Final Report Summary - FOFIND (“Development and human validation of new healthier food products using low-cost functional ingredients”.)
Executive Summary:
The project FOFIND will develop new affordable daily consumed functional food (biscuits, breads and juices) and nutritional supplements, using innovative low cost functional ingredients derived from milk and potato fibre. These new products will be directed to the general population with specific nutritional needs that control the symptoms of hypertension, hypercholesterol, osteoporosis, anemia, and digestive problems.
The main results of FOFIND will be (1) Functional ingredients derived from milk by-products with demonstrated activity (Lowpept plus phytosterols, CPPs plus calcium salts, CPPs plus iron salts); (2) Nutritional complement tablets with functional ingredients derived from milk by-products; (3) New biscuits with functional ingredients derived from milk by-products and potato fibre; (4) New bakery products with functional ingredients derived from milk by-products and potato fibre; and (5) New juices with functional ingredients derived from milk by-products.
Human testing will be carried out to demonstrate the functionality of the results: (i) Lowpept to prevent hypertension; (ii) Lowpept plus phytosterols to prevent cholesterol and hypertension; (iii) CPPs plus calcium salts to help the absorption of calcium and (iv) CPPs plus iron salts to prevent anemia.
FOFIND project will support SMEs breaking through these barriers, by means of the development of new added-value functional food and nutritional supplements to be implemented in a short term, which will allow SMEs to increase their competitiveness and will lead them to achieve new market niches and new consumer profiles. FOFIND will develop new functional ingredients and food aimed to improve the most prevalent nutritional needs in EU, providing added value to the final products and, ultimately, increasing the return of investment of food producer SMEs. In order to ensure project objectives, FOFIND consortium is formed by four SMEs and five RTDs from four EU countries (Spain, Poland, Sweden and Germany).
Currently the European context of the functional food ingredients exploitation is as follows: There are loads of potentially efficient molecules I the Research departments of companies, institutes and universities. To introduce those molecules in market it's necessary to fulfill the following conditions:
A) To demonstrate their effectiveness in clinical trials. In vitro or in vivo (animals) trials are not, logically, sufficient to that end.p
B) To demonstrate their lack of toxicity. This can be avoid in certain cases when the consumption of those molecules by the population is a well known reality.
C) In case the molecules are foreseen to be included in a food, the incorporation in the food must maintain the efficient molecular structure which allows the functional objective. Firstly in the production itself and lately during the shelf life of the product.
D) In case the molecules are foreseen to be included in a food (bread, biscuits, yogurt, etc.) the final product must be sensorially acceptable by the consumers.
E) When the molecules are foreseen to be incorporated to a food supplement (tablets, capsules, etc.) the former condition would not be applicable.
F) The production of the ingredient and the subsequent processes must be economically feasable.
G) When the product is foreseen to be sold in the retailing market, a health claim admitted by EFSA, written on the packaging, is necessary to be get significative sales. In other words, a product without health claim is practically impossible to get it sold. When the claim of the new ingredient itself, is not approved by EFSA, companies are currently including also in their products minerals or vitamins which have a health claim already approved by EFSA.
This project can be approached as two different sub-projects
1) Developments carried out by participants VINI (bread) and SGPlus (potato fiber functional to avoid cancer): There is no clinical trials (A). There's not either any health claim approved by EFSA (G) relative to cancer. VINI should state their ideas to commercialize their products with potato fiber.
As a reference, BISCUIT envisaged at the beginning to use potato fiber in their products but, after knowing the lack of clinical trials and claim, they considered impossible to commercialize and gave up to work with this ingredient within this project.
Developments made by VINI with INNAVES molecules: Results are totally useless. They have worked with an old ingredient which was not valid for the project and they were aware of that. They didn't fulfill the presence of target molecules after processing (C). The sensorial test they say to have done (D) is useless as the ingredient used was not valid. They were told that such work wouldn't be eligible.
2) Development carried out based on INNAVES molecules (casein hydrolysates) to be incorporated in biscuits (SME BISCUITS) and food supplement (tablets) (SME INNAVES):
As clinical trials haven't been carried out (A), the results of the project are totally useless for SMEs involved in the project. Partial results are irrelevant for companies and will end in folders on the shelves of institutes an universities. The main work of the project (human validation), included even in its title, hasn't been carried out. The objective of the program (benefit of SMEs) hasn't fulfilled at all.
Project Context and Objectives:
The general aim of the FOFIND proposal is to develop new affordable functional daily consumed foods (biscuits, breads and juices) and food supplements, using innovative low cost functional ingredients derived from milk and potato fibre. This new products will be directed to population with specific nutritional needs (hypertension and hypercholesterolemia patients, population with calcium and iron needs) and will comply with the legislative requirements for their commercialization. The main aspects of the project are:
1. Development of functional ingredients derived from milk subproducts:
i. Lowpept plus phytosterols (hypertension and hypercholesterolemia, metabolic syndrome)
ii. CPPs plus calcium salts (population with calcium needs, for example osteoporosis, children, healthier, postmenopausal woman, etc.)
iii. CPPs plus iron salts (population with iron needs, for example anaemia, pregnant woman, healthier, etc.)
iv. CPPs plus selenium salts (selenium is very antioxidant and contributes to normal spermatogenesis, to the normal function of the immune system, to the maintenance of normal hair, nails, etc.)
2. To demonstrate the functionality of functional ingredients derived from milk subproducts in humans:
i. Lowpept to prevent hypertension
ii. Lowpept plus phytosterols to prevent cholesterol and hypertension
iii. CPPs plus calcium salts to help the absorption of calcium
iv. CPPs plus iron salts to prevent anaemia
v. CPPs plus selenium salt
3. Development of affordable and high-quality nutritional supplements and functional food with different food matrix (juice, biscuit and bread)
4. Finally, the FOFIND is focused on a quick launching of the project results to the market that implies providing scientific evidences for an eventual approval by the EFSA.
The companies involved in the project consist of one biotechnological company in charge of the development of the functional ingredients and, accordingly, the food supplements (INNAVES), and three companies acting as end users (Biscuits, Vini and Jacoby), in charge of introducing the functional ingredients in their products (biscuits, bread and juices, respectively) in order to obtain a product with a higher value.
In this context, the aim of INNAVES is to develop food supplements and functional ingredients derived from milk proteins with different functionalities (antihypertensive properties, and enhance of mineral absorption), and jointly with phytosterols, and finally, to test their functionality by human trials.
CSIC is the leader of the Work package 1 (WP1: Development of functional ingredients derived from milk proteins). This WP is in charge of designing novel CPPs based products from milk by-products to improve mineral (calcium, iron and selenium) bioavailability. The impact of simulated gastrointestinal digestion and the stability of the ingredients when incorporated into the final food products will be also carried out. This group is also involved in the evaluation of the possible mechanism of action of the ingredients with antihypertensive (Lowpept®) effect by analyzing different plasma biomarkers in samples from the clinical studies.
At the same time, the aim of Biscuits, in collaboration with UVigo, is to develop functional biscuits with the functional ingredients derived from milk and milk sub-products provided by INNAVES. The objective of experiments carried out was study the incorporation of Fe, Ca and Se to casein hydrolisates in order to obtain functional ingredients to be used in the production of fortified biscuits. This study involved the way of incorporation of mineral salts to casein hydrolisates (including their encapsulation) the stability of the functional ingredients obtained and, finally, the evaluation of functional biscuits with ingredients added.
Similarly, Vini’s goal in the FOFIND project is to develop low-cost bakery products enriched with milk- and potato fibre-derived functional ingredients. Main activities include: elaboration of functional ingredients enriched bread recipe of suitable taste, smell and structure characteristics; studies on shelf-life and other properties; manufacturing bread needed for scientific evaluation.
At the same time, the aim of SGPlus is an in vitro evaluation of anticancer activity of water extracts obtained from bread supplemented with potato fiber (Potex). Eighteen different samples of bread were prepared. Three kinds of bread (graham, pytlowy and rye) were supplemented with potex. The test comprised - the initial screening of all samples for antiproliferative activity in colon carcinoma cell culture and selection of the most active bread sample. Technology for obtaining most effective water extracts was established first.
Regarding Jacoby and ILT, the aim is the incorporation of functional peptides partly in combination with minerals into juice based beverages (WP5). Juice provides a health-positive association to consumers. Since the sensory properties of both, peptides and minerals (iron) are problematic suitable formulation for the beverages have to be developed and must undergo sensory panel tests. Furthermore the beverages need to be stable for a period of several months without chilling in practice. A low pH-value and usually a thermal treatment is necessary therefore. Particularly these process condition have the potential to negatively affect the functional peptides. Hence, the stability tests and the therefore required analytical methods are the main objective. In addition the binding capacities of the minerals depend on the matrix properties and is hence an objective in WP5.
The project was based on a previous product called Lowpept®, based on a patent from CSIC and licenced by INNAVES, which consists on a food grade casein hydrolysate with antihypertensive properties. From this process, a high value by-product of hydrolysate casein was obtained, which consist of casein phosphopeptides (CPPs), and these CPPs are the basis for 3 other functional ingredients.
