation of several transcription factors and the expression of lipogenic enzymes. In this study, UMIL and HPF have proposed to investigate the lupin protein effects at LDL receptor. In particular, using a new In-Cell Western (ICW) assay developed in the framework of this project, the LDL protein variation level was detected after lupin peptide treatments directly at HepG2 cells. Furthermore, experiments were carried out in order to characterize functionally the lupin peptide effects. In particular, a LDL-uptake cell based assay was optimized and developed using the fluorescent LDL-DyLight 495 at HepG2 cells.
Literature indicates also that the LDL receptor up-regulation depends on the activation of several transcription factors, such as the Sterol regulatory element-binding protein (SREBP) 2, and the expression of lipogenic enzymes, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR). Immunoblotting experiments were performed in order to investigate the LDLr-SREBP2 pathway through the analysis of the LDLr, SREBP2 and HMGCoA reductase protein level variations after P and T treatment at HepG2 cell line. In particular, we have investigated the capability of P and T peptides to inhibit the activity of HMGCoAR performing direct experiments using the commercial catalytic domain of the enzyme. In addition, considering that several recent reports identify a crucial signaling pathway, via phosphatidylinositol-3-kinase (PI3K)/Akt, as an important player in the regulation of the LDLr (Luu et al, 2012), we have decided to investigate also these aspects. The PI3K/Akt pathway is known for its role in promoting cell growth, proliferation, and survival through increased glucose utilization and prevention of apoptosis. Akt has been recently implicated in a novel form of lipid metabolism regulation, through the SREBPs (Krycer et al., 2010). The physiological coordination between Akt and SREBPs pathways are needed to produce the lipids for new membrane synthesis, which in turn is required for growing and proliferating cells. Most of the works have been focused on SREBP-1c isoform, whereas it is important that new efforts are directed to explore and to investigate the link between Akt and SREBP2, which is very relevant in cholesterol biosynthesis.

