Final Report Summary - BIORICE (BIOtechnology for the recovery of valuable peptides from industrial RICE by-products and production of added value ingredients for nutraceuticals, functional foods and cosmetics)
Executive Summary:
The nutraceutical, functional food and cosmetic EU and worldwide markets are rapidly evolving sectors always in need to innovate by developing new classes of products. One of the most important market trends is the use of new bio-based ingredients obtained by environment-friendly extraction processes and testing methods. Of particular importance is the re-cycling and valorization of agro-food industry by-products as feedstocks for the isolation of bioactive molecules.
The BIORICE project aimed at filling the gap of knowledge of the involved SMEs on rice protein by-product pre-treatment, peptide isolation and relative bioactivity and safety testing. In particular, BIORICE research activities produced added value bioactive ingredients (semi-purified total protein digestates and peptide fractions) starting from protein by-products contained in the processing water of rice starch production stream as well as from commercially available rice proteins. Both types of rice proteins were pre-treated via biotechnological approaches and different small molecular weight peptide fractions were isolated by means of an eco-sustainable and not degrading technique. The protein products were tested by means of a wide range of in vitro and cell-based tests to characterize their bioactivity as well as their safety degree. A new method for in vitro evaluation of the skin sensitization potency (without the involvement of laboratory animals) based on human reconstructed epidermis (RHE) containing Langherans’ cells, was developed, validated and used to test the rice peptide fractions.
The optimized rice protein by-product and commercial protein hydrolyzation and fractionation via biotechnological and eco-sustainable tools, were scaled up and validated. To demonstrate the actual possibility of exploitation, the isolated peptide ingredients have been integrated into ready-to-use nutraceutical and cosmetic trail formulations. The economical feasibility and sustainability of products and protocols was evaluated by the SMEs, as well as their application in the target markets and the time to market after the end of the project. The SME partners produced specific business plans including projections on the exploitation and market routes as well as expected return on investment for each project result.
A number of project activities were designed and executed overall the project to specifically disseminate the foreground among a wide range of stakeholders. Both RTDs and SMEs actively promoted the project via conferences, fairs and exhibitions and a significant number of media dissemination actions were put in place (publications, press releases, posters, leaflets, website, project video).
The BIORICE project brought together 6 partners distributed in 3 EU Member States (Italy, France and Germany) and 1 Associated Country (Switzerland). In BIORICE RTD Performers made their multidisciplinary and complementary expertise in the areas of plant biotechnology, downstream processing and human tissue engineering available to SMEs operating in the nutraceutical, food and cosmetic markets.
The results achieved within BIORICE are expected to have a significant impact on the competitiveness of SME Participants that will be able to expand their business by adding to their product portfolio new bioactive ingredients and protocols enabling new product formulations applicable in food, cosmetic and nutraceutical sectors.
The BIORICE project was coordinated by a female researcher who devoted 60% of her time to the project, supported by a project management team with consolidated experience of managing over 250 FP7 projects, which included mostly female research advisors and project managers working full-time in the management of EU projects. In addition, also two CEOs of the involved SMEs are females.
Thanks to the BIORICE project two new jobs have been created. In particular, one biologist was employed as R&D manager in one of SMEs to carry out cosmetic trials and the product development after the end of the project, so ensuring successful knowledge transfer from academia to industry.
Project Context and Objectives:
Nutraceutical, functional food and cosmetic European and worldwide markets are rapidly evolving sectors always in need to develop new classes of products. One of the most important market trends is the use of new bio-based ingredients obtained by environment-friendly extraction processes and tested with more sustainable methodologies to comply with the existing EU regulations (7th Amendment to the EU Cosmetics Directive 2003/15/EC) that prohibit the commercialization of ingredients tested by using laboratory animals. In this view, of particular importance is the re-cycling and valorization of agro-food industrial by-products as sources for the isolation of bioactive molecules by means of optimized industrial biotechnological processes. In this respect BIORICE project aimed at filling the gap of knowledge of the involved SMEs on rice protein by-product pre-treatment, peptide isolation and their bioactivity and safety testing, allowing SMEs to integrate in their production lines and add to their portfolios new bioactive ingredients and protocols enabling innovative product formulations applicable in food, cosmetic and nutraceutical sectors.
The BIORICE project addressed specific problems and needs of the involved SMEs which work in different market sectors and operate at different levels in the supply chain. In particular, all involved SMEs faced the following specific technological problems that they could solve due to their lack of internal resources and proper expertise:
- Principium product range lacked of functional proteins and peptides. The company was interested in obtaining new natural ingredients of plant origin isolated by a new eco-sustainable biotechnological process (substituting the traditional solvent-based ones) allowing to by-pass the strict legislation constraints (i.e. REACH regulation) that at the moment strongly limit the product innovation. This will permit PRINC to penetrate new markets in the nutraceutical/functional food and cosmetic sectors.
- Farcoderm needed to standardize a new in vitro safety testing method to evaluate the occurrence of skin sensitization and the potency of chemicals/products to induce skin sensitization, allowing to better characterize the hazard of chemicals and products. This new protocol will be free of ethical issues complying with the existing EU regulations, which ban the use of laboratory animals.
- Carminia needed to enforce its portfolio of consultancy services aimed at favoring client companies to adopt advanced solutions in the biotech food sector specifically in the recycling of valuable products, in the implementation of technological efficiency and in the development of environmental sustainable industrial protocols.
The overall objective of the BIORICE project was to produce added value, eco-sustainable, bioactive and safe ingredients (semi-purified total protein digestates and peptide fractions) starting from protein by-products, derived from rice starch processing water, and from commercial proteins, and to demonstrate and validate at industrial level the new developed protocols and the application of the new products as ingredients in the food, nutraceutical and cosmetic sectors.
BIORICE had the following specific objectives:
Scientific and technological objectives:
• Develop innovative and sustainable, tailored biotechnological protocols to bioconvert rice protein by-products into new bioactive ingredients (semi-purified digestates and peptide fractions) and add or integrate them into existing industrial processes;
• Establish and optimize an innovative, not degrading and sustainable protocol to isolate small molecular weight peptides from the semi-purified digestates;
• Develop, optimize and validate an innovative protocol (using reconstructed human epidermis with Langherans’ cells) to characterize the safety of the new bioactive ingredients.
Social and policy objectives:
• Decrease the amount of disposed and discarded processing water by promoting a new destiny for protein organic residues. This will contribute to realize the objectives of environmental, socio-economic and industrial EU policy initiatives (Environmental Technology Action Plan and Lead Market Initiative on Bio-based products) and to the application of the objectives of EU Waste management directives and strategies such as the Thematic Strategy on the Prevention and Recycling of Waste, the Waste Framework Directive (2008/98/EC) and the Landfill Directive (1999/31/EC);
• Perform research on sustainable protein production will contribute to face to global increasing demand of new feed and food protein sources alternative to the animal ones (EU COST “Policy brief on sustainable protein supply”). This objective will also contribute to tackle the Horizon 2020 societal challenge “Food security, sustainable agriculture, healthy and sustainable marine and maritime research and the bioeconomy” (COM2011 (808) final) and the areas of innovative actions of the European Innovation Partnership (EIP) “Agricultural productivity and sustainability” (COM2012 (79) final);
• Avoid the use of laboratory animals to perform the safety tests of new ingredients. This objective will contribute to comply with the requirements of the EU Cosmetic Directive (76/768/EEC) 7th Amendment (2003/15/EC), that prohibits to put animal-tested cosmetics on the market in Europe after 2013.
Economic objectives:
• Generate natural ingredients with new and/or improved biological and safety characteristics allowing the SMEs to approach existing and new markets with added value products that have no ethical constraints with respect to the competitors;
• Improve the economical and environmental sustainability of the SMEs (such as by reducing the costs of processing water treatment) for a direct industrial and market exploitation of the processes and end-products, so to increase their competitiveness and financial benefits.
Enabling innovation-related objectives:
• Formulate project results into protectable form and patent them;
• Transfer knowledge from RTD performers to SMEs through case study products and create a generic design guide for product formulation and manufacturing processing options;
• Relay benefits of the developed technology and knowledge beyond the consortium to patent-users such as other SMEs or manufacturers;
• Disseminate the produced results through leaflets, advertising, presentation and participation to seminars and scientific conferences, publications in scientific peer-reviewed journals, newsletters, participation to exhibitions and trade shows, production of project video, CD-ROMS, open websites (project and products dedicated).
The technologies and methods developed and tested within the BIORICE project aimed at achieving the following objectives:
1. Bioconvert into value added compounds the rice protein by-products contained in the starch production processing water, by reducing their wasting routes and re-directing them as source of bioactive molecules into new biotechnological processes. Apply the optimised protocols also to commercial proteins.
2. Establish innovative, not disruptive, industry-driven and eco-sustainable protocols to purify and obtain new peptide ingredients. The developed protocols will avoid the use solvent-based extraction methods and better preserve the bioactivity and the biochemical profile of the initial rice constituents.
3. Establish a wide range of in vitro and cell-based tests to characterize the bioactivity and an innovative protocol to test safety (skin sensitization potency by human reconstructed epidermis containing Langherans’ cells) of the new products completely avoiding the use of laboratory animals.
4. Open the possibility for all the involved SMEs to improve their competitiveness against direct competitors by accessing new markets throughout the introduction of innovative ingredients, obtained via biotechnological and natural processes, having new and/or improved bioactivity, and consequently increase their financial benefit.