At the time the FOFIND project started, INNAVES was already searching for the latest technology which could greatly improve the obtaining of the functional ingredients as well as the possibilities to use them in the food matrices envisaged in the project. Such technology was based on the use of ultra and nano-filtration by using ceramic membranes. Consequently, during the project some trials with a small equipment of ceramic membranes were started and therefore a pilot plant of ceramic membranes were acquired by INNAVES. With the help of CSIC, which has been analyzing all the samples of the batches that INNAVES has produced with the new technology, we have realized that the previous by-product (the CPPs) obtained is no longer a by-product as now all the hydrolyzed casein is included in the Lowpept® product.
This circumstance has not changed the interest that INNAVES has in the products to enhance the mineral absorption (Fe, Ca and Se), and some interesting alternatives have arisen. For instance, as the new Lowpept® includes the CPPs obtained with the previous technology, INNAVES is performing some trials to bind iron salts (ferrous bis-glycinate) to Lowpept®, applying the same conditions obtained by CSIC for the previous CPPs.
On the other hand, the fact is that including the small patented peptides additionally to the CPPs (which is the new version of Lowpept®) may reinforce the absorption on the minerals through other two mechanisms of action that INNAVES and CSIC have discovered.
Project Results:
The progress of the work for each work package is going to report to follow. The detailed results can find in the deliverable of each participant
-Work package 1: Development of functional ingredients derived from milk proteins:
The characterization of the by-product obtained from Lowpept manufacture has been performed and the caseinophosphopeptides released from tryptic hydrolysis and simulated gastrointestinal digestion were identified. In order to develop protein-based formulations with capacity to improve mineral bioavailability, mixtures of the products provided by Innaves and different food-grade iron, calcium and selenium salts were assayed and the mineral binding ability was estimated. Based on the results obtained, three different enriched ingredients are proposed:
(i) By-product + ferrous bis-glycinate at a protein:Fe ratio 50:1
(ii) By-product + calcium chloride at a protein:Ca ratio 7:1
(iii) Lowpept® + sodium selenite in maltodextrine at a ratio of 1000 mg protein/35 μg Se
The functionality of the different ingredients developed in this project was evaluated by using different in vitro, ex vivo and animal assays. It was demonstrated that the by-product is an excellent source of caseinophosphopeptides. The peptides generated during the in vitro gastrointestinal digestion of this by-product are similar to those found in duodenum after in vivo oral ingestion of casein. The mineral enriched products based on Lowpept® or by-product showed good iron and calcium solubility under conditions that simulate the intestinal environment. The peptidomic study of porcine duodenal digests after ingestion of Lowpept® or the precursor casein revealed that in addition to the previously identified peptides, other regions, especially from β-casein, could be important for the antihypertensive effect. The gastric resistance of the previously identified peptides RYLGY and AYFYPEL was demonstrated and it was found a significant higher concentration of RYLGY and related peptides in the duodenal digests from pigs fed with Lowpept® in comparison with those fed with casein. A noteworthy increase of several plasma biomarkers was found in those animals which had received Lowpept® and in particular, an elevated concentration of L-arginine, which is a substrate for vascular nitric oxide production. The opioid activity of YFYPEL and other related sequences was proved in an ex vivo guinea pig ileum preparation and it could be demonstrated that the antihypertensive activity of Lowpept® and some active peptides is also mediated by the interaction with opioid receptors , since the effect can be reverted by an opioid antagonist. From the study with the ingredient based on the combination of Lowpept® and phytosterols, it was found that the hydrolysate was responsible for the antihypertensive activity and the improvement of other cardiovascular parameters, while the phytosterols decreased the atherogenic index and have a beneficial effect on the lipidic profile. These novel results, which some of them could deserve further research and protection, could be used to sustain health claims on the different ingredients regarding the maintenance of the health status.
-Work package 2: Up-scaling of milk proteins functional ingredients production process, the development of tablets to include the new ingredients and the validation of their functionality in humans.
At the time the FOFIND project started, INNAVES was already searching for the latest technology which could greatly improve the obtaining of the functional ingredients as well as the possibilities to use them in the food matrices envisaged in the project. Such technology was based on the use of ultra and nano-filtration by using ceramic membranes. Consequently, during the project some trials with a small equipment of ceramic membranes were started and therefore a pilot plant of ceramic membranes were acquired by INNAVES. With the help of CSIC, which has been analyzing all the samples of the batches that INNAVES has produced with the new technology, we have realized that the previous by-product (the CPPs) obtained is no longer a by-product as now all the hydrolyzed casein is included in the Lowpept® product.
This circumstance has not changed the interest that INNAVES has in the products to enhance the mineral absorption (Fe, Ca and Se), and some interesting alternatives have arisen. For instance, as the new Lowpept® includes the CPPs obtained with the previous technology, INNAVES is performing some trials to bind iron salts (ferrous bis-glycinate) to Lowpept®, applying the same conditions obtained by CSIC for the previous CPPs.
On the other hand, the fact is that including the small patented peptides additionally to the CPPs (which is the new version of Lowpept®) may reinforce the absorption on the minerals through other two mechanisms of action that INNAVES and CSIC have discovered.
During the first period, due to the new production process developed for Lowpept®, a new approach to the functional ingredients containing CPPs was been considered. This new approach is based on two mechanisms of action discovered for the small peptides, including RYLGY and AYFYPEL, already included in the patent which will potentially improve the enhancement of mineral absorption.
For this functional ingredient, the iron salt chosen was that recommended by CSIC and also by UVigo and ILT, and this is ferrous bisglycinate. In addition, in order to achieve a good yield in the up-scaling of this ingredient, different ways of binding the iron to the new version of Lowpept® have being carried out by the wet method was has been chosen. This method has been successfully applied to iron, calcium and selenium.
For the production of Lowpept® + phytosterols, a first screening of the commercialized phytosterols was carried out in order to choose the most suitable one to be mixed with a hydrolyzed casein. From the phytosterols already available in the market, five of them were selected based on the hydro-solubility and the daily dose. The new production process of Lowpept® has allowed concentrating the dose of Lowpept®, which has facilitated the incorporation of the necessary dose of phytosterols to the daily dose of Lowpept®.
For the Lowpept®+phytosterols tablets, several batches of tablets were made with both phytosterols mixed with Lowpept®, and one of phytosterols were chosen based on the final characteristic of the tablet (friability, broken resistance and dissolution). It should be noted that different excipients were proved in order to meet the best requirements and acceptability in the market. For example, at the beginning the poly vinil pirrolidone (PVP) (chemical origin) were used as an excipient, but we have changed it for xanthan gum (vegetal origin) because it is more natural than PVP.
After the ultrafiltration of the hydrolysis of casein, there are 2 main fractions: 1) the retentate from the micro-filtration and 2) the retentate from the nano-filtration (this fraction includes RYLGY and AYFYPEL). The binding process is based in these two fractions. The interest of binding RYLGY and AYFYPEL to the iron, calcium and selenium salts is that during other research projects currently conducted by Innaves, it was concluded that both peptides have other activities different from the antihypertensive activity, which are: (i) induction of the production of nitric oxide (NO) and (ii) α-agonist of opioid receptors. These two mechanisms could help to improve the absorption of minerals.
The results obtained dirung second period were:
1. New ratio protein:Fe more adequate for food supplement has been established, 30:1 (p/p). With this ratio, 1 gram of the functional ingredient will provide 23 mg of Fe, which is an adequate amount for a daily intake and can be formulated in 2 tablets.
2. For the application of the functional ingredient to the food matrix envisaged in the project, biscuits, bread and juices, some changes in the formulation can be made in order to adapt to the process and to the final product. For instance, 3 or 4 biscuits can be an adequate daily dose which means that a ratio of 50:1 could fit better in their process.
3. Five phytosterols were chosen based on the solubility and the daily dose. The phytosterols are very difficult to dissolve in water, and for this reason, the hydro solubility was the main criteria for the selection of the phytosterols.
4. After several batches, one formulation and one of the phytosterol was successfully chosen. The final formulation of tablets containing Lowpept®+phytosterols (named LOWDOS®) has been registered in the Sante Publique, Securite de la Chaine Alimentaire et Environnement, Direction Générale Animaux, Végétaux at Alimentatio, Brussels (Belgium), through the FOODSUP tool, in order to obtain the NUT/PL/AS number (NUT-AS number granted). This number will allow us to sell this product all over Europe. Both health claims for hypertension and cholesterolemia are accepted and will be written in the box.
2. The food supplement named IronPept®, which consists in the new version of Lowpept® bounded to an iron salt, has been successfully obtained.
3. The food supplement named CalciumPept®, which consists in the new version of Lowpept® bounded to a calcium salt, has already been obtained as a powder. The tablet can be produced, however, due to the high calcium RDA, the daily dose is more than 3 tablets per day, which is not desirable. Instead, an effervescent tablet is currently under development.
4. The food supplement named SeleniumPept® in powder has been successfully obtained at the laboratory. In addition, a tablet form is also under development, even though is not envisaged in the project.
