Flour fortification for nutritional and health improvement: A review

Deficiencies of micronutrients, essentially vitamins and minerals, have mainly cause several illnesses, especially in children and women worldwide. Governments and world organizations have focused great efforts to address these shortcomings and improve the health of the populations. This malnutrition can be combated by fortifying basic foods that are consumed daily. Thus, flours, especially wheat, maize and rice, are a popular and common food in the world's populations diet and are gaining considerable attention as a suitable vehicle for micronutrient fortification. There are some studies that show the effectiveness of flour fortification in combating micronutrient deficiencies and several diseases and the flour fortification strategies are directed to meet nutritional and health needs of the populations. The main points discussed in this review are food fortification, with great focus in flour fortification, health benefits, and legislative issues. This review also highlights multifaceted issues related to flour fortification to meet nutritional needs and to improve the health of vulnerable populations.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019), R.V.C. Cardoso grant (SFRH/BD/137436/2018). L. Barros and A. Fernandes also thank the national funding by FCT – Foundation for Science and Technology, P.I., through the institutional scientific employment program-contract for their contracts. This work is funded by the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020, within the scope of Project Mobilizador ValorNatural®; and to FEEI through the Rural Development Program (PDR2020).info:eu-repo/semantics/publishedVersio

Effectiveness of gamma and electron beam irradiation as preserving technologies of fresh Agaricus bisporus Portobello: a comparative study

The main objectives of the present work were as follows: (a) to develop an analytical procedure to quantify free sugars on chestnuts based on green-chemistry principles and (b) to evaluate the effect of hot air convective drying (50 °C along 10 hr) on the carbohydrate profile of sliced chestnuts (Longal and Judia varieties). The analytical method allowed the quantification of several sugars in chestnut extracts, with low detection limits and good precision. In fresh, Judia variety had higher sucrose, fructose, and glucose contents than Longal, together with amylose. Nevertheless, when applying hot air convective drying to chestnut slices, a significant increase in fructose contents was observed with time, particularly after 10 hr drying in Longal variety. Nonetheless, a significant increase in glucose content was observed on both varieties, probably related to starch thermal hydrolysis. Practical applications: An environment-friendly methodology for free sugars quantification, based on ethanol/water extraction (green solvents), ion-exchange high-performance liquid chromatography separation with water as mobile phase, and evaporative light scattering detection, was developed. The method allowed the quantification of raffinose, sucrose, glucose, and fructose in chestnut extracts, with detection limits of 6 μg for sucrose and 0.3 μg for other sugars. Good precision was achieved with intra-day and inter-day coefficients of variation below 5%. Regarding chestnuts' drying, hot air convective drying might be applied to chestnut slices but it may induce modifications on the carbohydrate composition of the samples, particularly when long drying periods are applied, requiring some attention because it might induce both visual and sensory changes.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), C2TN (UID/Multi/ 04349/2013), L. Barros and J. Barreira contracts, and A. Fernandes post-doc grant (SFRH/BPD/114753/2016). This work is funded by the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020, within the scope of Project Mobilizador ValorNatural®; and to FEEI through the Rural Development Program (PDR2020), within the scope of Project MicoCoating (PDR2020-101-031472). This work was also developed within the Coordinated Research Project D61024 “Development of New Applications of Machine Generated Food Irradiation Technologies” financed by the International Atomic Energy Agency (IAEA).info:eu-repo/semantics/publishedVersio

A case study on surplus mushrooms production: extraction and recovery of vitamin D

