Field of intervention of the project: 1) The proposal consists in forming an operational group in order to provide innovative solutions regarding the superior capitalization of the products and by-products from the agri-food sector (especially fruit) with an impact on the increase of the institutional performances of the partners. 2) Strengthening the links between agriculture, food production and forestry, on the one hand, research and innovation, on the other hand, including in order to better manage the environment and improve environmental performance. This field aims to strengthen the links between the practical needs of farmers and research activity. The project supports actions aimed at facilitating the exchange of expertise but also good practices and establishing a dialogue between farmers and the Romanian research community, more precisely by setting up an operational group set up by a Cooperative, a research and development partner, represented by the University. "Lower Danube" Galati and an SME. It also facilitates the inclusion of all partners in the knowledge exchange process. 3) Improving the competitiveness of primary producers by better integrating them into the agri-food chain through quality schemes, increasing the added value of agricultural products, promoting local markets in short supply chains, producer groups and organizations and of interprofessional organizations. The investment encourages the achievement of a higher level of competitiveness in the agri-food field, through a better processing of agricultural products, which will implicitly lead to the increase of the added value of agri-food products with a positive effect on producers and consumers.

Another activity within the project is the Development and validation of modern methods of encapsulation of bioactive compounds from the obtained extracts. As part of these activities, the formulation of new food products (with added value) was pursued, namely ice cream with sea buckthorn extract and microencapsulated powder. For the manufactured food product, tests were carried out regarding the stability evaluation of the microencapsulated bioactive compounds in the food matrix. Rosehip extract was used to obtain two microencapsulated active biopowders through the freeze-drying encapsulation technique. The two microencapsulated powders were used to formulate a food product (ice cream). In order to formulate food products with added value, rosehip extract was encapsulated in the matrix of soy protein isolate biopolymer, applying two variants of the experiments:Cousin. I. microencapsulation in a single cross-linking step (Figure 1, var. I) by the action of transglutaminase  at 50 degrees C, for 8 h (non-heat-treated powder).Cousin. II. microencapsulation by double cross-linking (Figure 1, var. II) made by heat treatment at 85 degrees C/20 min and by the action of transglutaminase at 50 degrees C, for 8 h (heat treated powder). the microencapsulated powder obtained without heat treatment before the cross-linking step by TG (powder obtained after lime I) has a higher encapsulation efficiency of 51.55%. The concentrations of bioactives are higher for the same experimental variant, respectively the powder obtained after the heat treatment of the IPS solution before the action of TG. This heat treatment allowed increasing the bioavailability of the proteins in the biopolymer matrix to become a substrate for the action of the TG enzyme.

Another bioactive powder obtained consisted of the use of sea buckthorn borhot extract (extraction was presented at PA11) in proportions of 2% and 8%. Thus, the extract was encapsulated by spray drying using MD+GA as biopolymers, obtaining powder 1 (with 2% extract) and powder 2 (with 8% extract). MD is a good microencapsulating agent due to its reduced viscosity at a content high dry matter, good solubility and oxidation stability. GA allows obtaining stable and low-viscosity emulsions. GA in combination with MD is used for encapsulation due to its emulsion stability with most oils, ability to form a film/film on the surface of microcapsules and oxidation protection, GA being recommended for encapsulation of oleoresins. A correlation was demonstrated between the amount of sea buckthorn extract added and the concentration of carotenoids determined in the encapsulated powders. Thus, in powder 2, carotenoid concentrations are much higher compared to powder 1, but the encapsulation efficiency was higher in powder 1 due to the inclusion of the extract in the emulsion (63.45%). The result is justified by the ability of GA to form very stable emulsions, and by combining with MD the bioactive compounds are retained in the matrix of the encapsulation material, avoiding the effect of oxidative factors. The different morphology of the particles is given by the type of biopolymer. Thus, after the spraying process, GA biopolymer presents deformed particles with multiple concavities on their surface due to the rapid evaporation of water from the emulsion. In powder 1 (MA:GA with 2% sea buckthorn oil), smaller spherical particles (5 – 8.56 micrometers) predominate.