Project Results:
1. Enzymatic and whole cell pre-treatment of rice protein by-products
Rice protein by-products coming from starch production were characterized and data regarding protein amount, dry weight, residual starch, pH, were collected. The protein by-products were stabilized by storage at -20°C. No indication of degradation or loss of activity was pointed out.
1.1 Enzymatic digestion protocol
Enzymatic laboratory scale digestion protocols were optimized leading to produce total semi-purified rice protein digestates. The activities performed included the screening of different protease enzymes and the development/optimisation of the process conditions. A selection of commercial proteases was used for enzymatic treatment of rice proteins: Alcalase, Neutrase, Flavourzyme, Protamex, Papain.
Several experiments were performed in order to optimize enzyme selection and enzyme/substrate ratio especially referring to protein hydrolysis yields. The protein digestates were analysed to assess the total content and the molecular mass distribution of proteins and peptides.
After several sets of experiments Alcalase and Protamex followed by Neutrase resulted to be the most efficient in digesting rice by-product proteins and in releasing peptides while Flavourzyme and Papain were not enough efficient and were not further utilized. The E/S ratio and time of incubation were also optimised.
With the aim of evaluating if residual rice starch, usually present in the by-product, could affect and to what extent the protease activity, further digestion experiments with Alcalase, Neutrase and Protamex were performed with and without the simultaneous presence of the a-amylases Fungamyl or Termamyl. Conclusions pointed out that the treatment with a-amylase did not affect protease and in addition seemed not necessary to improve yields of peptide released after protease hydrolysis.
A final laboratory scale protocol by using Alcalase and Protamex enzymes, was optimised and delivered for scale-up process trials.
1.2 Whole-cell digestion processes
During the BIORICE project, whole-cell processes for pre-treatment of stabilized rice protein by-products were performed by using microbial Lactobacillus strains.
In order to develop an efficient whole-cell rice protein fermentation process, 32 different Lactobacillus spp. strains were firstly screened. A range of growth conditions were explored to select the strains able to release the highest amount of small molecular weight peptides. The most representative bacteria candidates to be validated in the subsequent scaling up of the pilot fermentation process resulted to be L. rhamnosus C1122 (C1122), L. plantarum 82 (Lb82) and L. rhamnosus PRRH – PRLH (PRIN). These strains led to reach the highest peak of antioxidant activity and the most complex protein profile, after 72 hours of incubation. A substrate enrichment with MRS-like medium, containing sugars and salts, was supplied to improve process efficiency and increase bacterial ability to grow and produce complex mixtures of peptides.
In conclusion, both enzymatic and whole-cell microbial laboratory scale digestion protocols were optimized (project Result n° 1) leading to produce total semi-purified rice protein digestates (project Result n° 2). The semi-purified digestates, were delivered to partners for peptide fractionation and for biological activities testing. The protocols were utilized for the scale up optimization of the processes.
2. Scaling up of pre-treatment protocols for rice protein by-products.
The scale up of pre-treatment protocols for rice protein by-product was performed both by using commercial enzymes and whole microbial cells of Lactobacillus strains.
2.1. Enzymatic digestion
The enzymatic pre-treatment protocols with Alcalase and Protamex, initially developed at a laboratory scale, were preliminary scaled up to 1 and 2 L bioreactors to optimise all digestion parameters (pH, temperature, enzyme retention time, oxygen demand). As consequence of the obtained results, a 5°C decrease of the temperature process (leading to energy saving, an important factor from an industrial point of view) was selected for bioreactor hydrolysis. The protocols were further implemented in a 10 L scale bioreactor in view of a future industrial scale-up treatment.
Given the efficiency of the process when compared to laboratory scale, a reduction of the incubation time of the future industrial process, could also be foreseen, leading to a further consistent energy and time saving.
The bioactivity and safety characteristics of the total semi-purified digestates and the amount and profile of expected bioactive peptides, were assessed.
-Remypro N80+ scale-up experiments
An additional scale up digestion was performed with rice commercial proteins Remypro N80+ (Beneo-Remy Industries n.v. Belgium) to provide enough spray-dried fractions for trial product tests and also an alternative source of rice protein fractions. Enzymatic hydrolysis was performed with Alcalase enzyme and the digestate was subsequently processed via cross-flow filtration and freeze drying.
The final peptide fractions were tested for bioactivity and safety characteristics and utilised for the production of cosmetic prototypes.
-Whole-cell microbial digestion
The protocol for the treatment of rice protein by-product by means of selected microbial Lactobacillus strains, was initially scaled up at 1L digestion volume. MRS-like growing medium was adapted to integrate the by-product composition with some essential compounds to improve the growth of Lactobacillus strains. Before and during incubation aliquots of the control samples and of by-product inoculated with Lactobacillus strains, were sampled and analysed for protein concentration, peptide molecular weight distribution and antioxidant activity. The maximum amount of digested proteins and antioxidant activity was reached for all the samples after 72 hours of incubation. The control samples resulted to be more active than those incubated with the different bacterial strains.
The second scale-up step, on a 2 L volume, was performed at the same conditions of the 1 L scale-up, by inoculating the C1122 and Lb82 strains. The samples collected after 72 h incubation were analysed for protein concentration which resulted to be lower than that obtained at 1 L scale-up volume.
Semi-purified digestates were utilised for peptide fractionation via cross-flow filtration and obtained fractions were analysed for biological activity and safety profile.
3. Industry-driven recovery of the molecules.
3.1 Peptide isolation
Cost efficient separation processes were developed with the aim of isolating active peptide fractions with a molecular weight lower than 15 kDa. The results obtained pointed out that molecular distillation was not suitable for the technical requirements of the project. On the contrary, the peptide fractionation by their molecular size through cross-flow membrane filtration could significantly be demonstrated.
The selected cross-flow filtration technique was further optimised and validated by using both enzymatic and whole-cell microbiological digestates of rice starch protein by-products at laboratory and/or scale up volumes. This validated technology resulted to be suitable both for the technical and product requirements of the project (project Result n°3).
In particular for both enzymatic and whole-cell scale up digestates fractionation process led to recover the following final peptide fractions: R0.2 µm, R10, R5, R1, P1. The retentate (R) products were split after cross-flow filtration for biological activity determination, freeze drying and spray drying tests.
Additional scale-up digestion and fractionation experiments were performed with rice commercial protein Remypro N80+ in order to provide enough peptide fractions for spray drying test runs, for trial product tests and also to provide a possible source of rice protein fractions alternative to rice starch protein by-products. All the obtained peptide fractions were delivered to partners for bioactivity tests.
Some enzymatic fractions coming from both Protamex and Alcalase 10L scale up and from Remypro N80+ 20L Alcalase digestion as well as some microbial fractions from 1 L scale up of C1122, Lb82 and PRIN and control cultures, were sequenced by means of different mass spectrometry techniques. The obtained results indicated the presence of a large number of peptides in each fraction. A search for the biological activity of identified peptides was carried out and confirmation of peptide sequence in rice proteins and/or protease proteins, was also performed.
4. Peptide ingredient modification via freeze drying, spray drying and encapsulation.
Tests regarding spray drying, freeze drying and encapsulation of peptide fraction ingredients, were performed and the best technique for final ingredient stabilization and production, was selected.
In particular, the enzymatic digestates could be freeze dried to produce a powder, while the microbiological digestates could not be dried completely due to the organic acid content. Spray drying was applied to fractions from enzymatic scale up experiments both of rice by-products and Remypro N80+ proteins. Experiments were performed in parallel to freeze drying in order to compare the mass balances and a loss of product was observed for the spray drying technique. Encapsulation trial experiments, by using the PGSS technology (Particles from Gas Saturated Solutions) were performed to produce a shell material in order to cover the peptide fractions. Preliminary results have shown that the encapsulation of peptide ingredients is possible. The encapsulation trails will be completed after the end of the project due to the need of further implementation of the process. The encapsulated ingredients will then be used to perform trials of nutraceutical products.
5. Evaluation of biological activities by in vitro and cell-based tests and safety evaluation of the products.
The biological activities of semi-purified digestates and of the different peptide fractions isolated by cross-flow filtration, were assessed using a large range of in vitro and cell-based assays. Peptide fractions obtained from fractionation process of rice starch protein by products (treated with commercial enzymes or whole-cell Lactobacillus strains) and of Remypro N80+ rice commercial proteins, were tested.
The following conclusions could be reached for new ingredients derived from enzymatic pre-treatments of rice by-products and of Remypro N80+ proteins:
- Analyses on both semi-purified protein total liquid digestates and selected peptide fractions from flask scale enzymatic processes, indicated that Protamex and Alcalase treatments produced peptides with high bioactivities and no cytotoxicity nor irritation capacity, while lower levels of bioactivity were detected for Neutrase-derived samples.