The presented case study illustrates the possibility of adding value to the biological surplus remaining from the mushroom cultivation industry. In essence, the unused mushroom parts were submitted to UV-C irradiation, with the purpose of increasing the vitamin D2 content and validat-ing its extraction. Vitamin D2 concentration in three different mushroom species (Agaricus bisporus, A. bisporus Portobello, and Pleurotus ostreatus) was obtained by high-performance liquid chromatogra-phy (HPLC), by means of an ultraviolet (UV) detector. The method was validated using an A. bisporus Portobello sample, and its reproducibility and accuracy were confirmed. Independently of the UV-C irradiation dose, the effect on vitamin D2 concentration was significant, allowing it to increase from less than 4 µg/g dry weight (dw) to more than 100 µg/g dw in all mushroom species. These results are good indicators of the feasibility of industrial surplus mushrooms as sustainable vitamin D2 food sources, besides contributing to strengthen the circularity principals associated to the mushroom production chain.The authors are grateful to the Foundation for Science and Technology (Fundação para a Ciência e Tecnologia, Lisboa, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020), and R.V.C.C.’s PhD grant (SFRH/BD/137436/2018). The authors are also grateful to “Ponto Agricola” for providing the mushroom materials. A.F., J.C.M.B. and L.B. also thank the national funding by FCT, P.I. through the institutional scientific employment program contract. This work is funded by the ERDF through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®.info:eu-repo/semantics/publishedVersio

Physicochemical characterization and microbiology of wheat and rye flours

Seven types of wheat and rye flours were studied regarding their physical and chemical properties, as well as the presence of mycotoxins and microorganisms. The results revealed that flours presented moisture and ash contents below the recommended maximum limit. They also presented a low lipid content, which helps avoiding changes in the flours’ smell and taste. From the microbiological analysis, comparing the refined rye and wheat flours, the counting in the analysed microorganisms was not significantly different. The whole flours presented high contents in almost all the tested microorganisms, highlighting the molds counting for the whole wheat flour and the aerobic plate counting for the whole rye flour. None of the samples presented Salmonella spp. Aflatoxins and ochratoxin A were not detected in any of the flours. To the best of our knowledge, this is the first characterization of wheat and rye flours for Portuguese consumers.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros contract, A. Fernandes (SFRH/BPD/114753/2016) and S.A. Heleno (SFRH/BPD/101413/2014) pos-doc grants. This work is also funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®.info:eu-repo/semantics/publishedVersio

Combined effects of irradiation and storage time on the nutritional and chemical parameters of dried Agaricus bisporus Portobello mushroom flour

Abstract: Portobello variety of Agaricus bisporus mushrooms, appreciated for its taste, makes it desirable to be eaten fresh and also as flour in soups and gravies. Gamma and electron-beam radiation at four doses (1, 2, 5, and 10 kGy) were used to analyze its preservation effect on Portobello mushroom flour. A proximate analysis, as well as the impact on fatty acids, tocopherols, soluble sugars, organic acids, and ergosterol profiles, were performed every 3 months, during a storage period of 1 year. Gamma rays preserved mannitol (most abundant soluble sugar) over the 12 months, while electron beam radiation preserved organic acids. No significant changes were sought for any radiation type, and the slight changes extracted from the estimated marginal means reveal a tendency for irradiation as having preserving effects of nutrients and other important molecules. Thus, both irradiation types, up to 10 kGy are suitable for preservation of A. bisporus Portobello flour.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/AGR/00690/2020), C2TN (UID/Multi/04349/2013) for R.V.C. Cardoso’s grant (SFRH/BD/137436/2018), M. Carocho’s research contract. L. Barros and A. Fernandes also thank FCT’s funding through the institutional scientific employment program-contracts. This work is funded by the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020, within the scope of Project Mobilizador ValorNatural R . Acknowledgments are also due European Agricultural Fund for Rural Development (EAFRD), through the Rural Development Program (PDR2020), within the scope of Project MicoCoating (PDR2020-101-031472).info:eu-repo/semantics/publishedVersio

Combined effects of irradiation and storage time on the nutritional and chemical parameters of dried Agaricus bisporus Portobello mushroom flour