At activity 8.2. Participation in fairs / exhibitions / conferences, mixed teams from the operational group participated in the conferences: 1. Nutricon 2022 Conference – Food Quality & Safety. Health & Nutrition, June 8-10, 2022, Orhid, Macedonia, with the paper The potential of the plant by-product obtained at industrial scale and their valorization by supercritical carbon dioxide extraction, authors: Liliana Mihalcea, Adelina Ștefania Milea, Gigi Coman, Nicoleta Stănciuc, Iulia Cucolea, Mădălin George Dănilă.
2. Interdisciplinarity and Cooperation in Cross Border Research INTERNATIONAL SUMMER CONFERENCE, June 2-4, 2022, Chisinau, Republic of Moldova with the paper Supercritical Carbon Dioxide Extraction As Great Opportunity for the Romanian Entrepreneurs, authors: Liliana Mihalcea, Iulia Cucolea, Mădălin George Dănilă, Gigi Coman, Bogdan Păcularu, Adelina Ștefania Milea, Nicoleta Stănciuc. At activity 8.5. Dissemination of the project results through the publication of articles, mixed teams from the operational group published the scientific articles:
 During the reporting period 01.06.2022 – 31.10.2023, the team from the Lower Danube University in Galati developed the research-development-innovation (CDI) activity for submitting an invention patent application. Thus, the invention was submitted with the title Gluten-free biscuits with the addition of fruit shavings, and the method of obtaining them, according to the CBI registered at UDJG with no. 12080/24.04.2023 and at OSIM with no. A/00199/25.04.2023.

Currently, fruit products obtained by farmers are either sold as such, fresh or dried / dehydrated, or in the form of preparations. Regarding by-products, their main use is as feed or for obtaining alcohol by fermentation and distillation. In order to improve and increase farmers' incomes, this project is proposed so that the products and by-products resulting from the fruit sector are capitalized in value-added products. The innovative processes will be based on the extraction technology with supercritical liquids having as objective the capitalization of agri-food products and by-products from the fruit sector, in the form of food supplements.
The evolution trends on the operating horizon of the objective show a special interest on plant extracts among the population but also of food supplement producers because, this aspect is supported by the estimated growth of this market after improving the quality of plant products and due to the standardization of production. of extracts. In terms of economic competitiveness it is essential to promote advanced methods of processing all food waste because only in this way they can bring added value and can be marketed later.

Extraction with supercritical fluids, in particular with supercritical CO2 is an emerging processing technology being a viable alternative to conventional extraction with organic solvents. Supercritical CO2 offers multiple advantages at the industrial level regarding the intensification and simplification of the extraction processes, a clear consequence being the replacement of the classic solvents, the reduction of time and costs with utilities. In addition to maintaining the natural character of plant products by eliminating the presence of solvent residues in extracts, the proposed technology also offers the possibility of using extraction parameters that allow adjusting the technique according to the raw material used and bioactive compounds followed by it.
Consumer requirements for minimally processed products with natural sensory attributes and high nutritional properties involve the physico-chemical analysis of products to determine those compounds responsible for these attributes. Physico-chemical analysis of extracts involves the determination of antioxidant activity, concentrations of total phenolic compounds, total anthocyanins and flavonoids by spectro-photometric and chromatographic methods. Stability will be studied by evaluating the kinetics of thermal degradation of target bioactive compounds, applying fluorescence spectroscopy, chromatography and mass spectrometry techniques.

For over 20 years, the fruit sector has been in constant decline, marked by obvious structural problems of the sector and the low degree of adaptation to market requirements. Production technologies, equipment are uncompetitive and outdated, which leads to low productivity compared to the European average. Also, the investment capacity in reconversion, conditioning, storage, processing, packaging, transport is reduced.
Thus, starting from these aspects of the fruit sector, the results of the BIOPOWDER project contribute to the fulfillment of the set of measures proposed by the Fruit Sub-program in the following way:
1) Innovation is promoted through the establishment of an Operational Group, and the cooperation of actors in the sector is facilitated in order to market products in short chains.
2) The project is connected to the realities of the sector and aims to come with a contribution of know-how obtained from the implementation of the project, know-how that will reach farmers with beneficial effects on fruit growing.
3) Within the project will be applied and optimized a top technology that will lead to the development of 2 other innovative technologies.