The investigation at HepG2 cells was aimed at providing information on the mechanism through which lupin peptides may be responsible of the hypocholesterolemic activities previously observed in animal models and in humans. The main findings were the following: i) T peptides are able to directly interfere with the HMGCoAR activity, whereas P peptides are much less effective; ii) Nevertheless, both P and T peptides modulate the cholesterol metabolism at HepG2 cell line, through the up-regulation of the pathway involving the LDLR; iii) Both P and T peptide treatments increase the LDL-uptake.
HMGCoAR is the rate-controlling enzyme of cellular cholesterol biosynthesis pathway. For this reason, it constitutes the target of numerous investigations aimed at lowering the rate of cholesterol biosynthesis. Therefore, initially in vitro experiments were performed using the purified catalytic domain of the enzyme with the objective of clarifying whether lupin peptides were able to directly inhibit the activity of HMGCoAR. T peptides but not P peptides act as enzyme inhibitors. The LDLR expression and the receptor protein localization at cellular membranes are strictly correlated to the intracellular cholesterol biosynthesis pathway. In facts, the transcription of the LDLR and the genes required for cholesterol and fatty acid synthesis are controlled by membrane-bound transcription factors called SREBPs and the intracellular cholesterol acts with a negative feedback inhibition mechanism. The SREBP2 isoform is responsible for the LDLR and HMGCoAR transcription and the SREBP2 maturation is regulated by the intracellular cholesterol homeostasis. Thus, the up-regulation of LDLR represents a useful strategy to control plasma LDL cholesterol levels. Our findings demonstrate that, independently of the direct in vitro inhibition of the HMGCoAR activity, both lupin peptide mixtures are able to up-regulate the LDLR protein levels through an increase of mature SREBP2 protein. However, a detailed investigation of the LDLR pathway revealed that lupin peptides produce different effects on the HMGCoAR level. In particular, whereas P peptides are able to increase the enzyme protein level with a dose-dependent manner, T peptides have a different behavior. In fact, they up-regulate the enzyme protein levels in a significant way vs. the control at 0.5 mg/ml, the increase remains statistically significant, but it is smaller at 1.0 mg/ml, and it is finally abolished at 2.5 mg/ml. These evidences suggest that both peptide mixtures modulate the cholesterol metabolism pathway, through the induction of LDLR protein levels due to an increase of the SREBP2 mature form. Although the potency of induction is different, either P or T peptides are able to increase the LDLR through the SREBP2 activation.
Thus, their main difference consists in their capacity to interact with HMGCoAR. Precisely, comparing the results obtained treating with P and T peptides at 1.0 mg/mL, the HMGCoAR protein induction mediated by P peptides is much higher than that mediated by T peptides. The diverse behaviors of P and T peptides, possibly related to the diverse direct effects on the activity of HMGCoAR, lead to different consequences on the intracellular cholesterol biosynthesis. In particular, since the reduction of enzyme protein levels, the cholesterol biosynthesis in HepG2 cells treated with T peptides than in cells treated with P peptide mixture is reduced. For this reason, the intracellular cholesterol biosynthesis reduction might be balanced by an increased ability of cells treated with T peptides to uptake extracellular LDL for keeping constant the cholesterol homeostasis. In order to verify this hypothesis, LDL-uptake experiments were performed. As expected, the ability to uptake LDL of HepG2 cells treated with T peptides was ~30% higher than that of P treated cells, supporting our hypothesis. The increase of LDL clearance mediated by lupin peptides is not only correlated with the much higher increase of the LDLR protein level, but it is also regulated by their ability to modulate the cholesterol biosynthesis by affecting the HMGCoAR protein levels.
Recently studies were focused to link the Akt and the SREBP pathways, in facts, emerging evidences implicates Akt in the regulation of lipid metabolism through to activation of SREBPs. It has been shown that insulin-like growth factor-1, an inducer of Akt signalling, increases SREBP2 activation acutely. Our study provides evidence that Akt contributes to the regulation of cholesterol metabolism through activating SREBP2. Even if the precise target(s) of Akt still remains elusive and not clarified, we have focused part of our investigation on evaluating the effects of both lupin peptide mixtures on the PI3K/Akt pathway. Our study provides the experimental evidences that either P or T peptides from lupin protein are able to induce increases of Akt and GSK3β phosphorylation levels, which are completely abolished by the treatment with a well-known PI3K inhibitor, such as wortmannin. It has been shown that a constitutively active form of Akt activated the LDLR. Our findings clearly support this study, since both P and T increase the LDLR protein levels and induce an increased fluorescent LDL-uptake at HepG2 cells. Moreover, after treatment with both lupin peptides in presence of wortmannin, the LDL uptake is blocked vs. the P and T treatments alone, demonstrating that the inhibition of PI3K/Akt has general effects on cellular lipid homeostasis, although the precise Akt target(s) is(are) not definitely assigned yet.
In conclusion, this is the first report providing evidence that peptide mixtures obtained by the hydrolysis of lupin proteins are able to modulate the LDLR/SREBP2 pathway with the final effect of an increased LDL uptake. Since as indicated above, both P peptides and T peptides are complex mixtures, it appears very difficult to sort out which may be the peptides responsible for the observed activities. It is, however, possible to affirm that the diversity in the behaviours of the two peptide mixtures derives from their different composition. Further work will be necessary to investigate these aspects in detail in the future.



4: CONCEPT CREATION AND BUSINESS DEVELOPMENT FOR THE SMES

This was also a main objective successfully reached by the LUPICARP project. The RTD performer RUG has big experience in business creation in the area of innovative food products. Using innovative techniques, such as Fuzzy Front End, PESTEL analysis, Porter 5-forces analysis, ATAR analysis, and SWOT analysis, together with the SMEs, RUG developed numerous business cases for each company. Based on the results achieved in this area, SMEs identified an important approaches to increase their business: the creation of a “common brand”: each SME will sell its products under the common brand name “LupaVit”. The LupaVit concept exploits the health and the environmental benefits of lupin products and has the major advantage that these products will be recognized easily by consumers. This common brand has the potential to become a major strong brand in the European market of healthy food products.

Partner RUG has developed 9 different business cases for new lupin based products and businesses. In cooperation with SME-partners. These business cases consisted of a technical-economical evaluation of the proposed product and a market assessment. All business cases had at least the following elements:
1) Concept description.
2) Market positioning.
3) PESTEL analysis (macro evaluation). A PESTEL analysis gives a quick scan concerning Political, Economical, Societal, Technological, Ecological and Legal aspects of the business case.
4) Porter 5-forces analysis (meso evaluation) determines industry profitability by assessing power of competitors, suppliers, buyers, substitutes and potential new entrants.
5) Market size forecast consisting of assessments of total and specified markets (meso).
6) Sales potential analysis by ATAR model (micro) standing for Awareness, Trial, Availability and Repeat purchase.
7) Costs and financial analysis (Net Present Value, Return on Investment) and sensitivity analysis thereof.
8) Technological and other resources analysis (including technology roadmap).