- The peptide fractions showed different biological activities which were slightly variable depending on the type of enzymatic treatment and on the analysed fraction (fractions containing lower molecular weight peptides showed higher biological activities);
- Most of the fractions showed anti-aging effects;
- Cytotoxic and anti-proliferation effects seemed to be dependent on the concentration of the samples used during the tests, but for each fraction a not-toxic concentration was identified;
- Liquid and dried forms of each fraction were tested and in general the powders showed an equal or higher activity with respect to the corresponding liquid fractions. This result seems to be very promising in view of an industrial application of the ingredients (easy and cheap storage, high stability);
When rice protein by-product was pre-treated with whole-cell Lactobacillus strains, the following conclusions could be reached:
- Semi-purified protein total digestates tested at different incubation times, showed that antioxidant activity reached the highest values after 72 hours of incubation, with the control samples having double activity with respect to by-product incubated with bacterial strains;
- All the isolated peptide fractions had antioxidant activity, with low peptide molecular weight fractions showing the highest activity;
- Only the fractions derived from by-product itself showed anti-tyrosinase, anti-aging and not cytotoxic effects. Instead, the peptide fractions derived from by-product inoculated with Lactobacillus strains, have to be highly diluted to reach the not-toxic concentrations while they show very low or absent biological activities.
After biological activity and safety tests performed on liquid, freeze and spray dried fractions the most suitable peptide ingredients were selected by involved SME partner (Principium) according to the foreseen product applications (project Result n° 4).
6. Development, optimization and validation of an in vitro method to evaluate skin sensitization potency.
An engineered reconstructed human epidermis skin model including Langerhans’ cells was developed (project Result n°5). Monocytes and keratinocytes were co-cultivated in the liquid-air interface using standard operative procedure. The model was then validated against a series of chemicals classified by Local Lymph Node Assay (LLNA) as sensitizers. Chemical concentration, time of contact, and post-incubation time were optimised during the model development phase. Tissue viability by MTT assay and interleukins 12 and 18 and TNFa expression, were evaluated. Seventeen standard reference chemical compounds together with a false positive and a false negative compounds, permitted to further validate and fine-tune the skin experimental model. The same model was used to test 20 peptide fractions. All the BIORICE samples were considered safe and not toxic and resulted to be not sensitizers.
7. Integration of extracted ingredients into ready to-use formulation of trial nutraceuticals.
As originally planned, the maximum scale up volume tested for each optimized pre-treatment process was 10 L. However, the whole process of digestion, filtration and fraction freeze-dry gave peptide yields lower than those minimum requested (3 kg of powdered peptides) to be tested in the nutraceutical application and necessary to make galenic formulations by microencapsulation during the project. Nonetheless, in view of the future production of nutraceuticals, the microencapsulation procedure was tested on a small amount of rice peptides. The analytical tests in order to obtain a suitable peptide microencapsulate powder will be completed after the project end and the final encapsulated product will be used to perform trial products for the nutraceutical application.
8. Integration of extracted ingredients into ready to-use formulation of trial cosmetics
A large amount of Remypro N80+ proteins were processed by enzymatic protocols, giving around 300g of final peptide powdered fractions. For all the fractions a complete technical dossier was prepared by the SME Principium in order to present the product to the potential customers, in a cosmetic line called BIORICE. Particularly, a statement of non-dangerousness of the fractions was formulated (MSDS document) together with a technical data sheet, containing the main chemical and physical information on the ingredients (SDS document). Moreover, a product information data sheet (PIDS) containing all the information about peptide ingredient raw material identification, composition data, manufacturing process, storage, packaging and logistics, analytical data, toxicological information, product compliance with legislation and cosmetic information, was produced.
Taking into account the results of biological activity tests, the percentage of safety use of the fractions and the calculation for active ingredients final cost, the fractions were inserted in the prototype creams at 0.5% and 1%. Four different finished cosmetic creams, were produced: base cream; anti-age cream; whitening cream; anti-pain cream. The creams have been prepared also without the active (0%) to have a placebo check.
The creams were put in stability testing conditions and will be monitored for at least 6 months, looking at their organoleptic properties, to evaluate if any change in the appearance, color, smell and pH, occurs over time.
Potential Impact:
1. POTENTIAL IMPACT OF BIORICE PROJECT
As highlighted in the Europe 2020 Strategy, one of the prior challenges facing today’s society is the need for innovation. The BIORICE project involves different RTDs and SMEs having complementary areas of activities leading to a strong level of integration and increasing the possibility for SMEs of being innovative and competitive across the European Union markets and worldwide.
In the BIORICE project much effort was devoted to ensure that the project results delivered by RTD Performers will be easily and directly up-taken by the SME partners. The main impact achieved by the project is therefore on the innovation capacity and competitiveness of the SMEs involved, as envisaged by the “Research for the benefit of SMEs” funding scheme.
In particular, the innovative products (semi-purified digestates and peptide ingredients) as well as innovative processes (for treatment of processing water containing protein by-products and for extraction and isolation of new peptide ingredients) and methods (in vitro evaluation of the skin sensitization potency based on human reconstructed epidermis with Langherans’ cells) developed within BIORICE will allow the SMEs to deliver added value products to existing and new markets.
Both Carminia and Principium will be able to introduce new products into nutraceutical/functional food and cosmetic target markets. In particular, the total semi-purified hydrolysed proteins are believed to have great potential in the food market, given the increasing demand for plant proteins at EU level. The total hydrolysed proteins will have a market in particular in products both for vegans and vegetarians as well as they could be an excellent protein source for gluten free market sectors.
In addition, the new peptide ingredients extracted by means of green technologies will be easily exploitable in the nutraceutical and cosmetic target markets given their eco-sustainable origin.
On the basis of the economic feasibility assessment, each SME, in its exploitation plan, has estimated the economic impact that will be achieved thanks to the industrial application of the project results developed within BIORICE. In particular:
- Carminia has estimated an annual turnover between € 225,000 and € 300,000 for the total rice protein hydrolysates to be sold in the food market.
- Principium has estimated an annual turnover of € 500,000 for the new peptide ingredients to be commercialised in the cosmetic and nutraceutical markets;
- Farcoderm has estimated a potential for global sales of 30.9 M € for testing ingredients in the cosmetic field, considering that the list of the admitted ingredients includes more or less 20,600 compounds. A realistic estimation of RHE model global sales was calculated assuming an annual increase of market penetration of 2-25%.
In addition to that, the new products will have no ethical constraints because they will be tested on the basis of methods and processes that avoid the use of laboratory animals. This represents an important social contribution to the ethics policies of the European Union, included in the 7th Amendment to the EU Cosmetics Directive (Directive 2003/15/EC) that prohibits to market animal-tested cosmetics.
The optimized digestion protocols within BIORICE, have the potentiality of being exploited by other companies, such as other starch factories or similar industrial processes in the EU area, so improving their economical and environmental sustainability for a direct industrial and market exploitation of the processes and end-products, and increasing their competitiveness and financial benefits. SMEs could in fact be addressed to the production of high value compounds, while improving efficiency and reducing the overall environmental impacts (such reducing the amount and the environmental impact of processing water). This will contribute to realize the objectives of environmental, socio-economic and industrial EU policy initiatives (Environmental Technology Action Plan and Lead Market Initiative on Bio-based products) and to the application of the objectives of EU Waste management directives and strategies such as the Thematic Strategy on the Prevention and Recycling of Waste, the Waste Framework Directive (2008/98/EC) and the Landfill Directive (1999/31/EC).
The BIORICE project has also contributed to tackle the challenge of facing the global increasing demand of new feed and food protein sources alternative to animal proteins (whose production is largely environmentally unsustainable), in compliance with most recent EU policies such as the EU COST “Policy brief on sustainable protein supply”. In fact, the global demand for animal proteins is increasing rapidly due to the growing world population and reinforced by the increasing income per capita in industrialising countries. Conversely, the production of plant proteins is typically more sustainable than that of animal proteins, and an increase in the use of plant proteins, such as total digestates produced within BIORICE project, will therefore significantly contribute to a sustainable protein supply in the future.
Finally, thanks to the BIORICE project two new jobs have been created for junior researchers hired by two of the SMEs involved and, considered the limited lifespan and resources available for the BIORICE project as well as the size of the SMEs involved, this can be considered as a significant result.
2. DISSEMINATION ACTIVITIES PERFORMED DURING THE BIORICE PROJECT
The dissemination activities performed during the whole duration of BIORICE project have been largely consistent with the originally planned activities of the project proposal and were reported in the Interim Plan and in the Final Plan for the Use and Dissemination of Knowledge.
The Consortium partners, have used an array of media and tools in order to successfully disseminate the project to the relevant selected audience.
2.1. Publications
Publications, either in the form of Press Releases, informative papers or general divulgative papers have been published and have played a significant role in the dissemination during the whole project period. The project was also disseminated by means of various newspaper articles both in printed form and on-line on several websites.
Press Releases were aimed primarily at the local audience, general public, industrial stakeholders. This communication tool has proved particularly useful since it has disseminated the project at large to a wide public which would have been not easily identified via standardized methods of dissemination such as events and scientific conferences.
The logo of the BIORICE project is one of the first entries in Google images. A logo for BIORICE ingredients was produced by Principium and, after registration, will be included in the marketing and technical sheets of the future cosmetic and nutraceutical products on the market.
2.2. Website
The project website (www.biorice.eu) was launched publicly on the January 29th 2014 and since then acts as a platform with the aim of establishing an efficient and effective dissemination and communication tool for the BIORICE Consortium for the duration of the project.
It accounts also of a private area for which each partner has specific credentials (username and password) and in which the restricted documents relative to project were made available to the partners. The documents were uploaded by the Coordinator who was also the Administrator of the website restricted area. Each partner was assigned with a project dedicated e-mail address.