In recent years, emphasis has been placed on the revaluation of food supplements through a good knowledge of physicochemical properties through the development of extraction techniques and quality control means. The powder product is an ingredient for phytobotanical supplements and is intended for oral consumption (administration) by healthy people who require a higher exogenous intake due to specific nutritional requirements related to: physiological condition, such as pregnancy, lactation; age period, for example young children, adolescents, the elderly; intense physical activities - the case of performance athletes, professions based on physical effort, etc. Taking into account these aspects, the potential customers for the marketing of the powder are the manufacturers and distributors of food supplements.

Obtaining bioactive powders required the use of biopolymers in which extracts obtained from sea buckthorn were encapsulated. They were used as biopolymers: whey protein isolate : gum arabic = 7 : 3 (bioactive powder 1), soy protein isolate : gum arabic = 7 : 3 (bioactive powder 2). The powders were obtained by the spray drying method. Powder 1 turned out to have a higher percentage of carotenoids and a lower encapsulation efficiency (approx. 50%). The encapsulation efficiency is lower compared to lyophilization encapsulation methods due to the protein denaturation process, a situation that determines the reduction of the degree of retention of the lipophilic components and thus of the encapsulation efficiency. In powder 2, the encapsulation efficiency was higher (60.8%), thus a higher inclusion of carotenoids. The Faculty of Food Science and Engineering, in collaboration with SC Cromatec Plus SRL Bucharest and Romanațiul Cooperativa Agricola Urzica Dolj, together with the partners Innovative Cluster "For the Health of the Lower Danube" from Galați, SC IPA SA Bucharest, Enterprise Europe Network (EEN) and the S.M.U.R.D. Association. Galați organized on May 11, 2022 the workshop entitled Innovation & Entrepreneurial Education, a chance for the development of the agri-food sector in Romania. The Faculty of Food Science and Engineering, in collaboration with SC Cromatec Plus SRL Bucharest and Romanațiul Cooperativa Agricola Urzica Dolj, together with the partners Innovative Cluster "For the Health of the Lower Danube" from Galați, SC IPA SA Bucharest, Enterprise Europe Network (EEN) and the S.M.U.R.D. Association. Galați organized on May 11, 2022 the workshop entitled Innovation & Entrepreneurial Education

Partner 2, the Lower Danube University in Galati through the research group from the Faculty of Food Science and Engineering carried out the transfer of knowledge to the working group from the project leader, the information referring to: the constructive-functional description of an extraction installation with supercritical carbon dioxide, to the influencing parameters (extraction pressure, extraction temperature, carbon dioxide flow rate, extraction time, parameters set for separation/fractionation of extracted components, type of cosolvent used, particle sizes and initial moisture of the material solid subject to extraction). Representatives of the working group from the leader visited the location of partner 2 and carried out test experiments to be able to exploit and coordinate the extraction process on the installations that are purchased by the project leader. The 1st partner of the project (the farmer) participated in the preparation of the raw material for extraction, stages which included: sorting, washing, drying, shredding. The environmental factors that influence the humidity of plant materials, the working methods regarding the determination of humidity and the degree of shredding were discussed, so that the farmer will be able to use the information in the harvest and post-harvest stages. Extraction batches were made for sea buckthorn, including extract collection from the two separators. For the extraction process, the gravimetric yield was determined, which will allow farmers to analyze the productivity and quality of fruit products.