During the second period, the clinical trials will must be carry out. For this, it is necessary the authorised by the Galician Ethic Committee. The application must include the report of the trial as well as the technic sheets of the products to be tested. To that aim INNAVES manufactured 150 bottles (each one with 60 tablets) of the four products to test as follows:
1. Lowpept (2 tablets /day)
2. Lowpept plus phitoesterols (3 tablets/day)
3. CPPs with iron salts (2 tablets/day)
4. CPPs with calcium salts (4 tablets/day, however, another presentation for the consumer will be development before commercialization in order to avoid drop up of the consumers in long treatments as case with are focus on)
Besides, INNAVES manufactured the same quantity of bottles for placebo, without including our Active Ingredients. A code based on colours will allow the trial to be double bind. The batches to be used for the trials were biochemically and microbiologically analysed to check their fulfilment with the European Legislation. The technic sheets were issued.
The applications for the Committee was pending on the confirmation of the clinic which will carry out the trial which, in turn, is pending on the REA decision on the amendment letter. Without that information it can't be applied for. However, once we apply for, the authorization might be obtained within the term of one month. Regarding selenium tests will be run using biscuits instead of tablets. According with the legislation such products will be considered as enriched good instead of food supplements.
Work package 3: Development of functional biscuit
The main results obtained by UVIGO-BISCUITS during the period 1 were: optimum ratio Fe:protein was 1:5. At this ratio, a higher solubility of protein and iron was achieved, for both casein and hydrolysates. Hydrolysates from INNAVES increased the solubility of iron when they are used instead of casein. The linkage of iron to hydrolysates is not stable at high temperatures as well as in simulated gastric conditions suggesting that, previous encapsulation, could be considered to improve these results and to avoid lipid oxidation. Formulations F9 and F10 were used to obtain iron fortified biscuits resulting scarce differences with respect to control non fortified biscuits
Optimal conditions to link Ca to bydrolisates from INNAVES were: use calcium lactate at pH 7.5 for time 10 minutes. The incorporation of calcium into the biscuits resulted in harder biscuits.
The use of sodium selenate in a matrix of maltodextrin combined with H2 in a ratio 1:100 w:w, was selected as the selenium fortified biscuit of interest. Biscuits with Se provided not significant differences in sensorial test when they were compared with control biscuits
The new casein hidrolysate with low molecular size (CH300-0.1) revealed to be an ingredient with application for the encapsulation of iron, calcium and selenium. In addition, CH300-0.1 presented emulsifying capacity, similar to Tween 20 and slightly higher than lecithin and could be also used for the preparation of food emulsions and for the incorporation of phytosterols as functional ingredient. FTIR analysis reveal that the presence of cation in dissolution changes the configuration of the protein due to the linkage formed between the cation and the amide groups of protein. These changes were more obvious in presence of calcium and selenium.
In the second period, the deliverable provides
- The acceptability by consumers of biscuits containing functional ingredients (iron, calcium and selenium).
- Different formulations of minerals encapsulated or not with casein hydrolysates assayed.
- The stability of biscuits with minerals was evaluated and sensory analysis was performed.
- Organoleptic assays included objective characterization of functional biscuits (colour and textural properties) as well as subjective test including consumers trials.
According to results, the main conclusions are: The organoleptic characteristics of biscuits incorporating Fe or Ca were worse than those obtained with control series biscuits without minerals. These results were consistent in discriminant test and consumer trials. Encapsulation of Fe and Ca did not allow improve the sensorial properties of biscuits in relation with lyophilised minerals. Incorporation of selenium salt (encapsulated or not) did not modify the sensorial properties of the biscuits.
Work package 4: Development of functional bakery products
I. The aim of the reasearch
The aim of research made as a part of FOFIND for Vini was to obtain recipes and technology of production of bread containing:
1. Lowpept with ingredients defined by Innaves.
2. Potato fiber, the use of which would allow bread to have anti-cancer properties.
Introduction of such bread to the market is very important because the amount of diet related ilnesses is very high and there are deficiencies ion highly processed food.
II. The scope of research
Vini bakery scope research was established in
1. WP 1 task 3 Study of stability and compatibility of CCP – based in food matrices
2. WP 4 task To determine the technological aptitude of functional ingredients to be incorporated into the bread formulae.
Subtask 1.1 Highlight technological possibilities of the incorporation of heated and unheated potato fiber
3. WP 4 task 2 Incorporation of the ingredients into baking formulae
Subtask 2.1 Study of different formulae to incorporate the new ingredients
Subtask 2.2 Determination of baking technologies for incorporating the new ingredients
4. WP 4 task 3 Study of the stability of the functional ingredients in bread
Subtask 3.1 Kinetic stability analysis of the functional ingredient included in bread
Subtask 3.2 Production of bread and research performed by SGPlus
5 WP 4 task 4 Study of the sensory properties of functional bread
Results
1. Obtaining the recipe and technology needed for Lowpept bread production
2. Obtaing the recipe and technology needed to produce potato fiber bread with anti-cancer properties
3. Confirming carcinogenic properties of bread during in vitro and in vivo research
4. After sensory research we confirmed the high quality of bread and the acceptance of it by consumers
5. Patent application in Patent office of Sweden (application number 1451152-1, Awapatent reference number SE-21072185)
6. The signing of letters of intent by key buyers is a sign of their eagerness to introduce bread created by FOFIND research to the market.
7. On the basis of potato fiber bread created in a project of Medical University of Silesia - Public Health Department- a thesis is being written by Gabriela Potapowicz, titled " The analysis of quality of chosen properties of functional bread"- supervisor dr inż. Marek Kardas.
Work Package 5: Development of functional juices
- The minerals for the binding with CPP are characterized; a small choice is selected for further trials and shared with the partners. Two test recipies were develpoped
- Analytical methods for the identification of the functional peptides and two suitable methods for the binding capacity of iron and calcium to CPP´s are established and adapted to the methods of the partner CISC for comparison purposes.
- The binding of two selected ferrous salts and of calcium salt to the new CPP provided by the partner Innaves was examined (up to 80 % Ca binding, 50 % Fe binding). The technical processes for the binding of Ca and Fe is described.
- With the customers (SME) request five recipes with functional ingredients (Lowpept, CPP, calcium iron, selenium potassium) were developed.
- The stability of the functional peptides at different temperatures and with different technical equipment (flash pasteurisation, bottle pasteurisation, Ultra High Temperature Treatment (UHT) and autoclave) is tested. Extreme heat load lead to destructions. With moderate heat load conditions the peptides seem to stay stable. When employing same extents of (microbiologically) lethal heat loads but an different temperatures, higher temperature better to protect the peptides (in accordance to the different activation energies).
- The influence of the functional ingredients correspondent to the expectations. There is an effect on the turbidity, the ascorbic acid content, colour and sensory properties. Polyphenols seem not to be impacted.
- The long term stability in real beverages (customer tailored) in terms of the peptides and the beverage properties is acceptable.
Work package 6: In vivo and in vitro assays for anticarcinogenic activity validation
AD RP 1:
Bread is one of the most consuming products in the world. Besides the nutritional value, the bread might have different effects on the body, depending on the components that are included during processing and baking. The current project has focused on the anti-proliferative effects of bread supplemented with potato fiber (Potex) on colon cancer cells in vitro and in vivo models. To be able to perform in vitro study, 18 extracts from bread, both regular and supplemented with 1 or 2% Potex were tested. Two bread extracts one from Vini and one commercial showed highest anti-proliferative effect on colon carcinoma cells in vitro. Note! commercially available bread used as control was found to have also cytotoxic effect to normal colon cells, thus possibly being cancerogenic!!!
Ad RP 2
The best brad with anti-cancer properties develop by Vini in FOFIND project was studies in mice models with colonic carcinoma cells inoculation and tumour transplantation showed promising anticancer activity of the animal feed supplemened with potato fiber that underdone the heating process. Vini submit (2014-09-29) patent application for aforementioned discovery. Moreover, the total amount of leucocytes was decreased in the peripheral blood, indicating also an anti-inflammatory effect of potato-fiber processed in the bread. The latest is considered to be reviewed towards IPR protection during PCT submission. The MS6 was achieved we prove in vitro and in vivo the anticancer properties of potato fiber after processing in baking bread.
TASK 1. Preparation of water extracts and laboratory animal feed or water extracts from whole or bread shells and biscuits respectively (SGPlus + Vini)
PREPARATION OF WATER EXTRACTS FROM BREAD (SGPLUS)
3 different kinds of bread (18 different variation) were formulated and produced. Ready bread was chopped into pieces, dried and grinded into powder. 10-20 g of such bread powder was suspended in 200-400 ml of distilled water. The mixture was stirred by magnetic stirrer (active extraction) and left for some period without stirring (self extraction), totally for 24h at room temperature. Next the suspension was centrifuged (5000 rpm/ for 10 or 30min). Received supernatants were collected to the silicon tubes and evaporated under vacuum at water bath (80-100oC). The dried extracts were examined under microscope using round cover slips, weighed and saved for study in vitro.
In addition, solutions of 1% potex mixed water in proportions as in bread and 1% of potex mixed with water and yeast /leaven was also used for extractions.