The content of the website was regularly updated, upon request of each partner, as well as at the accomplishment of deliverables or after project meetings. The publications, posters, leaflets about BIORICE project were uploaded (download section) and highlighted on the main page of the website, together with the information (date, venue, organizing partner) regarding BIORICE technical meetings or partners’ participation to fairs, conferences and events. After the end of the project the website will be still on-line for 2 years during which it will be regularly updated.
2.3. Social networks
A BIORICE dedicated page was opened on Facebook (https://www.facebook.com/Biorice-372490549591970/?ref=bookmarks) showing pictures of the meetings and posting news on fairs and results of the project.
A BIORICE profile was also opened on Twitter (https://twitter.com/BIORICE_Europe) giving news about project meetings and contacting different European groups on research.
The link to both social networks pages was published on the main page of the BIORICE website.
2.4. Leaflets and Posters
Several leaflets and posters were used to present the project during the fairs and events to whom BIORICE partners took part. All these dissemination materials were made available in the Download section of the project website. Moreover, in the occasion of BIORICE presentation at EXPO (Milano, Italy, 10th September 2015), two new leaflets were specifically prepared.
Several posters, both aimed at general and/or at scientific public, were prepared and presented to exhibitions, conferences and fairs.
2.5. Events
During the whole project a good success in relation to the participation of the partners to different type of events such as conferences, exhibitions and fairs, was pointed out. All events had a wide geographic approach, targeting audiences in Europe and worldwide countries.
The project results were presented thoroughly and throughout these events, with the partners aiming at maximizing the visibility status of the project. The events helped to promote the project towards targeted audiences via personal fair stands and promotional material.
The dissemination activities performed approached an array of interested parties, coming from different fields and areas of interest. Universities and academic institutions, technology institutes and industrial partners are some examples of the type of audience approached. Specifically SME partners, it is notable to mention that they participate to conferences, fairs and exhibitions mainly targeted to customers. The well-received reactions of these audiences demonstrate that the BIORICE results are indeed innovative in various contexts.
A special emphasis should be put on BIORICE presentation at EXPO (Milano, Italy), on 10th September 2015, organized by Principium. All BIORICE partners, in particular SMEs, were invited to the exhibition; the partners disseminated the information of the event to their customers and to all possible stakeholders. The event was held at Italian Pavilion, in the Como Business Center.
2.6. Project video
On the occasion of the presentation of the BIORICE project to the EXPO exhibition, a dedicate video was shot under the supervision of Principium. The video was aimed at explaining to the general public the background of BIORICE and the main objectives proposed. A general focus on the achieved results, was also added. The video was uploaded on BIORICE website main page (www.biorice.eu) and on Principium website (www.principium-bsi.com).
2.7. Other activities
During participation at fairs, most importantly, the project’s outputs for Principium were explained to distributors, that have a direct contact with final customers, which will insert BIORICE ingredients into cosmetic and nutraceutical formulations.
A great opportunity for making contact with potential customers, was given by the networks in which Principium and Farcoderm are involved. In particular for cosmetics, Principium and Farcoderm participate to the HCI (Helvetic association for Cosmetic Ingredients) a Swiss network, member of the European EFfCI association; for nutraceuticals, Principium is part of the Sifood Association, an Italian network of food and nutraceutical companies. These networks represented a great chance to present BIORICE project to business partners and to discuss about the main pillars of the project: innovation and sustainability.
3. EXPLOITATION OF FOREGROUND
The exploitable results generated within the BIORICE project were acquired by SMEs in accordance with the DoW.
The exploitation of BIORICE project results, after the end of the project, will consist in:
a. a product development phase during which the SMEs will move from prototypes to saleable products;
b. an initial exploitation phase during which the SMEs will concentrate on establishing their market presence at the EU level;
c. a further expansion phase during which the markets in the target countries will be consolidated and results from sales obtained in the previous period will be used to penetrate new markets.
3.1 Principium exploitation Plan
From BIORICE project, Principium acquired the technological know-how for rice peptide isolation through cross-flow membrane filtration and the ownership of new peptide ingredients to be exploited in the cosmetic and nutraceutical markets.
-Time-to-market
After the end of BIORICE project, at least two steps will have to be considered before approaching the market.
First of all, the process has to be further standardized. For that, it will be necessary to spend at least 3 to 6 months analyzing different batches of by-products and making pilot batches, in order to compare their composition, activity and safety profile.
The second step will take at least one year and will be focused on additional testing of produced peptides. Principium will perform in vivo studies on active peptide fractions and will test their safety profile on humans. These steps will complete the product dossier.
In conclusion, it could be estimated that the selling of new BIORICE active peptides line will be in 2017. Once on the market, both in the nutraceutical and in the cosmetic fields, the products will be presented to fairs and to distributors. With a strong “green” background, the products could be sold worldwide, claiming innovation, sustainability and Italian origin of products.
-The Market
The new ingredients developed within BIORICE are targeted to the nutraceutical and cosmetic markets. These markets give huge potentialities in terms of innovation and insertion of active peptides with a safety tested profile. The developed ingredients, on the basis of their purity level and of their foreseen application, will be available for manufacturing companies that will formulate them into soft-drinks, tablets, capsules, sachet, bars, bakery products, dairy products, skin & body care lotions. These companies could be contract manufacturers in charge of the production for other or their own brand companies. The innovative character of the developed peptides, will allow Principium to face no direct competition for niche or premium products (targeting vegan or hypoallergenic products) but for the mass market, dominated by huge product volumes.
3.2 Farcoderm Exploitation Plan
Actually most of the data on skin sensitization rely on results obtained on cell cultures and on human exposure. With the entry into force of the 7th Amendment to the EU Cosmetics Directive (Directive 2003/15/EC) and the new regulation on the Registration, Evaluation, Authorisation and Restriction of Chemicals (Regulation No 1907/2006), there is an urgent and pressing need for alternative testing methods. The last trend is to develop in vitro complex models, in order to represent as much as possible the human conditions, but trying to remove all the variables and study the reaction to a product application.
From BIORICE project, Farcoderm enhanced its know-how in understanding the mechanism underlying skin sensitization and acquiring the ownership of a newly developed reconstructed human epidermis model (RHE) containing Langherans’ cells. This model will allow to standardize a new in vitro safety testing method to evaluate the occurrence of skin sensitization and the potency of chemicals/products to induce skin sensitization, and to better characterize the hazard of chemicals and products. This new protocol is free of ethical issues and complies with the existing EU regulations that ban the use of laboratory animals.
-Time-to-market
The RHE model represents for Farcoderm an interesting device to test the safety profile of an active molecule. This model will be completed with a reference scale, in order to give a value to the tested substance. Within BIORICE project, in order to build this reference scale, several standard molecules that give different grades of sensitization on human skin were applied on the RHE model and its reaction was monitored.
Farcoderm needs 6 more months, after the end of BIORICE project, to finally set up the model. In this time, other chemicals/endpoints will be tested and the reference scale will be further confirmed by using new substances. A fine tuning of the produced reference scale will be performed, in order to make the model a standard device for each chemical.
After this first step the model will be ready for the market. It will be a useful tool to test the safety profile of a new ingredient or formula. In fact it will be more detailed than a simple keratinocytes/fibroblast layer being more similar to an “ex vivo” model. The new method will be brought to the market in 2 years (time needed for EURL ECVAM validation phase) after the end of the project.
-The Market
The new testing method is targeted to raw material manufacturers (both cosmetic and pharmaceutical grade), cosmetic, medical device, pharmaceutical and textile industries. As the method will be patented and validated, the market share is assumed to be 100%. The main competitors are represented by testing service companies or academia that perform in vivo tests to evaluate skin sensitization. This method, anyway, has the good characteristic of being more precise of the in vitro cell cultures but less invasive than the in vivo testing.
3.3 Carminia Exploitation Plan
Carminia will enforce its portfolio of consultancy services to help client companies producing high value compounds (like proteins and other molecules), while improving efficiency and reducing the overall environmental impacts of production. Since Amideria Il Cervo has terminated the rice starch production and ceased from the project (March 2015), by entering the BIORICE project, Carminia was able to acquire the rights on the technical know-how for producing protein digestates, and the ownership of new total hydrolysed proteins to be sold in the food and beverage sector.
-Time-to-market
The protocol can be applied in a time span between 6 and 12 months, while, after this, adaptation to different producers is esteemed to require from one to two years considering experimental trials and validation. Regarding semi-purified digestates obtained during BIORICE, Carminia recently planned an investment to build a production site specifically dedicated to protein hydrolysis. The site will be in force during the first six month of 2016. This will allow the production of hydrolysed rice proteins derived directly from rice proteins.
-The Market
The future strategy is to directly address the starch factories in the EU area and propose the application of the BIORICE protocol using as substrate the slurry containing proteins and set up as set of demonstration steps in Carminia production plants facilities, so to demonstrate the effectiveness of the protocol. It would be as well possible to modify the protocol according the clients requirements. Regarding protein digestates these will be exploited by Carminia mainly in the food sector. Indeed these hydrolysed proteins could have a market in the production of reconstituted milk (based on soy or rice kernel) or creation of yogurt, as well in products both for vegans and vegetarians. In addition, the hydrolyzed rice proteins could be an excellent protein source for gluten free market sectors (bakery and sweets, ice cream ready meals).
