Fig tree bioresidues as natural preservatives in fig-based products

Mestrado de dupla diplomação com a Université Libre de TunisThe food processing and storage became imperative to provide the quotidian needs of humans. Therefore, the use of artificial additives started to be mandatory to decrease or postpone the nutritional value losses due to chemical, microbiological and enzymatic changes, thus maintaining the characteristics of the processed food for a longer period. Nevertheless, the use of synthetic additives has been highly studied due to several scientific alerts about their harmful effects that can include allergic problems, asthma, among others. This situation leads to a deep search for natural alternatives able to serve the same purpose. To do so, food industries started this mission through the extraction of natural compounds from microorganisms, animals, plants and agri-food bio-waste. Indeed, among the food industry, several sectors produce huge amounts of bio residues. Figs, for instance, are known for their richness in minerals and nutrients, which leads to their high consumption, thus generating several fig bio-wastes, which include fig leaves that could be useful for the recovery of bioactive compounds, such phenolic compounds, to act as natural preservatives, hence, valorizing fig-waste and promoting circular economy. Therefore, in the present work, fig leaves from five different varieties, namely Dauphine (Da), Longue d’Aout (La), Bourjassote Noire (Bn), Marseille (Ma) and Pasteliere (Pa) were screened to determine their phenolic content by HPLC-DAD-ESI-MS as well as their bioactivities, namely antioxidant through the TBARS and CAA assays, antimicrobial by the microdilution method, anti-inflammatory using RAW 264.7 mouse macrophage cell line and cytotoxic through the sulforhodamine B assay. The extraction of total phenols was optimized through response surface methodology and carried out by dynamic maceration and ultrasound assisted extraction, being their determination assessed by the Folin ciocalteu assay. Finally, the extracts obtained from the optimal conditions of ultrasound assisted extraction, used as a cost effective method, were mixed together searching for possible synergistic effects. When the leaves acted together, the antioxidant activity was higher, since the lowest EC50 recorded for Bn was 0.23±0.01 mg/mL, and for the mixture an EC50 of 0.12 ±0.01 mg/mL was achieved. Moreover, the mixture also revealed promising results regarding the antimicrobial activity by acting against all the tested bacteria and fungi strains. It was important to showcase that the mixture revealed activity against Pseudomonas aeruginosa with a MIC of 10 mg/mL. In addition, as the extracts showed no toxicity against normal cell line PLP2 with a GI50>400 μg/mL, a concentration of 10mg/mL was incorporated, after the cooking process, at 80°C and 50°C in two formulations of fig jams (extracts with honey and extracts without honey) to determine the efficiency of the developed extracts, acting as natural preservatives, and their stability in the final products. The fig jams were subjected to the evaluation of physical parameters (color, texture, aw and pH), nutritional (moisture, ash, fat using soxhlet, protein by the kjeldahl method, carbohydrates and energy) and chemical profiles as the free sugars by HPLC-RI and fatty acids through the GC-FID, and microbial load following ISO procedures. The low-sugar fig jam incorporated with natural fig leaf preservatives revealed that the incorporation did not change the overall appearance of the jams. Regarding the nutritional and chemical properties, the formulas presented low sugar, low protein content and high amount of carbohydrates, low fatty acids content with palmitic acid as the major compound. Furthermore, the different temperatures of incorporation showed no discernible changes over time, implying that the molecules of interest present in the extracts are not thermolabile.O processamento e armazenamento de alimentos tornou-se imperativo para suprir as necessidades quotidianas do ser humano. Assim, o uso de aditivos artificiais passou a ser