TASK 2. Studies in vitro to test anti-proliferative activity of potato fibre supplemented bread and biscuits covers extracts (SGPLUS)
TESTING THE ANTIPROLIFERATIVE ACTIVITY OF BREAD EXTRACTS. STUDY IN VITRO.
The aim of the study was an in vitro evaluation of anticancer activity of water extracts obtained from bread supplemented with potato fiber (Potex). The experiments are conducted in human colon carcinoma cell culture model. The antiproliferative activity of all bread extracts was tested in cell line (HT-29, European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Salisbury, UK).) by performing of MTT assay. Stock solutions (10 mg/ml) of each tested extract was freshly prepared in proper culture medium, then diluted to prepare working solutions used directly in the tests. HT-29 cells were grown in 1:1 mixture of DMEM and Nutrient mixture F-12 Ham (Ham’s F-12) (Sigma). Cell media was supplemented with 10% FBS (foetal bovine serum) (Sigma), penicillin (100u/ml) (Sigma) and streptomycin (100 μg/ml) (Sigma). Cultures were kept at 37oC (HT-29) in a humid atmosphere of 95% air and 5% CO2.
MTT test: HT-29 cells were plated on 96-well microplates at a density of 3 x 104 cells/ml. Next day the culture medium was removed and cells were exposed to serial dilutions of tested extracts in a fresh medium (0, 10, 50, 100, 250, 500, 1000 mg/ml). Cell proliferation was assessed after 96 h by MTT method in which the yellow tetrazolium salt (MTT) is metabolized by viable cells to purple formazan crystals. Formazan crystals were solubilized overnight in SDS buffer (10% SDS in 0.01N HCl) and the product was quantified spectrophotometrically by measuring absorbance at 570 nm wavelengths.
The anti-proliferative activity was recalculated as IC50 value – the concentration of the extract which is causing 50% of the anti-proliferative effect.
TASK 3. In vivo studies to test effect of feed preparation based on the bread /biscuits supplemented with potato fiber on colonic cancer growth.
IN VIVO ANTI-CANCER ACTIVITY RESEARCH OF A BREAD SUPPLEMENTED WITH POTATO FIBER
The aim of this task was to study the antitumor activity of the potato-fiber enriched bread added to the food for mice. In this study, 8-12 weeks old C57BL/6 female mice were used and experiment was conducted according to animal interdisciplinary principles and guidelines for the use of animals in research, marketing and education (permissions No 71/2009, 35/2013).
Design of the in vivo studies
Experiment A. On the day 0 of the study each mouse was inoculated subcutaneously (s.c.) into the right side region with 1x106 mouse colon cancer cells suspension MC38/0 cell line. The cells were cultured at 37°C in a humid atmosphere saturated with 5% CO2 in medium RPMI 1640 with 5% FBS (HyClone), 2mM L-glutamine and 1mM pyruvate. On the day of cells implantation cells were trypsinised, washed with culture medium, centrifuged (1300 rpm, +4C, 5 minutes), counted and suspended in Hanks medium and Matrigel in relation (75 μl of cell suspension for every 25 μl of Matrigel per mouse). Injection has been done under sterile conditions.
When the tumours became tangible, length and width parameters were measured 3 times a week and the tumour volume was calculated as
(a = shorter diameter, b = longer diameter)
until completion of the study.
Mice were fed with corresponding kinds of feed every day according to their demand and sacrificed by the cervical dislocation on the 45th day of the experiment. The tumours were cut out from all mice and the weight of tumours was measured. Also, a blood samples were collected from all mice for estimation of blood parameters.
Tested feed (experiment A):
1. Control
2.Group 2 - Feed with 60% breed and 1% potex
3.Group 3 - Feed with 30% breed and 1% potex
4. Group 4 - Feed with 15% breed and 1% potex
5. Group 5 - Feed with 60% breed and 1% potex, that has undergone baking
6. Group 6 - Feed with 30% breed and 1% potex, that has undergone baking
7. Group 7 - Feed with 15% breed and 1% potex, that has undergone baking
Experiment B. On the day 0 of the study 14 mice were inoculated subcutaneously (s.c.) into the right and left side region with 3.5x106 cancer cells MC38/EGFP-B (mouse colon cancer cell line transduced with green fluorescent protein gene). On the 24th day of the experiment these mice were sacrificed and tumours were cut out. Tumours were fragmented and received pieces were implanted into the colon of 33 mice fed with experimental fodder. After the surgery mice were monitored until the 71th day of the study.
Tested feed (experiment B):
1. Control – Feed with 60% bread
2. Group 2 - Feed with 60% bread and 1% potex
3. Group 3 - Feed with 60% bread and 1% potex, that has undergone baking
Results
Task 1
Repeated extractions from different bread types gave sufficient amount of extracts for following in vitro experiments.
Task 2
Testing in vitro showed that proliferation of HT-29 cells was decreased in a concentration-dependent manner for all tested samples as measured by means of the values after MTT assay.
Recalculated IC50 value (the concentration of compound which is necessary to induce 50% of inhibition) according to the method of Litchfield and Wilcoxon, showed that the most anti-proliferative activity was observed for extracts from bread supplemented with potato fiber (no 9, 6, 16 and 5).
Task 3
Results from tumour volume measurement after inoculation showed positive tendency to decreased tumour growth in the mouse consuming feed enriched with 60% of bread supplemented with 1% of Potex, that underwent heating process (Fig 1). Moreover, in study with tumour transplantation the group that was fed with 60% of bread supplemented with potex showed significant reduction of leukocyte number in the peripheral blood, restored to values of healthy mice.
Potential Impact:
The applications for the Committee was pending on the confirmation of the clinic which will carry out the trial which, in turn, is pending on the REA decision on the amendment letter. Without that information it can't be applied for. However, once we apply for, the authorization might be obtained within the term of one month. The final results and their potential impact, use, the socio-economic impact, the wider societal implications of the project, depend of result of trial assays that at the moment is pending.
European context of the functional food ingredients exploitation is as follows: There are loads of potentially efficient molecules I the Research departments of companies, institutes and universities. To introduce those molecules in market it's necessary to fulfill the following conditions:
A) To demonstrate their effectiveness in clinical trials. In vitro or in vivo (animals) trials are not, logically, sufficient to that end.
B) To demonstrate their lack of toxicity. This can be avoid in certain cases when the consumption of those molecules by the population is a well known reality.
C) In case the molecules are foreseen to be included in a food, the incorporation in the food must maintain the efficient molecular structure which allows the functional objective. Firstly in the production itself and lately during the shelf life of the product.
D) In case the molecules are foreseen to be included in a food (bread, biscuits, yogurt, etc.) the final product must be sensorially acceptable by the consumers.
E) When the molecules are foreseen to be incorporated to a food supplement (tablets, capsules, etc.) the former condition would not be applicable.
F) The production of the ingredient and the subsequent processes must be economically feasable.
G) When the product is foreseen to be sold in the retailing market, a health claim admitted by EFSA, written on the packaging, is necessary to be get significative sales. In other words, a product without health claim is practically impossible to get it sold. When the claim of the new ingredient itself, is not approved by EFSA, companies are currently including also in their products minerals or vitamins which have a health claim already approved by EFSA.
This project can be approached as two different sub-projects
1) Developments carried out by participants VINI (bread) and SGPlus (potato fiber functional to avoid cancer): There is no clinical trials (A). There's not either any health claim approved by EFSA (G) relative to cancer.
VINI should state their ideas to commercialize their products with potato fiber.
As a reference, BISCUIT envisaged at the beginning to use potato fiber in their products but, after knowing the lack of clinical trials and claim, they considered impossible to commercialize and gave up to work with this ingredient within this project.
Developments made by VINI with INNAVES molecules: Results are totally useless. They have worked with an old ingredient which was not valid for the project and they were aware of that. They didn't fulfill the presence of target molecules after processing (C). The sensorial test they say to have done (D) is useless as the ingredient used was not valid. They were told that such work wouldn't be eligible.
2) Development carried out based on INNAVES molecules (casein hydrolysates) to be incorporated in biscuits (SME BISCUITS) and juice (ILT) and food supplement (tablets) (SME INNAVES):
As clinical trials haven't been carried out (A), the results of the project are totally useless for SMEs involved in the project. Partial results are irrelevant for companies and will end in folders on the shelves of institutes a universities. The main work of the project (human validation), included even in its title, hasn't been carried out. The objective of the program (benefit of SMEs) hasn't fulfilled at all.
In opinion of SGPLUS, the obtained results in the WP 6 are definitive big step towards acceptation of the functional bread with anticancer activities. If implanted in production of bread it will make break thru population health.
Although the main objective of the project (Development and human validation of new healthier food products) was not carried out, the main dissemination activities were: 8 scientific publications and 25 dissemination activities (conferences, workshops, web sites, press releases, articles published in the popular press, presentations, posters).
List of Websites:
The web site can be reached through the following link: http://www.fofind-project.com/
It is divided in the following sections: FOFIND project, Consortium, News, Electronic newsletters, Events, Project results, Login and Contact. In addition, the website consists of a public and a private part. In the private part, each partner can login, and all the documentation, pictures, minutes regarding the project are reflected.