List of Websites:
www.biorice.eu
Project Coordinator: annalisa.tassoni2@unibo.it
The nutraceutical, functional food and cosmetic EU and worldwide markets are rapidly evolving sectors always in need to innovate by developing new classes of products. One of the most important market trends is the use of new bio-based ingredients obtained by environment-friendly extraction processes and testing methods. Of particular importance is the re-cycling and valorization of agro-food industry by-products as feedstocks for the isolation of bioactive molecules.
The BIORICE project aimed at filling the gap of knowledge of the involved SMEs on rice protein by-product pre-treatment, peptide isolation and relative bioactivity and safety testing. In particular, BIORICE research activities produced added value bioactive ingredients (semi-purified total protein digestates and peptide fractions) starting from protein by-products contained in the processing water of rice starch production stream as well as from commercially available rice proteins. Both types of rice proteins were pre-treated via biotechnological approaches and different small molecular weight peptide fractions were isolated by means of an eco-sustainable and not degrading technique. The protein products were tested by means of a wide range of in vitro and cell-based tests to characterize their bioactivity as well as their safety degree. A new method for in vitro evaluation of the skin sensitization potency (without the involvement of laboratory animals) based on human reconstructed epidermis (RHE) containing Langherans’ cells, was developed, validated and used to test the rice peptide fractions.
The optimized rice protein by-product and commercial protein hydrolyzation and fractionation via biotechnological and eco-sustainable tools, were scaled up and validated. To demonstrate the actual possibility of exploitation, the isolated peptide ingredients have been integrated into ready-to-use nutraceutical and cosmetic trail formulations. The economical feasibility and sustainability of products and protocols was evaluated by the SMEs, as well as their application in the target markets and the time to market after the end of the project. The SME partners produced specific business plans including projections on the exploitation and market routes as well as expected return on investment for each project result.
A number of project activities were designed and executed overall the project to specifically disseminate the foreground among a wide range of stakeholders. Both RTDs and SMEs actively promoted the project via conferences, fairs and exhibitions and a significant number of media dissemination actions were put in place (publications, press releases, posters, leaflets, website, project video).
The BIORICE project brought together 6 partners distributed in 3 EU Member States (Italy, France and Germany) and 1 Associated Country (Switzerland). In BIORICE RTD Performers made their multidisciplinary and complementary expertise in the areas of plant biotechnology, downstream processing and human tissue engineering available to SMEs operating in the nutraceutical, food and cosmetic markets.
The results achieved within BIORICE are expected to have a significant impact on the competitiveness of SME Participants that will be able to expand their business by adding to their product portfolio new bioactive ingredients and protocols enabling new product formulations applicable in food, cosmetic and nutraceutical sectors.
The BIORICE project was coordinated by a female researcher who devoted 60% of her time to the project, supported by a project management team with consolidated experience of managing over 250 FP7 projects, which included mostly female research advisors and project managers working full-time in the management of EU projects. In addition, also two CEOs of the involved SMEs are females.
Thanks to the BIORICE project two new jobs have been created. In particular, one biologist was employed as R&D manager in one of SMEs to carry out cosmetic trials and the product development after the end of the project, so ensuring successful knowledge transfer from academia to industry.
Project Context and Objectives:
Nutraceutical, functional food and cosmetic European and worldwide markets are rapidly evolving sectors always in need to develop new classes of products. One of the most important market trends is the use of new bio-based ingredients obtained by environment-friendly extraction processes and tested with more sustainable methodologies to comply with the existing EU regulations (7th Amendment to the EU Cosmetics Directive 2003/15/EC) that prohibit the commercialization of ingredients tested by using laboratory animals. In this view, of particular importance is the re-cycling and valorization of agro-food industrial by-products as sources for the isolation of bioactive molecules by means of optimized industrial biotechnological processes. In this respect BIORICE project aimed at filling the gap of knowledge of the involved SMEs on rice protein by-product pre-treatment, peptide isolation and their bioactivity and safety testing, allowing SMEs to integrate in their production lines and add to their portfolios new bioactive ingredients and protocols enabling innovative product formulations applicable in food, cosmetic and nutraceutical sectors.
The BIORICE project addressed specific problems and needs of the involved SMEs which work in different market sectors and operate at different levels in the supply chain. In particular, all involved SMEs faced the following specific technological problems that they could solve due to their lack of internal resources and proper expertise:
- Principium product range lacked of functional proteins and peptides. The company was interested in obtaining new natural ingredients of plant origin isolated by a new eco-sustainable biotechnological process (substituting the traditional solvent-based ones) allowing to by-pass the strict legislation constraints (i.e. REACH regulation) that at the moment strongly limit the product innovation. This will permit PRINC to penetrate new markets in the nutraceutical/functional food and cosmetic sectors.
- Farcoderm needed to standardize a new in vitro safety testing method to evaluate the occurrence of skin sensitization and the potency of chemicals/products to induce skin sensitization, allowing to better characterize the hazard of chemicals and products. This new protocol will be free of ethical issues complying with the existing EU regulations, which ban the use of laboratory animals.
- Carminia needed to enforce its portfolio of consultancy services aimed at favoring client companies to adopt advanced solutions in the biotech food sector specifically in the recycling of valuable products, in the implementation of technological efficiency and in the development of environmental sustainable industrial protocols.
The overall objective of the BIORICE project was to produce added value, eco-sustainable, bioactive and safe ingredients (semi-purified total protein digestates and peptide fractions) starting from protein by-products, derived from rice starch processing water, and from commercial proteins, and to demonstrate and validate at industrial level the new developed protocols and the application of the new products as ingredients in the food, nutraceutical and cosmetic sectors.
BIORICE had the following specific objectives:
Scientific and technological objectives:
• Develop innovative and sustainable, tailored biotechnological protocols to bioconvert rice protein by-products into new bioactive ingredients (semi-purified digestates and peptide fractions) and add or integrate them into existing industrial processes;
• Establish and optimize an innovative, not degrading and sustainable protocol to isolate small molecular weight peptides from the semi-purified digestates;
• Develop, optimize and validate an innovative protocol (using reconstructed human epidermis with Langherans’ cells) to characterize the safety of the new bioactive ingredients.
Social and policy objectives:
• Decrease the amount of disposed and discarded processing water by promoting a new destiny for protein organic residues. This will contribute to realize the objectives of environmental, socio-economic and industrial EU policy initiatives (Environmental Technology Action Plan and Lead Market Initiative on Bio-based products) and to the application of the objectives of EU Waste management directives and strategies such as the Thematic Strategy on the Prevention and Recycling of Waste, the Waste Framework Directive (2008/98/EC) and the Landfill Directive (1999/31/EC);
• Perform research on sustainable protein production will contribute to face to global increasing demand of new feed and food protein sources alternative to the animal ones (EU COST “Policy brief on sustainable protein supply”). This objective will also contribute to tackle the Horizon 2020 societal challenge “Food security, sustainable agriculture, healthy and sustainable marine and maritime research and the bioeconomy” (COM2011 (808) final) and the areas of innovative actions of the European Innovation Partnership (EIP) “Agricultural productivity and sustainability” (COM2012 (79) final);
• Avoid the use of laboratory animals to perform the safety tests of new ingredients. This objective will contribute to comply with the requirements of the EU Cosmetic Directive (76/768/EEC) 7th Amendment (2003/15/EC), that prohibits to put animal-tested cosmetics on the market in Europe after 2013.
Economic objectives:
• Generate natural ingredients with new and/or improved biological and safety characteristics allowing the SMEs to approach existing and new markets with added value products that have no ethical constraints with respect to the competitors;
• Improve the economical and environmental sustainability of the SMEs (such as by reducing the costs of processing water treatment) for a direct industrial and market exploitation of the processes and end-products, so to increase their competitiveness and financial benefits.
Enabling innovation-related objectives:
• Formulate project results into protectable form and patent them;
• Transfer knowledge from RTD performers to SMEs through case study products and create a generic design guide for product formulation and manufacturing processing options;
• Relay benefits of the developed technology and knowledge beyond the consortium to patent-users such as other SMEs or manufacturers;
• Disseminate the produced results through leaflets, advertising, presentation and participation to seminars and scientific conferences, publications in scientific peer-reviewed journals, newsletters, participation to exhibitions and trade shows, production of project video, CD-ROMS, open websites (project and products dedicated).
The technologies and methods developed and tested within the BIORICE project aimed at achieving the following objectives:
1. Bioconvert into value added compounds the rice protein by-products contained in the starch production processing water, by reducing their wasting routes and re-directing them as source of bioactive molecules into new biotechnological processes. Apply the optimised protocols also to commercial proteins.
2. Establish innovative, not disruptive, industry-driven and eco-sustainable protocols to purify and obtain new peptide ingredients. The developed protocols will avoid the use solvent-based extraction methods and better preserve the bioactivity and the biochemical profile of the initial rice constituents.
3. Establish a wide range of in vitro and cell-based tests to characterize the bioactivity and an innovative protocol to test safety (skin sensitization potency by human reconstructed epidermis containing Langherans’ cells) of the new products completely avoiding the use of laboratory animals.
4. Open the possibility for all the involved SMEs to improve their competitiveness against direct competitors by accessing new markets throughout the introduction of innovative ingredients, obtained via biotechnological and natural processes, having new and/or improved bioactivity, and consequently increase their financial benefit.
