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  • 28 Reads
Investigating culture media for obtaining lipolytic biocatalysts based on Rhizopus oryzae fungi.

Rhizopus oryzae is widely distributed in nature and can be isolated from different substrates such as decomposing vegetables, fruits and various soils. It is generally classified as GRAS filamentous fungi and commonly used in the production of oriental traditional food such as tempeh or peka. This microorganism has a great industrial potential due to the capability to synthesise enzymes (glucoamylases, cellulases and lipases) and organic acids (lactic acid, fumaric acid). The most studied enzymes of the fungi are lipases (ROL). Therefore, the aim of the study was the selection of growth medium content and initial pH rate, which would provide high lipase synthesis yield in 5 and 7 days shaken cultures. Two fractions of lipases were investigated in order to obtain lipase biocatalysts: extracellular enzymes present in supernatant and cell-bound lipases in biomass. There were used nutrient-rich media: YPG (1% yeast extract, 2% peptone, 2% glucose), YPO (1% yeast extract, 2% peptone, 2% olive oil), YMG (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 2% glucose), YMO (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 2% olive oil) and mineral media: SMG (1% peptone, 1.4% KH2PO4, 0.24% K2HPO4, 0.04% MgSO4, 2% glucose) and SMO (1% peptone, 1.4% KH2PO4, 0.24% K2HPO4, 0.04% MgSO4, 2% olive oil). Fungi biomass and supernatant were separated and used to measure lipase activity by a spectrophotometric method based on the hydrolysis of p-nitrophenyl laurate. The results showed that the highest lipase activity after 5 days of cultivation was reached in SMO medium for biomass and YMG for supernatant. The addition of citric acid slightly increased the activity of produced lipases.

  • Open access
  • 23 Reads
Acorns as a functional food for cardiovascular disease prevention: chemical characterization and bioactivity

Sustainability and circular economy of food production are among the main market trends that have led to the search for innovative solutions, such as using raw materials not typical of human food. In this sense, the exploitation of undervalued natural resources, particularly regarding low-cost and highly available vegetable matrices in Portugal, such as acorns, is possible. Furthermore, acorns are one of the most promising natural resources to be considered nationally and internationally due to their nutritional benefits. Hence, their valorization is an essential factor of sustainability nowadays. Therefore, this project aims to characterize acorns by analyzing their chemical composition and bioactivity toward inhibiting enzymes related to cardiovascular diseases.

After being collected in a forest, acorns were washed and dehydrated at 41°C for 72 h. After this process, the seed and pericarp of the fruit were separated. Finally, a proximate analysis was performed where moisture, ash, lipids and proteins were determined, and the values obtained in mass percentage, on a dry basis, were 9.95 ± 0.24, 1.92 ± 0.03, 3.83 ± 0.34 and 3.72 ± 0.07, respectively. Regarding acorn's bioactivities, α-glucosidase and α-amylase inhibition assays were performed. The concentration of acorn extract that inhibits 50% of the enzyme α-glucosidase is 29.8 µg/mL showing its great potential as an antidiabetic agent. However, no inhibition of the α-amylase was registered.

As future work, it is intended to perform further analyses of the chemical composition of the acorn, such as the content of carbohydrates, fibres, and minerals, among others, and study their biological characterization to assess its potential value as a functional food.


The authors are grateful for the financial support from REQUIMTE/LAQV—UIDB/50006/2020, UIDP/50006/2020 and LA/P/0008/2020, financed by FCT/MCTES and the project SYSTEMIC, "An integrated approach to the challenge of sustainable food systems: adaptive and mitigatory strategies to address climate change and malnutrition". The Knowledge hub on Nutrition and Food Security 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).

  • Open access
  • 159 Reads
Coffee flower as a promising novel food - Chemical characterization and sensory evaluation