The project FOFIND will develop new affordable daily consumed functional food (biscuits, breads and juices) and nutritional supplements, using innovative low cost functional ingredients derived from milk and potato fibre. These new products will be directed to the general population with specific nutritional needs that control the symptoms of hypertension, hypercholesterol, osteoporosis, anemia, and digestive problems.
The main results of FOFIND will be (1) Functional ingredients derived from milk by-products with demonstrated activity (Lowpept plus phytosterols, CPPs plus calcium salts, CPPs plus iron salts); (2) Nutritional complement tablets with functional ingredients derived from milk by-products; (3) New biscuits with functional ingredients derived from milk by-products and potato fibre; (4) New bakery products with functional ingredients derived from milk by-products and potato fibre; and (5) New juices with functional ingredients derived from milk by-products.
Human testing will be carried out to demonstrate the functionality of the results: (i) Lowpept to prevent hypertension; (ii) Lowpept plus phytosterols to prevent cholesterol and hypertension; (iii) CPPs plus calcium salts to help the absorption of calcium and (iv) CPPs plus iron salts to prevent anemia.
FOFIND project will support SMEs breaking through these barriers, by means of the development of new added-value functional food and nutritional supplements to be implemented in a short term, which will allow SMEs to increase their competitiveness and will lead them to achieve new market niches and new consumer profiles. FOFIND will develop new functional ingredients and food aimed to improve the most prevalent nutritional needs in EU, providing added value to the final products and, ultimately, increasing the return of investment of food producer SMEs. In order to ensure project objectives, FOFIND consortium is formed by four SMEs and five RTDs from four EU countries (Spain, Poland, Sweden and Germany).
Currently the European context of the functional food ingredients exploitation is as follows: There are loads of potentially efficient molecules I the Research departments of companies, institutes and universities. To introduce those molecules in market it's necessary to fulfill the following conditions:
A) To demonstrate their effectiveness in clinical trials. In vitro or in vivo (animals) trials are not, logically, sufficient to that end.p
B) To demonstrate their lack of toxicity. This can be avoid in certain cases when the consumption of those molecules by the population is a well known reality.
C) In case the molecules are foreseen to be included in a food, the incorporation in the food must maintain the efficient molecular structure which allows the functional objective. Firstly in the production itself and lately during the shelf life of the product.
D) In case the molecules are foreseen to be included in a food (bread, biscuits, yogurt, etc.) the final product must be sensorially acceptable by the consumers.
E) When the molecules are foreseen to be incorporated to a food supplement (tablets, capsules, etc.) the former condition would not be applicable.
F) The production of the ingredient and the subsequent processes must be economically feasable.
G) When the product is foreseen to be sold in the retailing market, a health claim admitted by EFSA, written on the packaging, is necessary to be get significative sales. In other words, a product without health claim is practically impossible to get it sold. When the claim of the new ingredient itself, is not approved by EFSA, companies are currently including also in their products minerals or vitamins which have a health claim already approved by EFSA.
This project can be approached as two different sub-projects
1) Developments carried out by participants VINI (bread) and SGPlus (potato fiber functional to avoid cancer): There is no clinical trials (A). There's not either any health claim approved by EFSA (G) relative to cancer. VINI should state their ideas to commercialize their products with potato fiber.
As a reference, BISCUIT envisaged at the beginning to use potato fiber in their products but, after knowing the lack of clinical trials and claim, they considered impossible to commercialize and gave up to work with this ingredient within this project.
Developments made by VINI with INNAVES molecules: Results are totally useless. They have worked with an old ingredient which was not valid for the project and they were aware of that. They didn't fulfill the presence of target molecules after processing (C). The sensorial test they say to have done (D) is useless as the ingredient used was not valid. They were told that such work wouldn't be eligible.
2) Development carried out based on INNAVES molecules (casein hydrolysates) to be incorporated in biscuits (SME BISCUITS) and food supplement (tablets) (SME INNAVES):
As clinical trials haven't been carried out (A), the results of the project are totally useless for SMEs involved in the project. Partial results are irrelevant for companies and will end in folders on the shelves of institutes an universities. The main work of the project (human validation), included even in its title, hasn't been carried out. The objective of the program (benefit of SMEs) hasn't fulfilled at all.
Project Context and Objectives:
The general aim of the FOFIND proposal is to develop new affordable functional daily consumed foods (biscuits, breads and juices) and food supplements, using innovative low cost functional ingredients derived from milk and potato fibre. This new products will be directed to population with specific nutritional needs (hypertension and hypercholesterolemia patients, population with calcium and iron needs) and will comply with the legislative requirements for their commercialization. The main aspects of the project are:
1. Development of functional ingredients derived from milk subproducts:
i. Lowpept plus phytosterols (hypertension and hypercholesterolemia, metabolic syndrome)
ii. CPPs plus calcium salts (population with calcium needs, for example osteoporosis, children, healthier, postmenopausal woman, etc.)
iii. CPPs plus iron salts (population with iron needs, for example anaemia, pregnant woman, healthier, etc.)
iv. CPPs plus selenium salts (selenium is very antioxidant and contributes to normal spermatogenesis, to the normal function of the immune system, to the maintenance of normal hair, nails, etc.)
2. To demonstrate the functionality of functional ingredients derived from milk subproducts in humans:
i. Lowpept to prevent hypertension
ii. Lowpept plus phytosterols to prevent cholesterol and hypertension
iii. CPPs plus calcium salts to help the absorption of calcium
iv. CPPs plus iron salts to prevent anaemia
v. CPPs plus selenium salt
3. Development of affordable and high-quality nutritional supplements and functional food with different food matrix (juice, biscuit and bread)
4. Finally, the FOFIND is focused on a quick launching of the project results to the market that implies providing scientific evidences for an eventual approval by the EFSA.
The companies involved in the project consist of one biotechnological company in charge of the development of the functional ingredients and, accordingly, the food supplements (INNAVES), and three companies acting as end users (Biscuits, Vini and Jacoby), in charge of introducing the functional ingredients in their products (biscuits, bread and juices, respectively) in order to obtain a product with a higher value.
In this context, the aim of INNAVES is to develop food supplements and functional ingredients derived from milk proteins with different functionalities (antihypertensive properties, and enhance of mineral absorption), and jointly with phytosterols, and finally, to test their functionality by human trials.
CSIC is the leader of the Work package 1 (WP1: Development of functional ingredients derived from milk proteins). This WP is in charge of designing novel CPPs based products from milk by-products to improve mineral (calcium, iron and selenium) bioavailability. The impact of simulated gastrointestinal digestion and the stability of the ingredients when incorporated into the final food products will be also carried out. This group is also involved in the evaluation of the possible mechanism of action of the ingredients with antihypertensive (Lowpept®) effect by analyzing different plasma biomarkers in samples from the clinical studies.
At the same time, the aim of Biscuits, in collaboration with UVigo, is to develop functional biscuits with the functional ingredients derived from milk and milk sub-products provided by INNAVES. The objective of experiments carried out was study the incorporation of Fe, Ca and Se to casein hydrolisates in order to obtain functional ingredients to be used in the production of fortified biscuits. This study involved the way of incorporation of mineral salts to casein hydrolisates (including their encapsulation) the stability of the functional ingredients obtained and, finally, the evaluation of functional biscuits with ingredients added.
Similarly, Vini’s goal in the FOFIND project is to develop low-cost bakery products enriched with milk- and potato fibre-derived functional ingredients. Main activities include: elaboration of functional ingredients enriched bread recipe of suitable taste, smell and structure characteristics; studies on shelf-life and other properties; manufacturing bread needed for scientific evaluation.
At the same time, the aim of SGPlus is an in vitro evaluation of anticancer activity of water extracts obtained from bread supplemented with potato fiber (Potex). Eighteen different samples of bread were prepared. Three kinds of bread (graham, pytlowy and rye) were supplemented with potex. The test comprised - the initial screening of all samples for antiproliferative activity in colon carcinoma cell culture and selection of the most active bread sample. Technology for obtaining most effective water extracts was established first.
Regarding Jacoby and ILT, the aim is the incorporation of functional peptides partly in combination with minerals into juice based beverages (WP5). Juice provides a health-positive association to consumers. Since the sensory properties of both, peptides and minerals (iron) are problematic suitable formulation for the beverages have to be developed and must undergo sensory panel tests. Furthermore the beverages need to be stable for a period of several months without chilling in practice. A low pH-value and usually a thermal treatment is necessary therefore. Particularly these process condition have the potential to negatively affect the functional peptides. Hence, the stability tests and the therefore required analytical methods are the main objective. In addition the binding capacities of the minerals depend on the matrix properties and is hence an objective in WP5.
The project was based on a previous product called Lowpept®, based on a patent from CSIC and licenced by INNAVES, which consists on a food grade casein hydrolysate with antihypertensive properties. From this process, a high value by-product of hydrolysate casein was obtained, which consist of casein phosphopeptides (CPPs), and these CPPs are the basis for 3 other functional ingredients.