Project Results:
1. Enzymatic and whole cell pre-treatment of rice protein by-products
Rice protein by-products coming from starch production were characterized and data regarding protein amount, dry weight, residual starch, pH, were collected. The protein by-products were stabilized by storage at -20°C. No indication of degradation or loss of activity was pointed out.
1.1 Enzymatic digestion protocol
Enzymatic laboratory scale digestion protocols were optimized leading to produce total semi-purified rice protein digestates. The activities performed included the screening of different protease enzymes and the development/optimisation of the process conditions. A selection of commercial proteases was used for enzymatic treatment of rice proteins: Alcalase, Neutrase, Flavourzyme, Protamex, Papain.
Several experiments were performed in order to optimize enzyme selection and enzyme/substrate ratio especially referring to protein hydrolysis yields. The protein digestates were analysed to assess the total content and the molecular mass distribution of proteins and peptides.
After several sets of experiments Alcalase and Protamex followed by Neutrase resulted to be the most efficient in digesting rice by-product proteins and in releasing peptides while Flavourzyme and Papain were not enough efficient and were not further utilized. The E/S ratio and time of incubation were also optimised.
With the aim of evaluating if residual rice starch, usually present in the by-product, could affect and to what extent the protease activity, further digestion experiments with Alcalase, Neutrase and Protamex were performed with and without the simultaneous presence of the a-amylases Fungamyl or Termamyl. Conclusions pointed out that the treatment with a-amylase did not affect protease and in addition seemed not necessary to improve yields of peptide released after protease hydrolysis.
A final laboratory scale protocol by using Alcalase and Protamex enzymes, was optimised and delivered for scale-up process trials.
1.2 Whole-cell digestion processes
During the BIORICE project, whole-cell processes for pre-treatment of stabilized rice protein by-products were performed by using microbial Lactobacillus strains.
In order to develop an efficient whole-cell rice protein fermentation process, 32 different Lactobacillus spp. strains were firstly screened. A range of growth conditions were explored to select the strains able to release the highest amount of small molecular weight peptides. The most representative bacteria candidates to be validated in the subsequent scaling up of the pilot fermentation process resulted to be L. rhamnosus C1122 (C1122), L. plantarum 82 (Lb82) and L. rhamnosus PRRH – PRLH (PRIN). These strains led to reach the highest peak of antioxidant activity and the most complex protein profile, after 72 hours of incubation. A substrate enrichment with MRS-like medium, containing sugars and salts, was supplied to improve process efficiency and increase bacterial ability to grow and produce complex mixtures of peptides.
In conclusion, both enzymatic and whole-cell microbial laboratory scale digestion protocols were optimized (project Result n° 1) leading to produce total semi-purified rice protein digestates (project Result n° 2). The semi-purified digestates, were delivered to partners for peptide fractionation and for biological activities testing. The protocols were utilized for the scale up optimization of the processes.
2. Scaling up of pre-treatment protocols for rice protein by-products.
The scale up of pre-treatment protocols for rice protein by-product was performed both by using commercial enzymes and whole microbial cells of Lactobacillus strains.
2.1. Enzymatic digestion
The enzymatic pre-treatment protocols with Alcalase and Protamex, initially developed at a laboratory scale, were preliminary scaled up to 1 and 2 L bioreactors to optimise all digestion parameters (pH, temperature, enzyme retention time, oxygen demand). As consequence of the obtained results, a 5°C decrease of the temperature process (leading to energy saving, an important factor from an industrial point of view) was selected for bioreactor hydrolysis. The protocols were further implemented in a 10 L scale bioreactor in view of a future industrial scale-up treatment.
Given the efficiency of the process when compared to laboratory scale, a reduction of the incubation time of the future industrial process, could also be foreseen, leading to a further consistent energy and time saving.
The bioactivity and safety characteristics of the total semi-purified digestates and the amount and profile of expected bioactive peptides, were assessed.
-Remypro N80+ scale-up experiments
An additional scale up digestion was performed with rice commercial proteins Remypro N80+ (Beneo-Remy Industries n.v. Belgium) to provide enough spray-dried fractions for trial product tests and also an alternative source of rice protein fractions. Enzymatic hydrolysis was performed with Alcalase enzyme and the digestate was subsequently processed via cross-flow filtration and freeze drying.
The final peptide fractions were tested for bioactivity and safety characteristics and utilised for the production of cosmetic prototypes.
-Whole-cell microbial digestion
The protocol for the treatment of rice protein by-product by means of selected microbial Lactobacillus strains, was initially scaled up at 1L digestion volume. MRS-like growing medium was adapted to integrate the by-product composition with some essential compounds to improve the growth of Lactobacillus strains. Before and during incubation aliquots of the control samples and of by-product inoculated with Lactobacillus strains, were sampled and analysed for protein concentration, peptide molecular weight distribution and antioxidant activity. The maximum amount of digested proteins and antioxidant activity was reached for all the samples after 72 hours of incubation. The control samples resulted to be more active than those incubated with the different bacterial strains.
The second scale-up step, on a 2 L volume, was performed at the same conditions of the 1 L scale-up, by inoculating the C1122 and Lb82 strains. The samples collected after 72 h incubation were analysed for protein concentration which resulted to be lower than that obtained at 1 L scale-up volume.
Semi-purified digestates were utilised for peptide fractionation via cross-flow filtration and obtained fractions were analysed for biological activity and safety profile.
3. Industry-driven recovery of the molecules.
3.1 Peptide isolation
Cost efficient separation processes were developed with the aim of isolating active peptide fractions with a molecular weight lower than 15 kDa. The results obtained pointed out that molecular distillation was not suitable for the technical requirements of the project. On the contrary, the peptide fractionation by their molecular size through cross-flow membrane filtration could significantly be demonstrated.
The selected cross-flow filtration technique was further optimised and validated by using both enzymatic and whole-cell microbiological digestates of rice starch protein by-products at laboratory and/or scale up volumes. This validated technology resulted to be suitable both for the technical and product requirements of the project (project Result n°3).
In particular for both enzymatic and whole-cell scale up digestates fractionation process led to recover the following final peptide fractions: R0.2 µm, R10, R5, R1, P1. The retentate (R) products were split after cross-flow filtration for biological activity determination, freeze drying and spray drying tests.
Additional scale-up digestion and fractionation experiments were performed with rice commercial protein Remypro N80+ in order to provide enough peptide fractions for spray drying test runs, for trial product tests and also to provide a possible source of rice protein fractions alternative to rice starch protein by-products. All the obtained peptide fractions were delivered to partners for bioactivity tests.
Some enzymatic fractions coming from both Protamex and Alcalase 10L scale up and from Remypro N80+ 20L Alcalase digestion as well as some microbial fractions from 1 L scale up of C1122, Lb82 and PRIN and control cultures, were sequenced by means of different mass spectrometry techniques. The obtained results indicated the presence of a large number of peptides in each fraction. A search for the biological activity of identified peptides was carried out and confirmation of peptide sequence in rice proteins and/or protease proteins, was also performed.
4. Peptide ingredient modification via freeze drying, spray drying and encapsulation.
Tests regarding spray drying, freeze drying and encapsulation of peptide fraction ingredients, were performed and the best technique for final ingredient stabilization and production, was selected.
In particular, the enzymatic digestates could be freeze dried to produce a powder, while the microbiological digestates could not be dried completely due to the organic acid content. Spray drying was applied to fractions from enzymatic scale up experiments both of rice by-products and Remypro N80+ proteins. Experiments were performed in parallel to freeze drying in order to compare the mass balances and a loss of product was observed for the spray drying technique. Encapsulation trial experiments, by using the PGSS technology (Particles from Gas Saturated Solutions) were performed to produce a shell material in order to cover the peptide fractions. Preliminary results have shown that the encapsulation of peptide ingredients is possible. The encapsulation trails will be completed after the end of the project due to the need of further implementation of the process. The encapsulated ingredients will then be used to perform trials of nutraceutical products.
5. Evaluation of biological activities by in vitro and cell-based tests and safety evaluation of the products.
The biological activities of semi-purified digestates and of the different peptide fractions isolated by cross-flow filtration, were assessed using a large range of in vitro and cell-based assays. Peptide fractions obtained from fractionation process of rice starch protein by products (treated with commercial enzymes or whole-cell Lactobacillus strains) and of Remypro N80+ rice commercial proteins, were tested.
The following conclusions could be reached for new ingredients derived from enzymatic pre-treatments of rice by-products and of Remypro N80+ proteins:
- Analyses on both semi-purified protein total liquid digestates and selected peptide fractions from flask scale enzymatic processes, indicated that Protamex and Alcalase treatments produced peptides with high bioactivities and no cytotoxicity nor irritation capacity, while lower levels of bioactivity were detected for Neutrase-derived samples.