The use of the flowers (blossoms) of the coffee plant (genus Coffea) has been neglected over the years, as the focus was primarily on cost-efficient production of coffee beans. Because of societal changes and economic pressures, there is an increasing demand for sustainability, so that the focus widened also toward the various by-products of the coffee production [1]. The coffee flower is a by-product because it can be harvested following pollination without any risk to bean production [2]. The coffee flower can be used as a whole or as floral water in some food and cosmetic products [1,3,4]. The flower can also be prepared as a tea with hot water infusion [1,5]. Another side-chain product in coffee plantations is the so-called coffee flower honey, which is rarely monofloral due to the short flowering period [6,7]. To date, there have been few studies on coffee flowers and their sensory characterization. In this work, various compounds in Coffea arabica, C. canephora and C. liberica flowers were identified and quantified by high performance liquid chromatography (HPLC) with diode array detection (DAD), nuclear magnetic resonance spectroscopy (NMR), and near-infrared (NIR) spectroscopy. Caffeine, chlorogenic acids, organic acids, trigonelline, and sugars were quantified. Additionally, sensory testing of coffee flower infusions according to the German norm DIN 10 809 was performed. With the acquired data, a principal component analysis (PCA) was performed in which hay, hops, sage, dried apricot, and honey were identified as major flavor descriptors in addition to the floral coffee flower flavors. The coffee flower is judged as a promising ingredient, which needs to be further assessed regarding its possible approval within the novel food regulation of the European Union.


[1] Lachenmeier, D.; Schwarz, S.; Rieke-Zapp, J.; Cantergiani, E.; Rawel, H.; Martín-Cabrejas, M.A.; Martuscelli, M.; Gottstein, V.; Angeloni, S. Coffee by-products as sustainable novel foods: Report of the 2nd International Electronic Conference on Foods—“Future Foods and Food Technologies for a Sustainable World.” Foods 2022, 11, 3. doi:10.3390/foods11010003.

[2] Lachenmeier, D.W.; Rajcic de Rezende, T.; Schwarz, S. An Update on Sustainable Valorization of Coffee By-Products as Novel Foods within the European Union. Biol. Life Sci. Forum 2021, 6, 37.

[3] Arrillaga, N.G. A new perfume oil from coffee flowers. Rev. Agr. Puerto Rico 1942, 34, 82−84.

[4] Berry-Caillet, V.; Husson, J.; Barro, L. Floral water from coffee flowers. Patent WO2022053 605A1, 10 September 2021.

[5] Bi, X.; Huang, J.; Hu, F.; Yang, Y.; Li, Y.; Lyu, Y.; Li, G.; Huang, W.; Zhang, X.; He H. et al. Preparation method of coffee flower tea. Patent CN110959719A, 16 December 2019.

[6] Chuttong, B.; Buawangpong, N.; Burgett, M. Honey, bees and coffee. Bee World 2015, 92, 80–83, doi:10.1080/0005772X.2015.1091230.

[7] Schiassi, M.; de Souza, V.; Lago, A.; Carvalho, G.; Curi, P.; Guimarães, A; Queiroz, F. Quality of honeys from different botanical origins. J. Food Sci. Technol. 2020, 58, 4167-4177, doi: 10.1007/s13197-020-04884-7.

  • Open access
  • 37 Reads
Meta-regression modelling of the inhibition diameter produced by extracts of Origanum, Syzygium and Citrus as a function of the minimum inhibitory concentration

Inhibition diameter (ID) and minimum inhibitory concentration (MIC) are important methodologies for antimicrobial susceptibility testing. In this work, the objective was to evaluate if a correlation between ID and MIC results exists.

Such relationship was assessed utilising in-vitro data collected through systematic literature search on the susceptibility of pathogens to extracts of Origanum, Syzygium and Citrus. Suitable studies were identified and the following information was retrieved: study identification, testing method (ID or MIC), dose applied (“LogDose”), pathogen, ID value (mm) and MIC value (“LogMIC”; ml/mg for extracts, μl/ml for essential oils). Then, a mixed-effect linear model with weights on ID produced by Origanum (n=145), Syzygium (n=10) and Citrus (n=7) extracts, as a function of the MIC, extract dose and bacterium, was built.

The estimate for “LogDose” (18.00 [SE=0.227]) indicates tendency for greater ID values as the dose applied increases (p<.0001), and the estimate for “LogMIC” (-5.554 [SE=0.181]) implies that this moderator and ID values are inversely correlated (p<.0001). The model also revealed different overall ID for distinct pathogens when the same extract is applied at the same dose (p<.0001): STEC appears as the most resistant pathogen, followed by C. jejuni, L. monocytogenes, S. aureus and Salmonella spp. (the latter two were not significantly different).

Whereas the goodness-of-fit (R=0.860) indicates a satisfactory correlation between predicted and observed values, the model hinted that other non-quantifiable sources may affect the ID measurements (heterogeneity analysis showed that moderators explain 47.7% of the between-studies variability). Hence, ID methodologies may not be appropriate to compare results from different studies, as the measurements could be affected by errors and variations in the protocols, impacting on the degree of extract diffusion within the agar matrix.