Portobello variety of Agaricus bisporus mushrooms, appreciated for its taste, makes it desirable to be eaten fresh and also as flour in soups and gravies. Gamma and electron-beam radiation at four doses (1, 2, 5, and 10 kGy) were used to analyze its preservation effect on Portobello mushroom flour. A proximate analysis, as well as the impact on fatty acids, tocopherols, soluble sugars, organic acids, and ergosterol profiles, were performed every 3 months, during a storage period of 1 year. Gamma rays preserved mannitol (most abundant soluble sugar) over the 12 months, while electron beam radiation preserved organic acids. No significant changes were sought for any radiation type, and the slight changes extracted from the estimated marginal means reveal a tendency for irradiation as having preserving effects of nutrients and other important molecules. Thus, both irradiation types, up to 10 kGy are suitable for preservation of A. bisporus Portobello flour.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/AGR/00690/2020), C2TN (UID/Multi/04349/2013) for R.V.C. Cardoso’s grant (SFRH/BD/137436/2018), M. Carocho’s research contract. L. Barros and A. Fernandes also thank FCT’s funding through the institutional scientific employment program-contracts. This work is funded by the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020, within the scope of Project Mobilizador ValorNatural R . Acknowledgments are also due European Agricultural Fund for Rural Development (EAFRD), through the Rural Development Program (PDR2020), within the scope of Project MicoCoating (PDR2020-101-031472).info:eu-repo/semantics/publishedVersio

UV-C radiation increases vitamin D2 content in Pleurotus ostreatus

Mushrooms have been traditionally recognized for their nutritional and medicinal value, and countless investigations have reported highly nutritious content as well as medicinal properties such as anticancer, antimicrobial, hypocholesterolemic, antioxidant, among others. These properties are attributed to bioactive compounds such as ergosterol (precursor of vitamin D2) and other steroids, phenolic compounds, and vitamins. Vitamin D2, in particular, plays an important role in many human metabolic processes. Mushrooms are the only non-animal food source of vitamin D2, which is also formed during UV exposure1. Since dietary sources are scarce, vitamin D deficiency can cause serious health problems; studies of vitamin D have received considerable attention in recent years, supported by the growing number of deficiency reports, e.g. rickets, osteoporosis, multiple sclerosis and cardiovascular disease, among others 2. The present work reports the effectiveness of UV-C radiation on increasing the content of vitamin D2 in sliced Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. samples. The irradiation was performed in a UV chamber at the intensity 0 (non-irradiated), 200, 800, 3200 mJ/cm2 and different exposure times: 0, 2, 6 and 10 min. Vitamin D2 was determined using high performance liquid chromatography coupled to a UV detector 2,3. It was verified a significant (p<0.050) interaction (exposure time x UVC) among factors, indicating that the effect of each UVC dose was modulated by exposure time and vice versa. Nonetheless, it is obvious that the application of UV-C radiation induced a clear increase in the quantity of vitamin D2, most likely due to the conversion of some of the ergosterol content naturally present in these mushrooms. In what concerns, exposure time, the adequate choice would be 6 min (125 μg/g DW of vitamin D2), as no significant increases were attained with the maximum assayed time (10 min). On the other hand, and despite the statistically significant differences, the advised UV-C dose would be 200 mJ/cm, particularly considering that this option would be less expensive, without relevantly compromising the increase in vitamin D2.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019), and R.V.C. Cardoso grant (SFRH/BD/137436/2018). L. Barros, J.C.M. Barreira, and A. Fernandes also thank the national funding by FCT - Foundation for Science and Technology, P.I. through the institutional scientific employment program-contract. Funding: This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®.info:eu-repo/semantics/publishedVersio

Effects of calcium silicate on the chemical and bioactive composition of Pleurotus ostreatus var. Florida