- The peptide fractions showed different biological activities which were slightly variable depending on the type of enzymatic treatment and on the analysed fraction (fractions containing lower molecular weight peptides showed higher biological activities);
- Most of the fractions showed anti-aging effects;
- Cytotoxic and anti-proliferation effects seemed to be dependent on the concentration of the samples used during the tests, but for each fraction a not-toxic concentration was identified;
- Liquid and dried forms of each fraction were tested and in general the powders showed an equal or higher activity with respect to the corresponding liquid fractions. This result seems to be very promising in view of an industrial application of the ingredients (easy and cheap storage, high stability);
When rice protein by-product was pre-treated with whole-cell Lactobacillus strains, the following conclusions could be reached:
- Semi-purified protein total digestates tested at different incubation times, showed that antioxidant activity reached the highest values after 72 hours of incubation, with the control samples having double activity with respect to by-product incubated with bacterial strains;
- All the isolated peptide fractions had antioxidant activity, with low peptide molecular weight fractions showing the highest activity;
- Only the fractions derived from by-product itself showed anti-tyrosinase, anti-aging and not cytotoxic effects. Instead, the peptide fractions derived from by-product inoculated with Lactobacillus strains, have to be highly diluted to reach the not-toxic concentrations while they show very low or absent biological activities.
After biological activity and safety tests performed on liquid, freeze and spray dried fractions the most suitable peptide ingredients were selected by involved SME partner (Principium) according to the foreseen product applications (project Result n° 4).
6. Development, optimization and validation of an in vitro method to evaluate skin sensitization potency.
An engineered reconstructed human epidermis skin model including Langerhans’ cells was developed (project Result n°5). Monocytes and keratinocytes were co-cultivated in the liquid-air interface using standard operative procedure. The model was then validated against a series of chemicals classified by Local Lymph Node Assay (LLNA) as sensitizers. Chemical concentration, time of contact, and post-incubation time were optimised during the model development phase. Tissue viability by MTT assay and interleukins 12 and 18 and TNFa expression, were evaluated. Seventeen standard reference chemical compounds together with a false positive and a false negative compounds, permitted to further validate and fine-tune the skin experimental model. The same model was used to test 20 peptide fractions. All the BIORICE samples were considered safe and not toxic and resulted to be not sensitizers.
7. Integration of extracted ingredients into ready to-use formulation of trial nutraceuticals.
As originally planned, the maximum scale up volume tested for each optimized pre-treatment process was 10 L. However, the whole process of digestion, filtration and fraction freeze-dry gave peptide yields lower than those minimum requested (3 kg of powdered peptides) to be tested in the nutraceutical application and necessary to make galenic formulations by microencapsulation during the project. Nonetheless, in view of the future production of nutraceuticals, the microencapsulation procedure was tested on a small amount of rice peptides. The analytical tests in order to obtain a suitable peptide microencapsulate powder will be completed after the project end and the final encapsulated product will be used to perform trial products for the nutraceutical application.
8. Integration of extracted ingredients into ready to-use formulation of trial cosmetics
A large amount of Remypro N80+ proteins were processed by enzymatic protocols, giving around 300g of final peptide powdered fractions. For all the fractions a complete technical dossier was prepared by the SME Principium in order to present the product to the potential customers, in a cosmetic line called BIORICE. Particularly, a statement of non-dangerousness of the fractions was formulated (MSDS document) together with a technical data sheet, containing the main chemical and physical information on the ingredients (SDS document). Moreover, a product information data sheet (PIDS) containing all the information about peptide ingredient raw material identification, composition data, manufacturing process, storage, packaging and logistics, analytical data, toxicological information, product compliance with legislation and cosmetic information, was produced.
Taking into account the results of biological activity tests, the percentage of safety use of the fractions and the calculation for active ingredients final cost, the fractions were inserted in the prototype creams at 0.5% and 1%. Four different finished cosmetic creams, were produced: base cream; anti-age cream; whitening cream; anti-pain cream. The creams have been prepared also without the active (0%) to have a placebo check.
The creams were put in stability testing conditions and will be monitored for at least 6 months, looking at their organoleptic properties, to evaluate if any change in the appearance, color, smell and pH, occurs over time.
Potential Impact:
1. POTENTIAL IMPACT OF BIORICE PROJECT
As highlighted in the Europe 2020 Strategy, one of the prior challenges facing today’s society is the need for innovation. The BIORICE project involves different RTDs and SMEs having complementary areas of activities leading to a strong level of integration and increasing the possibility for SMEs of being innovative and competitive across the European Union markets and worldwide.
In the BIORICE project much effort was devoted to ensure that the project results delivered by RTD Performers will be easily and directly up-taken by the SME partners. The main impact achieved by the project is therefore on the innovation capacity and competitiveness of the SMEs involved, as envisaged by the “Research for the benefit of SMEs” funding scheme.
In particular, the innovative products (semi-purified digestates and peptide ingredients) as well as innovative processes (for treatment of processing water containing protein by-products and for extraction and isolation of new peptide ingredients) and methods (in vitro evaluation of the skin sensitization potency based on human reconstructed epidermis with Langherans’ cells) developed within BIORICE will allow the SMEs to deliver added value products to existing and new markets.
Both Carminia and Principium will be able to introduce new products into nutraceutical/functional food and cosmetic target markets. In particular, the total semi-purified hydrolysed proteins are believed to have great potential in the food market, given the increasing demand for plant proteins at EU level. The total hydrolysed proteins will have a market in particular in products both for vegans and vegetarians as well as they could be an excellent protein source for gluten free market sectors.
In addition, the new peptide ingredients extracted by means of green technologies will be easily exploitable in the nutraceutical and cosmetic target markets given their eco-sustainable origin.
On the basis of the economic feasibility assessment, each SME, in its exploitation plan, has estimated the economic impact that will be achieved thanks to the industrial application of the project results developed within BIORICE. In particular:
- Carminia has estimated an annual turnover between € 225,000 and € 300,000 for the total rice protein hydrolysates to be sold in the food market.
- Principium has estimated an annual turnover of € 500,000 for the new peptide ingredients to be commercialised in the cosmetic and nutraceutical markets;
- Farcoderm has estimated a potential for global sales of 30.9 M € for testing ingredients in the cosmetic field, considering that the list of the admitted ingredients includes more or less 20,600 compounds. A realistic estimation of RHE model global sales was calculated assuming an annual increase of market penetration of 2-25%.
In addition to that, the new products will have no ethical constraints because they will be tested on the basis of methods and processes that avoid the use of laboratory animals. This represents an important social contribution to the ethics policies of the European Union, included in the 7th Amendment to the EU Cosmetics Directive (Directive 2003/15/EC) that prohibits to market animal-tested cosmetics.
The optimized digestion protocols within BIORICE, have the potentiality of being exploited by other companies, such as other starch factories or similar industrial processes in the EU area, so improving their economical and environmental sustainability for a direct industrial and market exploitation of the processes and end-products, and increasing their competitiveness and financial benefits. SMEs could in fact be addressed to the production of high value compounds, while improving efficiency and reducing the overall environmental impacts (such reducing the amount and the environmental impact of processing water). This will contribute to realize the objectives of environmental, socio-economic and industrial EU policy initiatives (Environmental Technology Action Plan and Lead Market Initiative on Bio-based products) and to the application of the objectives of EU Waste management directives and strategies such as the Thematic Strategy on the Prevention and Recycling of Waste, the Waste Framework Directive (2008/98/EC) and the Landfill Directive (1999/31/EC).
The BIORICE project has also contributed to tackle the challenge of facing the global increasing demand of new feed and food protein sources alternative to animal proteins (whose production is largely environmentally unsustainable), in compliance with most recent EU policies such as the EU COST “Policy brief on sustainable protein supply”. In fact, the global demand for animal proteins is increasing rapidly due to the growing world population and reinforced by the increasing income per capita in industrialising countries. Conversely, the production of plant proteins is typically more sustainable than that of animal proteins, and an increase in the use of plant proteins, such as total digestates produced within BIORICE project, will therefore significantly contribute to a sustainable protein supply in the future.
Finally, thanks to the BIORICE project two new jobs have been created for junior researchers hired by two of the SMEs involved and, considered the limited lifespan and resources available for the BIORICE project as well as the size of the SMEs involved, this can be considered as a significant result.
2. DISSEMINATION ACTIVITIES PERFORMED DURING THE BIORICE PROJECT
The dissemination activities performed during the whole duration of BIORICE project have been largely consistent with the originally planned activities of the project proposal and were reported in the Interim Plan and in the Final Plan for the Use and Dissemination of Knowledge.
The Consortium partners, have used an array of media and tools in order to successfully disseminate the project to the relevant selected audience.
2.1. Publications
Publications, either in the form of Press Releases, informative papers or general divulgative papers have been published and have played a significant role in the dissemination during the whole project period. The project was also disseminated by means of various newspaper articles both in printed form and on-line on several websites.
Press Releases were aimed primarily at the local audience, general public, industrial stakeholders. This communication tool has proved particularly useful since it has disseminated the project at large to a wide public which would have been not easily identified via standardized methods of dissemination such as events and scientific conferences.
The logo of the BIORICE project is one of the first entries in Google images. A logo for BIORICE ingredients was produced by Principium and, after registration, will be included in the marketing and technical sheets of the future cosmetic and nutraceutical products on the market.
2.2. Website
The project website (www.biorice.eu) was launched publicly on the January 29th 2014 and since then acts as a platform with the aim of establishing an efficient and effective dissemination and communication tool for the BIORICE Consortium for the duration of the project.
It accounts also of a private area for which each partner has specific credentials (username and password) and in which the restricted documents relative to project were made available to the partners. The documents were uploaded by the Coordinator who was also the Administrator of the website restricted area. Each partner was assigned with a project dedicated e-mail address.