The following concepts and business cases were analysed according to the format above. Two or three business cases were developed for each partner.
4a. LUPINE CUISINE, BUSINESS CASE FOR PARTNER P7-VEGA.
This concept is about lupin based meat analogues for hotel and catering industry for the Dutch market in order to accelerate societal acceptance of lupin based meat analogues. The product is launched by a major TopChef by using his name payback time of the investment is about 1.5 year thereby increasing acceptance of lupin based meat analogues.
In the world, an increasing amount of meat is consumed. Moreover, the amount consumed per person is more than the recommended amount, which results in health issues. The focus on health is increasing, which results in more awareness about nutrition. moreover, people tend to become more environmentally conscious. These aspects result in a trend that people become vegetarian or flexitarian – skipping meat for at least one day a week. The demand for meat analogues increases enormously and a large amount of companies respond to this. This study is performed for the Vegetarian Butcher, which is a start-up company specifically set up for the development of food, which is sustainable, healthy and tasteful. At this moment, it is researching the feasibility of products based on lupin beans. This report provides a guide for VEGA on how they can successfully market lupin based products. The research question in this report is defined as follows: “How could Vegetarian Butcher determine the best opportunity for lupin based products such that they can market a healthy, sustainable and tasty product within the boundaries of bearable risk?” First a methodology is chosen in order to come up with a method to cope with the Fuzzy Front End the project is dealing with. First opportunities should be identified and analysed where subsequently the vision and mission statement is identified, which is guidance for the project. The vision is stated as follows: “With passion we strive to improve people’s wellbeing by providing sustainable, tasteful and healthy products that improves the eating or drinking experience of people” Second, idea generation is generated in order to increase creativity and the amount of possible ideas. From all these ideas, two concepts are chosen for further research: lupin meat for the elderly market and lupin meat for young (trendy) adults. These ideas are further elaborated by a PESTEL analysis, the Porter five forces model and a SWOT analysis. From this is concluded to continue the young (trendy) adults’ by providing lupin meat at restaurants. For this option is chosen due to the fact that lower investment is needed and the fact that it fits the vision better. From here, this project is made more concrete and a name is given: Lupine Cuisine. Moreover a bankable business plan is made, which shows that the NPV after three years is 601,972 € and the payback period for the total investment amounts 141,000 € euro after 1.5 year. Overall advise to VEGA was to market Lupine Cuisine for the young adults in The Netherlands through restaurants, in order to keep investment at minimum due to economic decline. When proven a success, the market of Lupine Cuisine could be enlarged
4b. PARTY SNACK LIGHT, BUSINESS CASE FOR PARTNER P8-MEATLESS.
Party snack light is a product concept for the food industry. It is an assortment of hybrid meat products enriched with lupin protein inspired upon the traditional Dutch “bittergarnituur” (assorted appetizers). The concept is developed by the ingredient supplier of lupin based hybrid meats for B2B applications. License fees are part of the business case. The estimated Payback time of investments is about 2 years.

What is it? “Party Snacks Light” is a product concept for the Food industry. It is an assortment of hybrid meat snacks based on Lupin, a natural fiber, much like the traditional Dutch “bittergarnituur”.

Why believe in it?
- The product appeals to a large customer base, both B2B and B2C.
- It is a great way to promote meat substitutes.
- The product could include a health claim in the future, after more research on the beneficiary properties of the Lupin ingredient.
- Meat prices rise, which opens up the food market for meat substitutes.

How to exploit it? Meatless will develop the new business concept to a complete turn-key product that can be produced by larger production partners in the food industry. The main strategy is that Meatless will provide the product design and the manufacturing plans, as a package that B2B customers, being large food manufacturers, can implement without spending on R&D. Meatless will supply the product designs and plans to the customer, which will have an instant extra product to produce that is successful and provides revenue. Meatless will receive license fees for every produced product from the producer and it will have the exclusive rights as a supplier of ground Lupin as the key ingredient for a specified quantity of products at a set price. Meatless will become part of the supply chain of the manufacturer.