At the time the FOFIND project started, INNAVES was already searching for the latest technology which could greatly improve the obtaining of the functional ingredients as well as the possibilities to use them in the food matrices envisaged in the project. Such technology was based on the use of ultra and nano-filtration by using ceramic membranes. Consequently, during the project some trials with a small equipment of ceramic membranes were started and therefore a pilot plant of ceramic membranes were acquired by INNAVES. With the help of CSIC, which has been analyzing all the samples of the batches that INNAVES has produced with the new technology, we have realized that the previous by-product (the CPPs) obtained is no longer a by-product as now all the hydrolyzed casein is included in the Lowpept® product.
This circumstance has not changed the interest that INNAVES has in the products to enhance the mineral absorption (Fe, Ca and Se), and some interesting alternatives have arisen. For instance, as the new Lowpept® includes the CPPs obtained with the previous technology, INNAVES is performing some trials to bind iron salts (ferrous bis-glycinate) to Lowpept®, applying the same conditions obtained by CSIC for the previous CPPs.
On the other hand, the fact is that including the small patented peptides additionally to the CPPs (which is the new version of Lowpept®) may reinforce the absorption on the minerals through other two mechanisms of action that INNAVES and CSIC have discovered.
Project Results:
The progress of the work for each work package is going to report to follow. The detailed results can find in the deliverable of each participant
-Work package 1: Development of functional ingredients derived from milk proteins:
The characterization of the by-product obtained from Lowpept manufacture has been performed and the caseinophosphopeptides released from tryptic hydrolysis and simulated gastrointestinal digestion were identified. In order to develop protein-based formulations with capacity to improve mineral bioavailability, mixtures of the products provided by Innaves and different food-grade iron, calcium and selenium salts were assayed and the mineral binding ability was estimated. Based on the results obtained, three different enriched ingredients are proposed:
(i) By-product + ferrous bis-glycinate at a protein:Fe ratio 50:1
(ii) By-product + calcium chloride at a protein:Ca ratio 7:1
(iii) Lowpept® + sodium selenite in maltodextrine at a ratio of 1000 mg protein/35 μg Se
The functionality of the different ingredients developed in this project was evaluated by using different in vitro, ex vivo and animal assays. It was demonstrated that the by-product is an excellent source of caseinophosphopeptides. The peptides generated during the in vitro gastrointestinal digestion of this by-product are similar to those found in duodenum after in vivo oral ingestion of casein. The mineral enriched products based on Lowpept® or by-product showed good iron and calcium solubility under conditions that simulate the intestinal environment. The peptidomic study of porcine duodenal digests after ingestion of Lowpept® or the precursor casein revealed that in addition to the previously identified peptides, other regions, especially from β-casein, could be important for the antihypertensive effect. The gastric resistance of the previously identified peptides RYLGY and AYFYPEL was demonstrated and it was found a significant higher concentration of RYLGY and related peptides in the duodenal digests from pigs fed with Lowpept® in comparison with those fed with casein. A noteworthy increase of several plasma biomarkers was found in those animals which had received Lowpept® and in particular, an elevated concentration of L-arginine, which is a substrate for vascular nitric oxide production. The opioid activity of YFYPEL and other related sequences was proved in an ex vivo guinea pig ileum preparation and it could be demonstrated that the antihypertensive activity of Lowpept® and some active peptides is also mediated by the interaction with opioid receptors , since the effect can be reverted by an opioid antagonist. From the study with the ingredient based on the combination of Lowpept® and phytosterols, it was found that the hydrolysate was responsible for the antihypertensive activity and the improvement of other cardiovascular parameters, while the phytosterols decreased the atherogenic index and have a beneficial effect on the lipidic profile. These novel results, which some of them could deserve further research and protection, could be used to sustain health claims on the different ingredients regarding the maintenance of the health status.
-Work package 2: Up-scaling of milk proteins functional ingredients production process, the development of tablets to include the new ingredients and the validation of their functionality in humans.
At the time the FOFIND project started, INNAVES was already searching for the latest technology which could greatly improve the obtaining of the functional ingredients as well as the possibilities to use them in the food matrices envisaged in the project. Such technology was based on the use of ultra and nano-filtration by using ceramic membranes. Consequently, during the project some trials with a small equipment of ceramic membranes were started and therefore a pilot plant of ceramic membranes were acquired by INNAVES. With the help of CSIC, which has been analyzing all the samples of the batches that INNAVES has produced with the new technology, we have realized that the previous by-product (the CPPs) obtained is no longer a by-product as now all the hydrolyzed casein is included in the Lowpept® product.
This circumstance has not changed the interest that INNAVES has in the products to enhance the mineral absorption (Fe, Ca and Se), and some interesting alternatives have arisen. For instance, as the new Lowpept® includes the CPPs obtained with the previous technology, INNAVES is performing some trials to bind iron salts (ferrous bis-glycinate) to Lowpept®, applying the same conditions obtained by CSIC for the previous CPPs.
On the other hand, the fact is that including the small patented peptides additionally to the CPPs (which is the new version of Lowpept®) may reinforce the absorption on the minerals through other two mechanisms of action that INNAVES and CSIC have discovered.
During the first period, due to the new production process developed for Lowpept®, a new approach to the functional ingredients containing CPPs was been considered. This new approach is based on two mechanisms of action discovered for the small peptides, including RYLGY and AYFYPEL, already included in the patent which will potentially improve the enhancement of mineral absorption.
For this functional ingredient, the iron salt chosen was that recommended by CSIC and also by UVigo and ILT, and this is ferrous bisglycinate. In addition, in order to achieve a good yield in the up-scaling of this ingredient, different ways of binding the iron to the new version of Lowpept® have being carried out by the wet method was has been chosen. This method has been successfully applied to iron, calcium and selenium.
For the production of Lowpept® + phytosterols, a first screening of the commercialized phytosterols was carried out in order to choose the most suitable one to be mixed with a hydrolyzed casein. From the phytosterols already available in the market, five of them were selected based on the hydro-solubility and the daily dose. The new production process of Lowpept® has allowed concentrating the dose of Lowpept®, which has facilitated the incorporation of the necessary dose of phytosterols to the daily dose of Lowpept®.
For the Lowpept®+phytosterols tablets, several batches of tablets were made with both phytosterols mixed with Lowpept®, and one of phytosterols were chosen based on the final characteristic of the tablet (friability, broken resistance and dissolution). It should be noted that different excipients were proved in order to meet the best requirements and acceptability in the market. For example, at the beginning the poly vinil pirrolidone (PVP) (chemical origin) were used as an excipient, but we have changed it for xanthan gum (vegetal origin) because it is more natural than PVP.
After the ultrafiltration of the hydrolysis of casein, there are 2 main fractions: 1) the retentate from the micro-filtration and 2) the retentate from the nano-filtration (this fraction includes RYLGY and AYFYPEL). The binding process is based in these two fractions. The interest of binding RYLGY and AYFYPEL to the iron, calcium and selenium salts is that during other research projects currently conducted by Innaves, it was concluded that both peptides have other activities different from the antihypertensive activity, which are: (i) induction of the production of nitric oxide (NO) and (ii) α-agonist of opioid receptors. These two mechanisms could help to improve the absorption of minerals.
The results obtained dirung second period were:
1. New ratio protein:Fe more adequate for food supplement has been established, 30:1 (p/p). With this ratio, 1 gram of the functional ingredient will provide 23 mg of Fe, which is an adequate amount for a daily intake and can be formulated in 2 tablets.
2. For the application of the functional ingredient to the food matrix envisaged in the project, biscuits, bread and juices, some changes in the formulation can be made in order to adapt to the process and to the final product. For instance, 3 or 4 biscuits can be an adequate daily dose which means that a ratio of 50:1 could fit better in their process.
3. Five phytosterols were chosen based on the solubility and the daily dose. The phytosterols are very difficult to dissolve in water, and for this reason, the hydro solubility was the main criteria for the selection of the phytosterols.
4. After several batches, one formulation and one of the phytosterol was successfully chosen. The final formulation of tablets containing Lowpept®+phytosterols (named LOWDOS®) has been registered in the Sante Publique, Securite de la Chaine Alimentaire et Environnement, Direction Générale Animaux, Végétaux at Alimentatio, Brussels (Belgium), through the FOODSUP tool, in order to obtain the NUT/PL/AS number (NUT-AS number granted). This number will allow us to sell this product all over Europe. Both health claims for hypertension and cholesterolemia are accepted and will be written in the box.
2. The food supplement named IronPept®, which consists in the new version of Lowpept® bounded to an iron salt, has been successfully obtained.
3. The food supplement named CalciumPept®, which consists in the new version of Lowpept® bounded to a calcium salt, has already been obtained as a powder. The tablet can be produced, however, due to the high calcium RDA, the daily dose is more than 3 tablets per day, which is not desirable. Instead, an effervescent tablet is currently under development.
4. The food supplement named SeleniumPept® in powder has been successfully obtained at the laboratory. In addition, a tablet form is also under development, even though is not envisaged in the project.