Mushrooms are low-calorie foods with good quality proteins, vitamins and minerals, besides holding potential for some medicinal applications. In fact, mushrooms could be a source of many different nutraceuticals such as steroids, phenolic compounds, and others. Thus, they might be used directly in diet and promote health, taking advantage of the additive and synergistic effects of all the bioactive compounds present1. The edible mushroom Pleurotus ostreatus var. Florida known as "Hiratake" is one of the most consumed mushrooms in the world2, mainly due its easy cultivation, economic potential, nutritional quality, as well as therapeutic and biological properties. Silicon (Si) is known to play an important role in the mineral nutrition supplementation of mushrooms and plants, including increased productivity, through the availability of nutrients, increased biomass and resistance to biotic and abiotic stresses3. In this study, cultivated Pleurotus ostreatus var. Florida was supplemented with calcium silicate (0.5, 1, 2 and 4 %) and the effects of this supplementation on chemical and bioactive composition were evaluated. Ergosterol and vitamin D2 were determined by high performance liquid chromatography coupled to a UV detector; organic acids and phenolic compounds were determined by ultrafast liquid chromatography coupled to a photodiode array detector4. The supplementation with calcium silicate exerted remarkably positive effects in the evaluated parameters. Specifically, higher vitamin D2 contents were obtained in samples treated with 1% and 2% (866 and 862 μg/100 g dw). A similar increase was obtained in organic acids (5.15 g/100 g dw), considering the three identified compounds (oxalic, malic and fumaric acids) in samples treated with 0.5% of calcium silicate. The calcium silicate supplementation also increased the total phenolic compounds (protocatechuic acid, p-coumaric acid and cinnamic acid) relatively to the control sample. Calcium silicate supplementation was effective in improving the chemical profiles of P. ostreatus. Therefore, this practice may represent an effective way to increase the compounds of interest in different mushrooms.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019), and R.V.C. Cardoso grant (SFRH/BD/137436/2018). L. Barros, João C.M. Barreira, and A. Fernandes also thank the national funding by FCT - Foundation for Science and Technology, P.I. through the institutional scientific employment program-contract. Funding: This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®, and for the contract of M. Carocho; and to European Agricultural Fund for Rural Development (EAFRD), through the Rural Development Program (PDR2020), within the scope of Project MicoCoating (PDR2020-101-031472).info:eu-repo/semantics/publishedVersio

Optimization and recovery of Vitamin D2 from spurplus production of Agaricus bisporus Portobello

During mushroom production a percentage as high as 20% of surplus can be generated. These unused mushrooms have high nutritional value and valuable chemical compounds. In this sense, finding innovative alternatives to valorizing this surplus mushroom production needs to be explored [1,2]. Irradiation of mushrooms surplus to obtain vitamin D2 is a sustainable strategy to increment vitamin D availability. Under this perspective, the objective of this study was setting the UV-C irradiation and extraction conditions that maximize vitamin D2 contents in surplus mushrooms production (Agaricus bisporus Portobello). The bioactive effects and potential toxicity of vitamin D2-enriched extracts were also evaluated. The surplus production from A. bisporus Portobello were supplied by the Ponto Agrícola, Baião, North of Portugal. The irradiation was performed using an ultraviolet (UV-C at 200 mJ/cm2, 800 mJ/cm2 and 3200 mJ/cm2) radiation chamber with different exposure times: 0, 2, 6 and 10 min. Sensitivity and linearity of the high performance liquid chromatography (HPLC) coupled to ultraviolet detector (UV) were determined and the method was validated by the instrumental precision, repeatability and accuracy, and the extracts rich in vitamin D2 were also quantified by HPLC-UV. The cytotoxicity of the vitamin D2 extract were evaluated using three tumoral cell lines (MCF-7 - breast adenocarcinoma, NCI-H460 - non-small cell lung cancer and AGS - gastric cancer) and one non-tumoral cell line of bone origin (h-FOB 1.19 - human osteoblasts). The chromatographic method revealed a great reproducibility and accuracy, thus being the method validated. Independently of the UV-C irradiation dose, the effect in the conversion of vitamin D2 concentration was very high, allowing it to increase from ~3 μg/g dw to more than 100 μg/g dw in A. bisporus portobello. The extract enriched in vitamin D2 presented an effective activity in AGS (82 μg/mL) tumoral cell line and a moderate activity in NCI-H460 (293 μg/mL) and CaCo (377 μg/mL) tumoral cell lines. Furthermore, the extract did not show cytotoxicity against the non-tumor bone cell h-FOB 1.19 (GI50> 400 μg/mL). Accordingly, development food applications of mushroom extracts enriched in vitamin D2, from surplus mushroom production, can be considered and valorized, supporting and adding value to the agriculture sector or pharmaceutical industries.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020), and R.V.C. Cardoso’s PhD grant (SFRH/BD/137436/2018). The authors are also grateful to “Ponto Agricola” for providing the mushrooms material. L. Barros and A. Fernandes also thank the national funding by FCT, P.I. through the institutional scientific employment program-contract. This work is funded by the ERDF through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®info:eu-repo/semantics/publishedVersio

Ganoderma Lucidum: Uma fonte importante de ingredientes bioativos multifuncionais