The key activities of Meatless are thus:
- On-demand R&D in food products as a speciality
- Delivering value to the B2B customer through innovation
- Delivering value to the B2B customer through integration in the supply chain
- Supplying the key base ingredient: ground Lupin
Lupin in the fast food business, business case for partner MEATLESS. The concept is about lupin based hybrid and pure meat analogues for the fast food market. Recent years have seen a change in the fast food markets where consumers demanding for healthier and environmental friendly food. Specific benefits such as the health claim, positive environmental effects and cost-price reduction are important drivers for creating a strong market position for lupin based meat analogues.
Health claims with hybrid and pure meat analogues, for partner MEATLESS. The strategy is based upon creating awareness of the health effects and environmental benefits by establishing strategic alliances with industrial catering for enabling consumer acceptance and hence super market penetration. The industrial catering is a new market for this kind of products. Net present Value calculations are € 4.1 and € 2.7 million after 6 years for hybrid and pure meat analogues respectively. The combined NPV is calculated as € 6.8 million after 6 years.
This report holds a research performed for MEATLESS and possibilities for the exploitation of their lupin products. MEATLESS has the vision of developing better dietary and nutritional concepts for the society and the ambition to be a frontrunner in the application of plant fibre in meat, fish and meat-free products. The case study presented is about the exploitation of the health claim obtained by the LUPICARP alliance of four companies working with lupin. The board of management requested a strategy for a possible increase of the exploitation of Meatless products.
Raised cholesterol levels is a rising problem in the Netherlands, 11 to 12% of the Dutch population suffers from a high cholesterol level. The products of MEATLESS based on lupin beans can lower cholesterol significantly. Therefore, a strategic alliance was formed with the name LUPICARP to obtain a health claim based on article 14 ‘risk reduction claim’ from the European Food Safety Authority.
The methodology is based on the model of Koen et al (2001) for the New Concept Development model. The first and second frontend elements out of the NCD model Opportunity Identification and Opportunity Analysis are done by combining preliminary market assessment and market research into the internal and external market analysis. The NCD activities are linked to the Porter Six Forces model, PESTEL, consumer and risk analysis and a market assessment and research. These are supplemented with a technical, financial and competencies report of MEATLESS and the suppliers.
From the research, the lupin product of MEATLESS haves large potentials, especially when the health claim is in place. The concepts we identified differentiate from most of the products in the market because of the several benefits (health, environment) which are associated with lupin. To cope with incorporated risks a market strategy of selective distribution using two sales channels, supermarkets and catering companies, is proposed. Cooperation with catering companies is very important, if this succeeds a very stable and highly profitable market channel is created. The supermarkets however present a lower risk of entering this specific channel, but it is more difficult to get a reasonable market share. A premium price strategy is advised for both supermarket products and in the alliances with caterers. Products to continue were pure meatless and hybrid minced meat. Convincing customers to try the product is an essential step, since they might be apprehensive at first. The primary channel for this at first should be the catering company. To cross the ‘chasm’ the advice is to periodically scale up promotion activities coupled to targeted channels like health magazines and insurance companies. The case calculation of the Net Present Value (NPV) of hybrid was about 4.1mln and for pure meatless 2.7 mln. In interpreting the NPV, it is important to mention that the sensitivity in unit sales and unit price is rather high. The latter further justifies the application of a premium price strategy, while unit sales are an important factor to stabilize through contracts.
The advice is obtain strategic alliances with caterers to increase sales and the awareness of the supermarket products and increase the trial rate and first consumer experience. In this report a pluralistic vision and integrated methodology is formulated on the exploitation of MEATLESS to increase sales and take steps to become a frontrunner in the application of plant fibre in meat and meat free products. The health claim to be attained through LUPICARP is an important foundation for this strategy.
4c. LUPO ‘CATERING HEALTH’ WITH PROTEIN POWDERS, BUSINESS CASE PARTNER P6-DOMINAE.
Lupo consists of lupin powder with high protein content which can be easily processed into food products and menus produced by industrial caterers. It is a B2B (business to business) marketing concept where close relations with customers are created. Payback period is about 1.25 year and the initial investment is about € 300k.