obrigatório para diminuir as perdas de valor nutricional por alterações químicas, microbiológicas e enzimáticas, mantendo assim as características do alimento processado por mais tempo. No entanto, o uso de aditivos artificiais tem sido bastante avaliado devido a vários estudos científicos que alertaram sobre os seus efeitos nocivos, que podem incluir problemas alergénicos, asma, entre outros. Esta situação leva à busca exaustiva por alternativas naturais capazes de servir o mesmo propósito. Para tal, a indústria alimentar iniciou essa missão através da extração de compostos naturais de microrganismos, animais, plantas ou de bio resíduos/desperdício alimentar. De facto, na indústria alimentar, diversos setores produzem grandes quantidades de resíduos, como por exemplo na produção de figos, que podem ser úteis para a recuperação de compostos bioativos para atuar como conservantes naturais. Os figos, por exemplo, são conhecidos pela sua riqueza em minerais e nutrientes e, por isso, o seu consumo aumentou significativamente, levando também à inevitável produção de bioresíduos que incluem folhas de figueira. Assim, no presente trabalho, foram exploradas folhas de figueira de cinco variedades diferentes, nomeadamente Dauphine, Longue d'Aout, Bourjassote Noire, Marseille e Pasteliere. Estas variedades foram analisadas relativamente ao seu perfil fenólico por HPLC-DAD-ESI- MS, bem como as suas bioatividades, nomeadamente propriedades antioxidantes pelos métodos de TBARs e CAA, antimicrobianas pelo ensaio de microdiluição, anti-inflamatórias utilizando macrógafos de rato e citotóxicas pelo método da sulforrodamina B. A extração dos compostos fenólicos foi otimizada pela metodologia de superfície de resposta e realizada por maceração dinâmica e extração assistida por ultrassons, sendo a sua determinação feita pelo ensaio de Folin ciocalteu. Finalmente, os extratos obtidos nas condições ótimas de extração pela tecnologia de ultrassons, utilizada por ser mais rápida, com um custo mais baixo, foram misturados de forma a analisar possíveis efeitos sinérgicos. Quando avaliada a atividade antioxidante dos extratos de folhas em conjunto, esta foi mais forte, já que a menor EC50 registrada para Bn (0,23 ± 0,01 mg/mL), e para a mistura foi EC50 de 0,12 ± 0,01 mg/mL. Além disso, a mistura apresentou resultados promissores quanto à atividade antimicrobiana, pois foram capazes de inibir todas as estirpes de bactérias e fungos testados. De realçar, que, quando em conjunto, os extratos revelaram atividade contra Pseudomonas aeruginosa com valor de CMI de 10 mg/mL. Como os extratos não apresentaram toxicidade na linha celular PLP2 com GI50>400 μg/mL, estes foram incorporados em duas formulações de compota de figo (com e sem mel), de forma a atuarem como conservantes naturais. Os produtos finais foram avaliados quanto às suas propriedades físicas (cor, textura, aw e pH), perfil nutricionais (humidade, cinzas, gordura por soxhlet, proteína pelo método kjeldahl, hidratos de carbono e energia), e químico, como os açúcares livres por HPLC-RI e ácidos gordos por GC-FID, e aindacarga microbiana. Após o processo de cocção, as diferentes propriedades foram avaliadas em duas temperaturas de incorporação (80°C e 50°C), de forma a determinar a eficiência dos extratos desenvolvidos e a sua estabilidade nos produtos finais. A compota de figo com baixo teor de açúcar incorporada com extratos conservantes naturais obtidos a partir de folhas de figueira, não revelou alterações na aparência geral das compotas. Em relação às propriedades nutricionais e químicas, as formulações apresentaram baixo teor de açúcar, baixo teor de proteína, alta quantidade de hidratos de carbono, e baixo teor de ácidos gordos (ácido palmítico como composto majoritário). Além disso, as diferentes temperaturas de incorporação não apresentaram alterações discerníveis ao longo do tempo, implicando que as moléculas de interesse presentes nos extratos não são termolábeis.European Regional Development Fund (ERDF) through the Competitiveness and Internationalization Operational Program for financial support to the project 100% Figo (POCI-01-0247- FEDER-064977)