Rosehip fruits were characterized physico-chemically, dried, ground, then subjected to extraction with supercritical carbon dioxide under the conditions: for extraction / pressure of 350 bar and temperature 45 degrees C, for fractionation 1 / pressure 100 bar and temperature 45 degrees C; at fraction 2, pressure 50 bar and temperature 20 degreesC. The extraction time was set at 120 min from the specialized literature. The supercritical carbon dioxide mass flow rate was 0.42 kg carbon dioxide/min. Since the extraction yield increases exponentially with decreasing particle sizes of the solid material, advanced grinding of whole, lyophilized rosehip fruits has been achieved. This is explained by the fact that small sizes increase the contact surface of the solid material particles with the extraction solvent. Supercritical carbon dioxide-assisted extraction was performed in the presence of cosolvent, namely 5% ethyl alcohol with a concentration of 96%. The addition of cosolvent has the role of increasing the probability of extraction of non-polar compounds because supercritical carbon dioxide is polar. Fraction 2 has a rich profile of bioactive compounds consisting of: caffeic acid, p-coumaric acid, chlorogenic acid with concentration values ​​between 8.55-15.75 mg/100 g s.u. fruit, kaempferol (16.79±0.15 mg/100 g s.u. fruit) and quercetin (14.57±0.48 mg/100 g s.u. fruit).Gallic acid was identified only in fraction 2, and (+)-catechin was the major compound in both analyzed fractions (1 and 2), with a higher concentration in fraction

Supercritical carbon dioxide extraction of whole rosehip fruits after drying and grinding allowed an extraction yield of 11.85%. The extraction parameters were specified at PA13, and the extraction was carried out in the presence of cosolvent, namely 5% ethyl alcohol with a concentration of 96%. In order to transfer the results regarding the stability of the bioactive compounds from the extracts, it was decided to follow this characteristic for 90 days. Thus, the extracts were kept at a temperature of 4 degrees C, the stability being analyzed for hydrophilic compounds (flavonoids, polyphenols) and lipophilic compounds. The rosehip extract showed concentrations of total polyphenols (CPT) with values ​​between 2.28 ± 0.05 and 2.89 ± 0.26 mg gallic aci/g extract. During storage at 40C, a 25% reduction in flavonoid concentrations was observed for the mixture extract (both fractions). The study of carotenoids in lipophilic extracts is justified due to their association with antioxidant activity in both situations (in vitro and in vivo). Supercritical fluid extraction resulted in obtaining fractions with different concentrations of carotenoid compounds. Thus, the highest values ​​of the concentrations of lipophilic compounds are found in the fraction obtained at 100 bar and 45 degreesC (fraction 1), while in the fraction obtained at 50 bar (fraction 2), the concentrations were lower. From the point of view of maintaining the concentrations of bioactive compounds, an acceptable stability of the extracts stored at a temperature of 4 degrees C is observed, the decrease in concentrations of lipophilic compounds being in the range of 3-15%.

The bioactive powders obtained by encapsulating rosehip extract were used to formulate a food product. Due to the preponderance of lipophilic compounds in rosehip extract, it was decided to obtain ice cream using as main ingredients: whipping cream (300 g), whole milk powder (98 g), lactose-free milk (600 g), microencapsulated powder (2%). 4 ice cream samples were obtained, as follows: control ice cream (ÎM), ice cream with non-encapsulated rosehip extract (ÎE), ice cream with powder obtained after var.I (Î V.I.) and ice cream with powder obtained after var.II (Î V .II.). All ice cream assortments were used to fill wafers obtained from a gluten-free dough with the addition of shavings obtained from supercritical carbon dioxide extraction. For all powders, the concentrations of bioactive compounds were determined and stability was monitored for 43 days when stored at -18 degrees C. The concentrations of lipophilic compounds, namely carotenoids and lycopene, follow the same trend, namely a slight decrease after 43 days of storage. The total carotenoid concentration and the lycopene concentration show an increase only in the case of the ÎE sample, the one with a non-encapsulated oil extract content. The concentration difference between the samples is given by their composition. As expected, samples enriched with extract or powder show much higher values, reaching values ​​of approximately 2.5 mg carotenes/g and 1.5 mg/g, respectively. The concentration of lycopene varies approximately 100 µg/g, in the case of the control sample up to values ​​of approximately 400 µg/g, in the case of samples with added powders (Î V.I and Î V.II) and over 800 µg/g in the case of the sample with addition of non-encapsulated extract (EE). 