Ganoderma lucidum {Curtis) P. Karst. é um cogumelo medicinal muito conhecido, cuja compos1çao bioativa {compostos fenólicos, triterpenoides e polissacáridos) tem sido relacionada com efeitos farmacológicos e fisiológicos (por exemplo, propriedades imunomoduladoras, citotóxicas, anti-inflamatórias, antimicrobianas e antioxidantes). No presente trabalho, os corpos frutíferos de G. ludicum foram caracterizados quanto à sua composição química (terpenoides e triterpenos, polissacáridos e tocoferóis), sendo posteriormente avaliadas diferentes propriedades bioativas (e.g., antioxidante, anti-inflamatória, antitirosinase, citotóxica e antimicrobiana) no extrato etanólico obtido por Soxhlet. O extrato foi também caracterizado quanto à sua composição em ácidos fenólicos e triterpenoides por HPLC-DAD-ESI/MS. Por fim, o extrato bioativo foi incorporado numa formulação cosmética semi-sólida, cujas propriedades bioativas e físico-químicas foram também avaliadas. A espécie G. lucidum demonstrou ser uma boa fonte de tocoferóis (141 ± 7 (lg/100 g massa seca, ms), sobretudo pela contribuição das isoformas a e õ. Os conteúdos em terpenoides, triterpenoides e polissacáridos foram 27,2 ± 0,7 mg equiv. linalol/g ms, 5,6 ± 0,5 mg equiv. ácido ursólico/g ms e 15,4 ± 0,2 mg equiv. amido/g ms, respetivamente. O extrato etanólico apresentou uma elevada atividade antioxidante (valores de EC50 para a atividade captadora de radicais livres - 0,73 ± 0,01 mg/ml e poder redutor - 0,15 ± 0,01 mg/ml), antitirosinase (EC50 = 2,81 ± 0,01 mg/ml), citotóxica (valores de Gl 50 para as linhas celulares MCF7: 61 ± 4 (lg/ml; NCI -H460: 64 ± 3 (lg/ml; HepG2: 68,44 ± 0,08 (lg/ml; Hela: 73 ± 2 (lg/ml) e antimicrobiana (MICs até 10 mg/ml contra Escherichia coli, Enterococcus faecalis, MRSA- Staphylococcus aureus resistente à meticilina, MSSA- Staphylococcus aureus suscetível à meticilina). O extrato não exibiu toxicidade para células não tumorais {GI 50> 400 ~g/ml). A análise do extrato, realizada por HPLC-DAD-ESI/MS, revelou a presença de três ácidos fenólicos {ácidos protocatéquico, p-hidroxibenzoico e siringíco) e 26 ácidos triterpénicos, sendo os ácidos ganodéricos C2, A e H os compostos mais abundantes. A formulação cosmecêutica preparada com o extrato manteve todas as bioatividades (atividades antioxidantes, antitirosinase e antimicrobiana). Os parâmetros de cor avaliados através do sistema L* a* b* foram de 70,3 ± 0,6; 12,1 ± 0,5 e 32,9 ± 0,9, respetivamente para L (luminosidade), a (verde-vermelho) e b (azul-amarelo). A formulação apresentou um pH de 4,61 ± 0,02, que é considerado desejável para a manutenção da função de barreira da pele. Em suma, o extrato etanólico de G. lucidum incorporado em formulações tópicas revelou ter um grande potencial para garantir as diferentes funções fisiológicas da pele. No entanto, as questões relacionadas com a estabilidade, permeabilidade, eficácia e biodisponibilidade tópica serão abordadas em estudos futuros.POCI-01-0145-FEDER-006984 (LA LSRE-LCM), financiado pelo FEDER, através do POCI-COMPETE2020 e FCT; Projeto NORTE-01-0145- FEDER-000006, financiado pelo NORTE2020 sob o PT2020, através do FEDER; FCT e FEDER sob o PT2020 pelo apoio financeiro ao CIMO (UID/AGR/00690/2013); Projeto mobilizador ValorNatural® financiado pelos Fundos Europeus Estruturais e de Investimento (FEEI) através do Programa Operacional Regional Norte 2020.info:eu-repo/semantics/publishedVersio