Non-alkaloid nitrogen-containing compounds from fungi

Fungi represent an ancient ubiquitous and interesting kingdom that can be classified into Phycomycota, Oomycota, Microsporidiomycota, Zygomycota, Ascomycota, Basidiomycota, and Deuteromycota divisions. These microorganisms are considered as a reservoir of bioactive compounds that can be exploited in crucial fields: food, pharmaceutical, and agricultural industries. Non-alkaloid nitrogenated compounds are considered a prolific bouquet composed of proteins such as enzymes playing an essential role in the synthesis of compounds that are involved in vital life processes, lytic enzymes possessing depleting effects, antibiotics, toxins applied as antimicrobials and antifungals, immunosuppressive molecules, etc. Moreover, a number of species found in phyla like Oomycota and Microsporidiomycota have been and are still used as biological control agents for crops as a counterpart to chemical products.The research leading to these results was supported by MICINN supporting the Ramón y Cajal grant for M.A. Prieto (RYC-2017-22891), the post-doctoral grant of M. Fraga- Corral (ED481B-2019/096), and L. Cassani (ED481B-2021/152), and the pre-doctoral grant of A.G. Pereira (ED481A-2019/0228). Authors are grateful to Ibero-American Program on Science and Technology (CYTED-AQUA-CIBUS, P317RT0003), to the Bio Based Industries Joint Undertaking (JU) under grant agreement No 888003 UP4HEALTH Project (H2020-BBI-JTI-2019). The JU receives support from the European Union’s Horizon 2020 research and innovation program and the Bio Based Industries Consortium. The project SYSTEMIC Knowledge hub on Nutrition and Food Security, has received funding from national research funding parties in Belgium (FWO), France (INRA), Germany (BLE), Italy (MIPAAF), Latvia (IZM), Norway (RCN), Portugal (FCT), and Spain (AEI) in a joint action of JPI HDHL, JPI-OCEANS and FACCE-JPI launched in 2019 under the ERA-NET ERA-HDHL (n° 696295). The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to the CIMO (UIDB/00690/2020). I. Oliveira thanks FCT for her PhD grant (BD/06017/2020). M. Carocho thanks FCT for his individual scientific contract (CEEC-IND/00831/2018).info:eu-repo/semantics/publishedVersio

FIG (Ficus Carica L.) Bioresidues as sources of bioactive compounds and natural pigments for the food industry

The 17 goals of sustainable development address several topics, such as: (2) Zero hunger and sustainable agriculture; (9) Industry, Innovation, and Infrastructure; (12) Responsible consumption and production; that are essential for the promotion of the circular economy, product development and conscious production [1]. Fig is a food matrix, cultivated in Portugal and valued by the Portuguese people for consumption in natura, being also used in wines, liqueurs, and jams. As this fruit is very appreciated and consumed, it´s cultivation leads to the production of tons of leaves, usually discarded [2]. Therefore, in the present work, the leaves of five fig varieties (Figure 1), namely Dauphine (Da), Longue d'Aout (La), Pasteliere (Pa), Marseille (Ma) and Bourjassote Noire (Bn), were nutritionally and chemically characterized to detect possible bioactive molecules. The antioxidant and antimicrobial, activities were also analyzed, to provide the food industry with natural additives in alternative to the artificial ones; and at the same time, promote the circular economy. Regarding the nutritional profile of the five leaves, La sample exhibited the highest amount in proteins (18.0±0.6 g/100g dw), while Pa revealed the highest content in fats (2.2±0.1 g/100g dw). The highest moisture content was presented by Da leaves (17.3±0.1 g/100g fw), and for the ashes, La sample was the one that presented the highest value (14.18±0.06 g/100g fw). Concerning the organic acids, these molecules were most abundant in Ma leaves, where it was possible to identify oxalic, malic and citric acids with a total of 139.6±0.4 mg/g dw. For the soluble sugars profiling, in all samples it was possible to identify five sugars, namely, fructose, glucose, sucrose, trehalose, and raffinose in different concentrations; however, Da leaves revealed the higher amount (17±1 g/100g dw). Tocopherols where also analyzed, and in all samples, three of the four isoforms where detected, being Pa sample standing for the predominance of these compounds (4.14±0.05 mg/100 g dw). For the bioactive analysis, different assays were performed, and Pa sample showed the strongest antioxidant potential for the TBARS assay, with an EC50 value of 105±5 mg/mL. For the antimicrobial activity assay, Da leaf extract was the one displaying the best results, by presenting Minimum Inhibitory Concentrations (MIC) ranging from 1.25 to 10 mg/mL against the tested bacterial strains. On the other hand, for the antifungal activity, the samples present very similar profiles, with the exception of the Pa sample, that present the lowest MIC of 5 mg/mL for Aspergillus fumigatus. In general, these leaf extracts can be used in the food industry namely in pastry and bakery products as promising sources of bioactive compounds, and at the same time, this reuse of biowaste promotes circular economy, and reduces the impact of biowaste resulting from the fig industry, thus meeting some of the goals of sustainable development.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to the CIMO (UIDB/00690/2020). S.Heleno and M. Carocho thank FCT for their individual employment program–contract (CEEC-IND/00831/2018, CEECIND/03040/2017), and L. Barros also thanks to the national funding by FCT through the institutional scientific employment program–contract for her contract. the European Regional Development Fund (ERDF) through the Competitiveness and Internationalization Operational Program for financial support to the project 100% Figo (POCI-01-0247-FEDER-064977) and for C. Shirashi PhD grant.info:eu-repo/semantics/publishedVersio