During the second period, the clinical trials will must be carry out. For this, it is necessary the authorised by the Galician Ethic Committee. The application must include the report of the trial as well as the technic sheets of the products to be tested. To that aim INNAVES manufactured 150 bottles (each one with 60 tablets) of the four products to test as follows:
1. Lowpept (2 tablets /day)
2. Lowpept plus phitoesterols (3 tablets/day)
3. CPPs with iron salts (2 tablets/day)
4. CPPs with calcium salts (4 tablets/day, however, another presentation for the consumer will be development before commercialization in order to avoid drop up of the consumers in long treatments as case with are focus on)
Besides, INNAVES manufactured the same quantity of bottles for placebo, without including our Active Ingredients. A code based on colours will allow the trial to be double bind. The batches to be used for the trials were biochemically and microbiologically analysed to check their fulfilment with the European Legislation. The technic sheets were issued.
The applications for the Committee was pending on the confirmation of the clinic which will carry out the trial which, in turn, is pending on the REA decision on the amendment letter. Without that information it can't be applied for. However, once we apply for, the authorization might be obtained within the term of one month. Regarding selenium tests will be run using biscuits instead of tablets. According with the legislation such products will be considered as enriched good instead of food supplements.
Work package 3: Development of functional biscuit
The main results obtained by UVIGO-BISCUITS during the period 1 were: optimum ratio Fe:protein was 1:5. At this ratio, a higher solubility of protein and iron was achieved, for both casein and hydrolysates. Hydrolysates from INNAVES increased the solubility of iron when they are used instead of casein. The linkage of iron to hydrolysates is not stable at high temperatures as well as in simulated gastric conditions suggesting that, previous encapsulation, could be considered to improve these results and to avoid lipid oxidation. Formulations F9 and F10 were used to obtain iron fortified biscuits resulting scarce differences with respect to control non fortified biscuits
Optimal conditions to link Ca to bydrolisates from INNAVES were: use calcium lactate at pH 7.5 for time 10 minutes. The incorporation of calcium into the biscuits resulted in harder biscuits.
The use of sodium selenate in a matrix of maltodextrin combined with H2 in a ratio 1:100 w:w, was selected as the selenium fortified biscuit of interest. Biscuits with Se provided not significant differences in sensorial test when they were compared with control biscuits
The new casein hidrolysate with low molecular size (CH300-0.1) revealed to be an ingredient with application for the encapsulation of iron, calcium and selenium. In addition, CH300-0.1 presented emulsifying capacity, similar to Tween 20 and slightly higher than lecithin and could be also used for the preparation of food emulsions and for the incorporation of phytosterols as functional ingredient. FTIR analysis reveal that the presence of cation in dissolution changes the configuration of the protein due to the linkage formed between the cation and the amide groups of protein. These changes were more obvious in presence of calcium and selenium.
In the second period, the deliverable provides
- The acceptability by consumers of biscuits containing functional ingredients (iron, calcium and selenium).
- Different formulations of minerals encapsulated or not with casein hydrolysates assayed.
- The stability of biscuits with minerals was evaluated and sensory analysis was performed.
- Organoleptic assays included objective characterization of functional biscuits (colour and textural properties) as well as subjective test including consumers trials.
According to results, the main conclusions are: The organoleptic characteristics of biscuits incorporating Fe or Ca were worse than those obtained with control series biscuits without minerals. These results were consistent in discriminant test and consumer trials. Encapsulation of Fe and Ca did not allow improve the sensorial properties of biscuits in relation with lyophilised minerals. Incorporation of selenium salt (encapsulated or not) did not modify the sensorial properties of the biscuits.
Work package 4: Development of functional bakery products
I. The aim of the reasearch
The aim of research made as a part of FOFIND for Vini was to obtain recipes and technology of production of bread containing:
1. Lowpept with ingredients defined by Innaves.
2. Potato fiber, the use of which would allow bread to have anti-cancer properties.
Introduction of such bread to the market is very important because the amount of diet related ilnesses is very high and there are deficiencies ion highly processed food.
II. The scope of research
Vini bakery scope research was established in
1. WP 1 task 3 Study of stability and compatibility of CCP – based in food matrices
2. WP 4 task To determine the technological aptitude of functional ingredients to be incorporated into the bread formulae.
Subtask 1.1 Highlight technological possibilities of the incorporation of heated and unheated potato fiber
3. WP 4 task 2 Incorporation of the ingredients into baking formulae
Subtask 2.1 Study of different formulae to incorporate the new ingredients
Subtask 2.2 Determination of baking technologies for incorporating the new ingredients
4. WP 4 task 3 Study of the stability of the functional ingredients in bread
Subtask 3.1 Kinetic stability analysis of the functional ingredient included in bread
Subtask 3.2 Production of bread and research performed by SGPlus
5 WP 4 task 4 Study of the sensory properties of functional bread
Results
1. Obtaining the recipe and technology needed for Lowpept bread production
2. Obtaing the recipe and technology needed to produce potato fiber bread with anti-cancer properties
3. Confirming carcinogenic properties of bread during in vitro and in vivo research
4. After sensory research we confirmed the high quality of bread and the acceptance of it by consumers
5. Patent application in Patent office of Sweden (application number 1451152-1, Awapatent reference number SE-21072185)
6. The signing of letters of intent by key buyers is a sign of their eagerness to introduce bread created by FOFIND research to the market.
7. On the basis of potato fiber bread created in a project of Medical University of Silesia - Public Health Department- a thesis is being written by Gabriela Potapowicz, titled " The analysis of quality of chosen properties of functional bread"- supervisor dr inż. Marek Kardas.
Work Package 5: Development of functional juices
- The minerals for the binding with CPP are characterized; a small choice is selected for further trials and shared with the partners. Two test recipies were develpoped
- Analytical methods for the identification of the functional peptides and two suitable methods for the binding capacity of iron and calcium to CPP´s are established and adapted to the methods of the partner CISC for comparison purposes.
- The binding of two selected ferrous salts and of calcium salt to the new CPP provided by the partner Innaves was examined (up to 80 % Ca binding, 50 % Fe binding). The technical processes for the binding of Ca and Fe is described.
- With the customers (SME) request five recipes with functional ingredients (Lowpept, CPP, calcium iron, selenium potassium) were developed.
- The stability of the functional peptides at different temperatures and with different technical equipment (flash pasteurisation, bottle pasteurisation, Ultra High Temperature Treatment (UHT) and autoclave) is tested. Extreme heat load lead to destructions. With moderate heat load conditions the peptides seem to stay stable. When employing same extents of (microbiologically) lethal heat loads but an different temperatures, higher temperature better to protect the peptides (in accordance to the different activation energies).
- The influence of the functional ingredients correspondent to the expectations. There is an effect on the turbidity, the ascorbic acid content, colour and sensory properties. Polyphenols seem not to be impacted.
- The long term stability in real beverages (customer tailored) in terms of the peptides and the beverage properties is acceptable.
Work package 6: In vivo and in vitro assays for anticarcinogenic activity validation
AD RP 1:
Bread is one of the most consuming products in the world. Besides the nutritional value, the bread might have different effects on the body, depending on the components that are included during processing and baking. The current project has focused on the anti-proliferative effects of bread supplemented with potato fiber (Potex) on colon cancer cells in vitro and in vivo models. To be able to perform in vitro study, 18 extracts from bread, both regular and supplemented with 1 or 2% Potex were tested. Two bread extracts one from Vini and one commercial showed highest anti-proliferative effect on colon carcinoma cells in vitro. Note! commercially available bread used as control was found to have also cytotoxic effect to normal colon cells, thus possibly being cancerogenic!!!
Ad RP 2
The best brad with anti-cancer properties develop by Vini in FOFIND project was studies in mice models with colonic carcinoma cells inoculation and tumour transplantation showed promising anticancer activity of the animal feed supplemened with potato fiber that underdone the heating process. Vini submit (2014-09-29) patent application for aforementioned discovery. Moreover, the total amount of leucocytes was decreased in the peripheral blood, indicating also an anti-inflammatory effect of potato-fiber processed in the bread. The latest is considered to be reviewed towards IPR protection during PCT submission. The MS6 was achieved we prove in vitro and in vivo the anticancer properties of potato fiber after processing in baking bread.
TASK 1. Preparation of water extracts and laboratory animal feed or water extracts from whole or bread shells and biscuits respectively (SGPlus + Vini)
PREPARATION OF WATER EXTRACTS FROM BREAD (SGPLUS)
3 different kinds of bread (18 different variation) were formulated and produced. Ready bread was chopped into pieces, dried and grinded into powder. 10-20 g of such bread powder was suspended in 200-400 ml of distilled water. The mixture was stirred by magnetic stirrer (active extraction) and left for some period without stirring (self extraction), totally for 24h at room temperature. Next the suspension was centrifuged (5000 rpm/ for 10 or 30min). Received supernatants were collected to the silicon tubes and evaporated under vacuum at water bath (80-100oC). The dried extracts were examined under microscope using round cover slips, weighed and saved for study in vitro.
In addition, solutions of 1% potex mixed water in proportions as in bread and 1% of potex mixed with water and yeast /leaven was also used for extractions.