4d. SAVORY LUPIN-SARDIN FISH SNACKS, BUSINESS CASE FOR P9-NATURAL CRUNCH.
This concept is developed for production facilities of SME “Natural Crunch”. Savoury lupin-sardine fish snacks is a healthy product by combination of the lupin proteins and the fish oil rich in omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and precursors of certain eicosanoids that are known to reduce inflammation in the body. Payback time of the investments is about two years with an ROI of a factor 2, each invested euro will result in two euros return. Health issues are becoming increasingly severe throughout the entire world. More and more people suffer from overweight, obesity, diabetes or strokes and the main cause of this is an unhealthy diet. An answer to these problems could be the introduction of lupin based products, as they can be a healthier substitute for high protein products like meat and dairy.
A business plan was presented to Natural Crunch for launching a new product based on lupin proteins and the following vision is kept in mind throughout the entire investigation: “We strive for Natural Crunch to make people familiar with healthy innovative food products based on lupin proteins.” To achieve this vision, analyses of both external and internal factors were made to further investigate the possibilities. With this, a possible segment; people with obesity or overweight, and a possible product; dried lupin based sardines were identified. Another opportunity arises during this investigation where elderly people might be a possible secondary market. Next, a marketing strategy was drawn up in order to secure, serve, and retain customers for the firm’s product. Promotion will mainly focus on a healthy life style, and the connection to the health aspect of lupins should be made very clear. Moreover, a relationship with an insurance company or with a doctor recommending the product, can boost sales even further. Other alliances can be formed to create a stronger and more effective market presence. These may include alliances with the government and supplier.
To analyse the financial aspects of the business plan, the cash inflows and outflows were estimated. On the basis of the market size and target cost, the NPV and return on investment (ROI) are determined. An NPV of around € 1.4 million was found and this can be considered a good investment. However, due to these high investments it takes relatively long to become profitable (in the 9th quarter). The ROI is quite high, with a ratio of 2.02. This means that every Euro spent will provide more than two euro’s in return. A bubble chart shows that the probability of success is relativity high too (between 0.55 and 0.85).
To conclude, the introduction of lupin based dried sardines will be a good investment for NATURAL CRUNCH as they already possess the necessary technologies for production and the financial plan shows great potential. Also, the awareness of lupin protein as a substitute for animal protein might help the increasing health problems the world is facing. By producing dried sardines with lupin protein, Natural Crunch is able to make people familiar with healthy and innovative food products, which was the vision of this entire investigation.
4e. LUPIN SNACK, BUSINESS CASE FOR P9-NATURAL CRUNCH.
This report provides an answer on the question: “How can Natural Crunch manage to successfully introduce Lupine VitaSnacks in the Spanish snacks market?” As analysed, lupin is a good alternative to the now unsustainable soy. It has good characteristics like: a low fat amount, high values of protein and high values of fibre. Due to this certain health claims are made. For instance, a lupin-rich diet lowers the cholesterol-level. Natural Crunch is a Spanish based company creating healthy snacks under the brand name ‘VitaSnacks’. The scope of the project is given by the above mentioned. A creativity session led to the vision of creating a Pringles-like potato chip product, but using lupine beans instead of potatoes. The market analysis focused on the Spanish savoury-snack market shows signs of an increase in size. Also, the organic food market worldwide is rapidly increasing in size as well. The lupin chip product fits the current strategy and product line. Therefore, the distribution lines do not have to change. The supply chain analysis shows the possibilities of growing the raw ingredient close to the production facility, increasing the product’s sustainable image. Health claims of the product increase the potential on the market. The potential is further increased by the financial analysis. Though based on assumptions it shows that when a certain market share is reached a positive Net Present Value (NPV) can be achieved. The NPV is, however, really sensitive to the amounts sold and the selling price. In a scenario planning external and internal factors are analysed which can have positive or negative impacts on the success of the product. Most important is found to be the taste of the chip. Moderate risks are the risk of not obtaining the before mentioned health claims and the risk of a decrease in domestic consumer spending. In the section ‘technology roadmap’ it is recognized that the product can be differentiated later in time. It is proposed to enter the market with a one-flavour product. In time certain flavours can be added to better suit market segments. Another conclusion of the section is that when competitors enter the market, Natural Crunch has to research new technology to keep the company at a state-of-the-art level. The paper concludes with an interesting product which has the potential to be prosperous on today’s Spanish savoury market.