The content of the website was regularly updated, upon request of each partner, as well as at the accomplishment of deliverables or after project meetings. The publications, posters, leaflets about BIORICE project were uploaded (download section) and highlighted on the main page of the website, together with the information (date, venue, organizing partner) regarding BIORICE technical meetings or partners’ participation to fairs, conferences and events. After the end of the project the website will be still on-line for 2 years during which it will be regularly updated.
2.3. Social networks
A BIORICE dedicated page was opened on Facebook (https://www.facebook.com/Biorice-372490549591970/?ref=bookmarks) showing pictures of the meetings and posting news on fairs and results of the project.
A BIORICE profile was also opened on Twitter (https://twitter.com/BIORICE_Europe) giving news about project meetings and contacting different European groups on research.
The link to both social networks pages was published on the main page of the BIORICE website.
2.4. Leaflets and Posters
Several leaflets and posters were used to present the project during the fairs and events to whom BIORICE partners took part. All these dissemination materials were made available in the Download section of the project website. Moreover, in the occasion of BIORICE presentation at EXPO (Milano, Italy, 10th September 2015), two new leaflets were specifically prepared.
Several posters, both aimed at general and/or at scientific public, were prepared and presented to exhibitions, conferences and fairs.
2.5. Events
During the whole project a good success in relation to the participation of the partners to different type of events such as conferences, exhibitions and fairs, was pointed out. All events had a wide geographic approach, targeting audiences in Europe and worldwide countries.
The project results were presented thoroughly and throughout these events, with the partners aiming at maximizing the visibility status of the project. The events helped to promote the project towards targeted audiences via personal fair stands and promotional material.
The dissemination activities performed approached an array of interested parties, coming from different fields and areas of interest. Universities and academic institutions, technology institutes and industrial partners are some examples of the type of audience approached. Specifically SME partners, it is notable to mention that they participate to conferences, fairs and exhibitions mainly targeted to customers. The well-received reactions of these audiences demonstrate that the BIORICE results are indeed innovative in various contexts.
A special emphasis should be put on BIORICE presentation at EXPO (Milano, Italy), on 10th September 2015, organized by Principium. All BIORICE partners, in particular SMEs, were invited to the exhibition; the partners disseminated the information of the event to their customers and to all possible stakeholders. The event was held at Italian Pavilion, in the Como Business Center.
2.6. Project video
On the occasion of the presentation of the BIORICE project to the EXPO exhibition, a dedicate video was shot under the supervision of Principium. The video was aimed at explaining to the general public the background of BIORICE and the main objectives proposed. A general focus on the achieved results, was also added. The video was uploaded on BIORICE website main page (www.biorice.eu) and on Principium website (www.principium-bsi.com).
2.7. Other activities
During participation at fairs, most importantly, the project’s outputs for Principium were explained to distributors, that have a direct contact with final customers, which will insert BIORICE ingredients into cosmetic and nutraceutical formulations.
A great opportunity for making contact with potential customers, was given by the networks in which Principium and Farcoderm are involved. In particular for cosmetics, Principium and Farcoderm participate to the HCI (Helvetic association for Cosmetic Ingredients) a Swiss network, member of the European EFfCI association; for nutraceuticals, Principium is part of the Sifood Association, an Italian network of food and nutraceutical companies. These networks represented a great chance to present BIORICE project to business partners and to discuss about the main pillars of the project: innovation and sustainability.
3. EXPLOITATION OF FOREGROUND
The exploitable results generated within the BIORICE project were acquired by SMEs in accordance with the DoW.
The exploitation of BIORICE project results, after the end of the project, will consist in:
a. a product development phase during which the SMEs will move from prototypes to saleable products;
b. an initial exploitation phase during which the SMEs will concentrate on establishing their market presence at the EU level;
c. a further expansion phase during which the markets in the target countries will be consolidated and results from sales obtained in the previous period will be used to penetrate new markets.
3.1 Principium exploitation Plan
From BIORICE project, Principium acquired the technological know-how for rice peptide isolation through cross-flow membrane filtration and the ownership of new peptide ingredients to be exploited in the cosmetic and nutraceutical markets.
-Time-to-market
After the end of BIORICE project, at least two steps will have to be considered before approaching the market.
First of all, the process has to be further standardized. For that, it will be necessary to spend at least 3 to 6 months analyzing different batches of by-products and making pilot batches, in order to compare their composition, activity and safety profile.
The second step will take at least one year and will be focused on additional testing of produced peptides. Principium will perform in vivo studies on active peptide fractions and will test their safety profile on humans. These steps will complete the product dossier.
In conclusion, it could be estimated that the selling of new BIORICE active peptides line will be in 2017. Once on the market, both in the nutraceutical and in the cosmetic fields, the products will be presented to fairs and to distributors. With a strong “green” background, the products could be sold worldwide, claiming innovation, sustainability and Italian origin of products.
-The Market
The new ingredients developed within BIORICE are targeted to the nutraceutical and cosmetic markets. These markets give huge potentialities in terms of innovation and insertion of active peptides with a safety tested profile. The developed ingredients, on the basis of their purity level and of their foreseen application, will be available for manufacturing companies that will formulate them into soft-drinks, tablets, capsules, sachet, bars, bakery products, dairy products, skin & body care lotions. These companies could be contract manufacturers in charge of the production for other or their own brand companies. The innovative character of the developed peptides, will allow Principium to face no direct competition for niche or premium products (targeting vegan or hypoallergenic products) but for the mass market, dominated by huge product volumes.
3.2 Farcoderm Exploitation Plan
Actually most of the data on skin sensitization rely on results obtained on cell cultures and on human exposure. With the entry into force of the 7th Amendment to the EU Cosmetics Directive (Directive 2003/15/EC) and the new regulation on the Registration, Evaluation, Authorisation and Restriction of Chemicals (Regulation No 1907/2006), there is an urgent and pressing need for alternative testing methods. The last trend is to develop in vitro complex models, in order to represent as much as possible the human conditions, but trying to remove all the variables and study the reaction to a product application.
From BIORICE project, Farcoderm enhanced its know-how in understanding the mechanism underlying skin sensitization and acquiring the ownership of a newly developed reconstructed human epidermis model (RHE) containing Langherans’ cells. This model will allow to standardize a new in vitro safety testing method to evaluate the occurrence of skin sensitization and the potency of chemicals/products to induce skin sensitization, and to better characterize the hazard of chemicals and products. This new protocol is free of ethical issues and complies with the existing EU regulations that ban the use of laboratory animals.
-Time-to-market
The RHE model represents for Farcoderm an interesting device to test the safety profile of an active molecule. This model will be completed with a reference scale, in order to give a value to the tested substance. Within BIORICE project, in order to build this reference scale, several standard molecules that give different grades of sensitization on human skin were applied on the RHE model and its reaction was monitored.
Farcoderm needs 6 more months, after the end of BIORICE project, to finally set up the model. In this time, other chemicals/endpoints will be tested and the reference scale will be further confirmed by using new substances. A fine tuning of the produced reference scale will be performed, in order to make the model a standard device for each chemical.
After this first step the model will be ready for the market. It will be a useful tool to test the safety profile of a new ingredient or formula. In fact it will be more detailed than a simple keratinocytes/fibroblast layer being more similar to an “ex vivo” model. The new method will be brought to the market in 2 years (time needed for EURL ECVAM validation phase) after the end of the project.
-The Market
The new testing method is targeted to raw material manufacturers (both cosmetic and pharmaceutical grade), cosmetic, medical device, pharmaceutical and textile industries. As the method will be patented and validated, the market share is assumed to be 100%. The main competitors are represented by testing service companies or academia that perform in vivo tests to evaluate skin sensitization. This method, anyway, has the good characteristic of being more precise of the in vitro cell cultures but less invasive than the in vivo testing.
3.3 Carminia Exploitation Plan
Carminia will enforce its portfolio of consultancy services to help client companies producing high value compounds (like proteins and other molecules), while improving efficiency and reducing the overall environmental impacts of production. Since Amideria Il Cervo has terminated the rice starch production and ceased from the project (March 2015), by entering the BIORICE project, Carminia was able to acquire the rights on the technical know-how for producing protein digestates, and the ownership of new total hydrolysed proteins to be sold in the food and beverage sector.
-Time-to-market
The protocol can be applied in a time span between 6 and 12 months, while, after this, adaptation to different producers is esteemed to require from one to two years considering experimental trials and validation. Regarding semi-purified digestates obtained during BIORICE, Carminia recently planned an investment to build a production site specifically dedicated to protein hydrolysis. The site will be in force during the first six month of 2016. This will allow the production of hydrolysed rice proteins derived directly from rice proteins.
-The Market
The future strategy is to directly address the starch factories in the EU area and propose the application of the BIORICE protocol using as substrate the slurry containing proteins and set up as set of demonstration steps in Carminia production plants facilities, so to demonstrate the effectiveness of the protocol. It would be as well possible to modify the protocol according the clients requirements. Regarding protein digestates these will be exploited by Carminia mainly in the food sector. Indeed these hydrolysed proteins could have a market in the production of reconstituted milk (based on soy or rice kernel) or creation of yogurt, as well in products both for vegans and vegetarians. In addition, the hydrolyzed rice proteins could be an excellent protein source for gluten free market sectors (bakery and sweets, ice cream ready meals).
List of Websites:
www.biorice.eu
Project Coordinator: annalisa.tassoni2@unibo.it