TASK 2. Studies in vitro to test anti-proliferative activity of potato fibre supplemented bread and biscuits covers extracts (SGPLUS)
TESTING THE ANTIPROLIFERATIVE ACTIVITY OF BREAD EXTRACTS. STUDY IN VITRO.
The aim of the study was an in vitro evaluation of anticancer activity of water extracts obtained from bread supplemented with potato fiber (Potex). The experiments are conducted in human colon carcinoma cell culture model. The antiproliferative activity of all bread extracts was tested in cell line (HT-29, European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Salisbury, UK).) by performing of MTT assay. Stock solutions (10 mg/ml) of each tested extract was freshly prepared in proper culture medium, then diluted to prepare working solutions used directly in the tests. HT-29 cells were grown in 1:1 mixture of DMEM and Nutrient mixture F-12 Ham (Ham’s F-12) (Sigma). Cell media was supplemented with 10% FBS (foetal bovine serum) (Sigma), penicillin (100u/ml) (Sigma) and streptomycin (100 μg/ml) (Sigma). Cultures were kept at 37oC (HT-29) in a humid atmosphere of 95% air and 5% CO2.
MTT test: HT-29 cells were plated on 96-well microplates at a density of 3 x 104 cells/ml. Next day the culture medium was removed and cells were exposed to serial dilutions of tested extracts in a fresh medium (0, 10, 50, 100, 250, 500, 1000 mg/ml). Cell proliferation was assessed after 96 h by MTT method in which the yellow tetrazolium salt (MTT) is metabolized by viable cells to purple formazan crystals. Formazan crystals were solubilized overnight in SDS buffer (10% SDS in 0.01N HCl) and the product was quantified spectrophotometrically by measuring absorbance at 570 nm wavelengths.
The anti-proliferative activity was recalculated as IC50 value – the concentration of the extract which is causing 50% of the anti-proliferative effect.
TASK 3. In vivo studies to test effect of feed preparation based on the bread /biscuits supplemented with potato fiber on colonic cancer growth.
IN VIVO ANTI-CANCER ACTIVITY RESEARCH OF A BREAD SUPPLEMENTED WITH POTATO FIBER
The aim of this task was to study the antitumor activity of the potato-fiber enriched bread added to the food for mice. In this study, 8-12 weeks old C57BL/6 female mice were used and experiment was conducted according to animal interdisciplinary principles and guidelines for the use of animals in research, marketing and education (permissions No 71/2009, 35/2013).
Design of the in vivo studies
Experiment A. On the day 0 of the study each mouse was inoculated subcutaneously (s.c.) into the right side region with 1x106 mouse colon cancer cells suspension MC38/0 cell line. The cells were cultured at 37°C in a humid atmosphere saturated with 5% CO2 in medium RPMI 1640 with 5% FBS (HyClone), 2mM L-glutamine and 1mM pyruvate. On the day of cells implantation cells were trypsinised, washed with culture medium, centrifuged (1300 rpm, +4C, 5 minutes), counted and suspended in Hanks medium and Matrigel in relation (75 μl of cell suspension for every 25 μl of Matrigel per mouse). Injection has been done under sterile conditions.
When the tumours became tangible, length and width parameters were measured 3 times a week and the tumour volume was calculated as
(a = shorter diameter, b = longer diameter)
until completion of the study.
Mice were fed with corresponding kinds of feed every day according to their demand and sacrificed by the cervical dislocation on the 45th day of the experiment. The tumours were cut out from all mice and the weight of tumours was measured. Also, a blood samples were collected from all mice for estimation of blood parameters.
Tested feed (experiment A):
1. Control
2.Group 2 - Feed with 60% breed and 1% potex
3.Group 3 - Feed with 30% breed and 1% potex
4. Group 4 - Feed with 15% breed and 1% potex
5. Group 5 - Feed with 60% breed and 1% potex, that has undergone baking
6. Group 6 - Feed with 30% breed and 1% potex, that has undergone baking
7. Group 7 - Feed with 15% breed and 1% potex, that has undergone baking
Experiment B. On the day 0 of the study 14 mice were inoculated subcutaneously (s.c.) into the right and left side region with 3.5x106 cancer cells MC38/EGFP-B (mouse colon cancer cell line transduced with green fluorescent protein gene). On the 24th day of the experiment these mice were sacrificed and tumours were cut out. Tumours were fragmented and received pieces were implanted into the colon of 33 mice fed with experimental fodder. After the surgery mice were monitored until the 71th day of the study.
Tested feed (experiment B):
1. Control – Feed with 60% bread
2. Group 2 - Feed with 60% bread and 1% potex
3. Group 3 - Feed with 60% bread and 1% potex, that has undergone baking
Results
Task 1
Repeated extractions from different bread types gave sufficient amount of extracts for following in vitro experiments.
Task 2
Testing in vitro showed that proliferation of HT-29 cells was decreased in a concentration-dependent manner for all tested samples as measured by means of the values after MTT assay.
Recalculated IC50 value (the concentration of compound which is necessary to induce 50% of inhibition) according to the method of Litchfield and Wilcoxon, showed that the most anti-proliferative activity was observed for extracts from bread supplemented with potato fiber (no 9, 6, 16 and 5).
Task 3
Results from tumour volume measurement after inoculation showed positive tendency to decreased tumour growth in the mouse consuming feed enriched with 60% of bread supplemented with 1% of Potex, that underwent heating process (Fig 1). Moreover, in study with tumour transplantation the group that was fed with 60% of bread supplemented with potex showed significant reduction of leukocyte number in the peripheral blood, restored to values of healthy mice.
Potential Impact:
The applications for the Committee was pending on the confirmation of the clinic which will carry out the trial which, in turn, is pending on the REA decision on the amendment letter. Without that information it can't be applied for. However, once we apply for, the authorization might be obtained within the term of one month. The final results and their potential impact, use, the socio-economic impact, the wider societal implications of the project, depend of result of trial assays that at the moment is pending.
European context of the functional food ingredients exploitation is as follows: There are loads of potentially efficient molecules I the Research departments of companies, institutes and universities. To introduce those molecules in market it's necessary to fulfill the following conditions:
A) To demonstrate their effectiveness in clinical trials. In vitro or in vivo (animals) trials are not, logically, sufficient to that end.
B) To demonstrate their lack of toxicity. This can be avoid in certain cases when the consumption of those molecules by the population is a well known reality.
C) In case the molecules are foreseen to be included in a food, the incorporation in the food must maintain the efficient molecular structure which allows the functional objective. Firstly in the production itself and lately during the shelf life of the product.
D) In case the molecules are foreseen to be included in a food (bread, biscuits, yogurt, etc.) the final product must be sensorially acceptable by the consumers.
E) When the molecules are foreseen to be incorporated to a food supplement (tablets, capsules, etc.) the former condition would not be applicable.
F) The production of the ingredient and the subsequent processes must be economically feasable.
G) When the product is foreseen to be sold in the retailing market, a health claim admitted by EFSA, written on the packaging, is necessary to be get significative sales. In other words, a product without health claim is practically impossible to get it sold. When the claim of the new ingredient itself, is not approved by EFSA, companies are currently including also in their products minerals or vitamins which have a health claim already approved by EFSA.
This project can be approached as two different sub-projects
1) Developments carried out by participants VINI (bread) and SGPlus (potato fiber functional to avoid cancer): There is no clinical trials (A). There's not either any health claim approved by EFSA (G) relative to cancer.
VINI should state their ideas to commercialize their products with potato fiber.
As a reference, BISCUIT envisaged at the beginning to use potato fiber in their products but, after knowing the lack of clinical trials and claim, they considered impossible to commercialize and gave up to work with this ingredient within this project.
Developments made by VINI with INNAVES molecules: Results are totally useless. They have worked with an old ingredient which was not valid for the project and they were aware of that. They didn't fulfill the presence of target molecules after processing (C). The sensorial test they say to have done (D) is useless as the ingredient used was not valid. They were told that such work wouldn't be eligible.
2) Development carried out based on INNAVES molecules (casein hydrolysates) to be incorporated in biscuits (SME BISCUITS) and juice (ILT) and food supplement (tablets) (SME INNAVES):
As clinical trials haven't been carried out (A), the results of the project are totally useless for SMEs involved in the project. Partial results are irrelevant for companies and will end in folders on the shelves of institutes a universities. The main work of the project (human validation), included even in its title, hasn't been carried out. The objective of the program (benefit of SMEs) hasn't fulfilled at all.
In opinion of SGPLUS, the obtained results in the WP 6 are definitive big step towards acceptation of the functional bread with anticancer activities. If implanted in production of bread it will make break thru population health.
Although the main objective of the project (Development and human validation of new healthier food products) was not carried out, the main dissemination activities were: 8 scientific publications and 25 dissemination activities (conferences, workshops, web sites, press releases, articles published in the popular press, presentations, posters).
List of Websites:
The web site can be reached through the following link: http://www.fofind-project.com/
It is divided in the following sections: FOFIND project, Consortium, News, Electronic newsletters, Events, Project results, Login and Contact. In addition, the website consists of a public and a private part. In the private part, each partner can login, and all the documentation, pictures, minutes regarding the project are reflected.