4f. LUPAVIT: A COMMON COMMUNICATION AND MARKETING STRATEGY
In this frame, based on the results achieved by LUPICARP, SMEs identified two overall approaches: the first one is the “common brand” approach; each SME will sell its products under the common brand name “LupaVit”. The LupaVit concept exploits the positive approved health effects when the EU claim is successful and the environmental benefits from lupin cropping in the EU. The major advantage is that these products will be recognized easily by consumers. This common brand has the potential to become a major strong brand in the European market of healthy food products.
The communication to consumers can be straight forward about the health claim: “30 grams lupin protein a day lowers LDL cholesterol significantly”. The communication message will be: ”The LupaVit products will serve you 30 grams lupin protein a day. This products contains xx% of your daily intake of lupin proteins.” SME’s will lose part of their individual identity, which is the major disadvantage of this approach. The second approach is the “common branded unique selling point”, the LupaVit “inside” strategy. This approach allows SME’s to retain their identity and have the advantages of the common branding at the same time.
The concept is based upon the second approach where companies retain their own identify but will grasp the advantages of a common branding strategy by exploring the LupaVit “inside” approach. The communication message will be: ”LupaVit lupin proteins are beneficial for preventing cardiovascular diseases and high blood pressures. This LupaVit approved product will serve you x grams lupin protein a day. This products contains x% of your daily intake of lupin proteins.” Note the final communication message has to be approved by the dedicated national authorities.
This strategy allows SME’s to build gradually the knowledge for beneficial LupaVit consumption and disseminate this through this common brand. The LupaVit domain names www.lupavit.com and www.lupavit.eu are secured for these purposes and the LupaVit brand will be deposited at the office of harmonization of internal markets https://oami.europa.eu/ohimportal/en/home when the health claim is submitted to EU.

Potential Impact:
For the SME members of the LUPICARP consortium there are clear and demonstrable benefits to be gained from the implementation of the project. In order to make an economical assessment, it is useful to discuss the current situation of the international market of foods that provide health benefits. The worldwide market volume for healthy foods is currently estimated to be up to 62 billion US dollars and experiencing strong growth. Consumers have shown greater interest towards this market especially in US, Japan and Europe, where the 90% of the consumers consider the improvement of their health condition a priority. One example for a rapidly growing sector is the market for plant-based desserts and drinks (dairy-free products) and meat substitutes (meat-free products).
It will be possible to promote the lupin products for their health benefits, because, even though the clinical study has only partially confirmed the hypocholesterolemic activity, some important published papers confirm that a portfolio of lupin foods has favourable hypocholesterolemic effects.
It is also important to underline that the European citizens pay greater and greater attention to the issue of food production sustainability “from the field to the fork”. In fact, plant proteins have a much better environmental sustainability vs. animal proteins: the water and soil needed for producing 1 kg plant protein are much lower than those needed for producing 1 kg animal protein: the production of 1 kg meat protein consumes about 4 kg plant proteins. In addition, whereas plants consume CO2 for biosynthesizing carbohydrates, lipids and proteins, animals metabolize the same nutrients producing CO2 and other waste materials. In view of the increasing world population, decreasing availability of arable soil and environmental pollution, it is clear that any activity that may favour the use of plant proteins has a great ethical impact. Grain legumes are the most important source of plant proteins and soy is the most cultivated grain legume. In 2012, the turnover of the European non-GMO soy market was about 550 million euros, with an 18% increase vs. 2010 (data provided by the European Natural Soy Association – ENSA). This sustained growth relies on: a) a greater awareness among European consumers of the environmental and health benefits of plant based foods as a part of the daily diet; b) the continuous innovation of soy foods and other plant-based foods in Europe; c) the high-quality standard of EU soy foods guaranteeing sale, nutritious and tasty products.
Lupin, containing 35-40% protein, is the only legume that may compete with soybean for food production. The SMEs have worked actively to develop the market of lupin products: either taking profits of the increasing awareness of the EU consumers for the environment, or improving their innovation potential and the quality of their products. The dissemination of the results of the project may additionally increase their business.
The partnership with the RTD performers has brought credibility in the promotion towards consumers and is pivotal in shaping the consumer view, following clear and concise explanations on the properties of the lupin food products and their considerable environmental advantages and health benefits.

Dissemination. The LUPICARP Consortium considers the dissemination of project results a strategic issue and a solid base for the exploitation phase. It is important to underline that some results of the project, in particular those related to the clinical trial, had to be kept confidential for the subsequent exploitation. This has greatly impaired the possibility to disseminate the results of the projects and has delayed the publication of papers. In any cases, all project results that did not required confidentiality, in particular the parts related to the nutritional properties of the lupin foods, the composition of lupin proteins and how they are damaged by the processing and the mechanism of the hypocholesterolemic activity as investigated at molecular level in HepG2 cells were disseminated actively during the period of the project and will be actively disseminated also after the end of the project by presentations in national and international congresses. Two manuscripts have been submitted for publication to Food Chemistry and Agricultural and Food Chemistry, two high-reputation peer-review journal of the area. A video-clip and reports containing the non-confidential results of the project are also available at the web-site.

List of Websites:
www.lupicarp.eu
Prof. Anna Arnoldi
University of Milan
anna.arnoldi@unimi.it or lupicarp@unimi.it