The extract obtained by supercritical carbon dioxide extraction from sea buckthorn showed a complex profile of carotenoids. The main carotenoids identified were zeaxanthin, lycopene and beta-carotene. The results indicated that during the successive extraction, the amount of cosolvent (ethyl alcohol-water mixture) induced a decrease in the concentration of lycopene causing a reduction in the extraction efficiency of lipophilic compounds. Thus, the research group recommends the addition of a cosolvent consisting only of ethyl alcohol or vegetable oils (sunflower oil, canola oil). The research group used blueberries to obtain a bioactive powder. In this sense, aqueous extracts rich in anthocyanins were obtained and inoculated with a culture of lactic bacteria, Lactobacillus casei, in a percentage of 2%. . The obtained bioactive powder was obtained with maltodextrin and pectin, resulting in an encapsulation efficiency of 81.33%. This high percentage is explained by the increased stability and bioavailability of anthocyanins given by the character of the polysaccharide used. The results were disseminated through a paper presented at the Inovaliment 2021 Scientific Conference, held between November 22 and 26, 2021, in online format, organized by the Scientific Senate of the Dan Voiculescu Foundation for the Development of Romania, in partnership with the Forum of Romanian Inventors.

The objective of the project is to offer innovative technologies (superfluid extraction and microencapsulation) regarding the capitalization of agri-food products and by-products. Following the implementation, the members of the cooperative will be provided with the technologies, the implementation method and all the know-how necessary for their implementation. The main results are:
1) Identification of problems at the level of partner 1 (farmer) regarding the capitalization of by-products from fruit processing
2) Development and optimization at pilot level of the supercritical CO2 extraction technique for the capitalization of by-products from the fruit sector
3) Physical and phyto-chemical investigation of the extracts obtained at pilot level in order to quantify qualitatively and quantitatively the biologically active compounds that will be the main source in obtaining functional composites by micro and nanoencapsulation
4) Development of cost-effective, reproducible encapsulation methods for biologically active compounds.
5) Evaluation of the stability and bioavailability of bioactive compounds in the extracts and powders with added value obtained and validation of encapsulation technologies.
6) Development of at least two innovative technologies for obtaining value-added food products and their patenting.
7) Technology transfer in the operational group.

The project contributes to the IEP objective of increasing productivity and sustainable management of resources by increasing productivity in agriculture
1) Increasing productivity in fruit farms - GO farmers, but not only, will have the opportunity to learn the technologies resulting from the project and methods of processing products and waste in the fruit sector, leading to increased value added products in this sector and having a positive impact on farmers' own activity.
2) The increase of farmers' productivity is closely related to the increase of their competitiveness in the market, the project stimulating the creation of processing units at the level of farms (through the technological transfer and know-how resulting from the implementation of the project).
3) The development of the processing sector will ensure both the maintenance of jobs and the creation of new ones.
4) The farmers' access to the research results will help them in diversifying the agricultural activities and capitalizing on the resources they have in the fruit sector: fruits and vegetable waste. All this will support and transform rural communities into attractive places to live.
5) The diversification of farmers' activities will lead to new opportunities to increase their incomes.

The stability studies of the obtained bioactive powders with rosehip extract were carried out by keeping them for 30 days at room temperature, then reanalyzed in order to evaluate the phytochemical and antioxidant profile. In the case of carotenoids and lycopene, their concentrations remained approximately 60% for both sample N and sample T. However, the antioxidant activity (micromoles Trolox equivalent/g extract) in both samples had a stability of over 90%, decreasing moderately from values ​​of 52.45±0.47, respectively 45.39±0.86, to values ​​of 47.26±3.86, respectively 44.09±2.03 , thus signaling the fact that other compounds besides those determined spectrophotometrically may be responsible for the antioxidant activity. Regarding the efficiency of carotenoid encapsulation, a better rate of preservation and protection of the compounds could be noted in the N variant, generating a percentage of 51.17±1.62%, in contrast to the 43.25±2.97% value obtained for the T variant. The heat treatment at 85°C for 20 minutes, applied before the enzymatic cross-linking on the T sample, may have generated a denaturation of the soy protein, which could explain the lower encapsulation efficiency values. In the case of hydrophilic compounds (flavonoids), an accelerated decrease in concentration can be observed after one month of storage. Flavonoids are sensitive polyphenolic compounds, their stability largely depending on encapsulation efficiency. On the other hand, the polyphenols kept a percentage of approximately 92% of the concentration in the case of the powder obtained through a single cross-link step, respectively 84% for the case heat-treated prior to the TG action (so double cross-linked process).

The supercritical carbon dioxide extraction of sea buckthorn was carried out at the pilot plant in the Faculty of Food Science and Engineering. In the studies of the research group from partner 2, the optimization of the extraction assisted with supercritical carbon dioxide indicated that the two independent variables temperature (T) and pressure (p) have a determining influence on the extraction roughness. Thus, the parameters were established: the extraction pressure of 30 MPa and the extraction temperature of 40 degreeC. The extraction time was considered to be 82 min according to the specialized literature. The parameters set during the separation were: for the S40 separator, pressure 15 MPa, temperature 40 degree C, and for the S45 separator, pressure 5 MPa and temperature 22 degree C. In order to make better use of the borhot, the extraction was carried out in two stages for the meal obtained with the addition of co-solvent (ethyl alcohol-water mixture), demonstrating the improvement of the extraction of carotenoids. Finally, the gravimetric yield of supercritical carbon dioxide extraction was approx. 10%. In addition, an increase in total carotenoid concentration by 11.63% was obtained due to the addition of 10% cosolvent ((ethyl alcohol-water mixture).
The highest concentration increase was obtained for the beta-carotene compound, respectively by 12.37% (the experiment with the addition of cosolvent compared to the experiment without cosolvent). The results demonstrate and validate the efficiency of supercritical carbon dioxide extraction for obtaining carotenoid-rich oils with applications in the food, cosmetic, nutraceutical or medicine industries.

The technologies proposed to be developed through the project:
Extraction with supercritical fluids, in particular with supercritical CO2 is an emerging processing technology being a viable alternative to conventional extraction with organic solvents. Supercritical CO2 offers multiple advantages at the industrial level regarding the intensification and simplification of the extraction processes, a clear consequence being the replacement of the classic solvents, the reduction of time and costs with utilities. In addition to maintaining the natural character of plant products by eliminating the presence of solvent residues in extracts, the proposed technology also offers the possibility of using extraction parameters that allow adjusting the technique according to the raw material used and bioactive compounds followed by it.
. Physico-chemical analysis of extracts involves the determination of antioxidant activity, concentrations of total phenolic compounds, total anthocyanins and flavonoids by spectro-photometric and chromatographic methods. Stability during extraction will be studied by evaluating the thermal degradation kinetics of the target bioactive compounds, applying fluorescence spectroscopy, chromatography and mass spectrometry techniques. 

There are 2 categories of consumers who buy food supplements. In the first category there are consumers of all ages and from all social and professional categories, who inherit sensitivities and biological data that are reflected in difficulties of growth and development, then in difficulties of the profession, and later in the avalanche of inconveniences, dissatisfaction and suffering caused by aging. Also in this first category must be placed those who practice sports and carry out competitive actions, very demanding and pushed to forms of high risk and stress, impossible to sustain without a consistent intake of food supplements. Consumers in this category are advised by specialists (doctors, nutritionists, coaches) who follow the evolution of the person advised and make the necessary corrections in the administration of supplements.
The second category is composed of consumers with very obvious imitation behavior, sensitive to fashion, egocentric, eccentric, etc. They ensure the strong upward trend in the demand for nutritional supplements, especially in developed countries. In most cases, consumers in this category do not turn to specialists, administering their supplements according to their own beliefs and recipes. It is estimated that in developed countries, about a third of the population is in this situation.