This meta-regression provides insight on the effectiveness of Origanum, Syzygium and Citrus against various organisms, which can be useful to select biopreservatives for pathogen control.

  • Open access
  • 49 Reads
Evaluation of the physicochemical and textural properties of binary protein-polysaccharide hydrogels

The aim of this research was to evaluate the physicochemical and textural properties of the binary hydrogels obtained using plant-based protein and prebiotic polysaccharide. The concentration levels of both biopolymers (pea protein and psyllium husk) were calculated using the DOE statistical tool, resulting in 10 combinations (variants). The hydrogels were obtained using the thermo-mechanical induction technique (pea protein concentration ranging from 10 to 15% and psyllium husk concentration ranging from 1.5 to 2%). The obtained hydrogels were then analyzed in terms of their volumetric gelling index, water holding capacity, microrheology, texture and spreadability, and color parameters.

Based on the conducted research it was found that the volumetric gelling index and water holding capacity of each hydrogel variant was equal to 100%, meaning that they all developed a gel structure causing them to have a high physical stability. In the case of microrheology parameters, the value of solid-liquid balance (SLB) index was below 0.5 (except in for the hydrogel containing 10% pea protein and 1.5% psyllium husk) which means that the analyzed systems had more solid-like properties due to their gel structure (G’ > G”). The elasticity index value was the highest in the case of the hydrogel containing the maximal concentration of both biopolymers. Furthermore, the variant containing 15% pea protein and 2% psyllium husk had significantly the highest values of texture (0.88 N) and spreadability (24.48 N*s). The total color difference DE was below 3.5 meaning that no clear color difference between the hydrogels was noticed.

The physicochemical and textural properties of the obtained binary hydrogels can be controlled by modulating the concentration levels of both pea protein and psyllium husk. In terms of the analyzed properties, the most optimal variant was the one containing 12.5% pea protein and 1.5% psyllium husk. Such binary hydrogels can be used as a structural matrix in plant-based functional food development, by modulating the texture attributes and helping to fortify such foods by acting as a delivery system for nutrients and bioactive ingredients.

  • Open access
  • 33 Reads
Characterization and biological analysis of avocado seed and peel extracts for the development of new therapeutical strategies.

The avocado, Persea americana, is one of the most produced and consumed tropical fruits worldwide, so more than 1.5 million tonnes of waste are generated per year, due to by-products such as the seed or peel [1]. Their revalorisation as sources of bioactive compounds, or directly as high added-value products, would considerably reduce the negative impact on the environment and would promote the circular economy, taking advantage of their incredible phenolic richness and all the health-promoting properties associated with their presence [2]. The antioxidant and anti-inflammatory profile is the most important, beneficial for reducing oxidative stress and, thus, a lot of detrimental processes, such as neuroprotection through blocking acetylcholinesterase in neuronal synapses [3].

Therefore, in order to determine their potential, a series of in vitro tests were carried out: identification of phenolic compounds with HPLC-ESI-qTOF-MS; quantification of the phenolic content and the antioxidant capacity through methodologies based on proton/electron transfer; analysis of the ability to scavenge different reactive oxygen species (ROS); and evaluation of the inhibitory capacity on different enzymes related to oxidative stress, aging and neurodegeneration.

Results reflected two matrices rich in flavonoids (quercetin, catechin, epicatechin, etc.), phenolic acids (quinic, citric and chlorogenic acid), and procyanidins with elevated degree of polymerisation, which explain the high antioxidant and ROS scavenging activity. Specifically, avocado peel was concluded as the most phenolic-rich extract with higher antioxidant activity; however, both extracts exerted beneficial effects inhibiting some of the enzymes tested. Avocado seed and peel are pointed out as suitable options for the development of new medical, cosmetical and food strategies.

[1] Ayala, T. et al. (2014). Springer.
[2] Jyoti, D. et al. (2019). Antiox.
[3] Ameer, K. et al. (2016). Adv. Neurobiol.

  • Open access
  • 76 Reads
Chlorogenic acids profile of Coffee arabica by-products (cascara and silverskin): a comparison with green and roasted beans

Coffee, one of the most traded commodities in the world, has several compounds with health-promoting properties [1,2]. The presence of chlorogenic acids (CGA) is not only responsible for its bitter and astringent taste, but also for its anti-inflammatory, antitumoral, and antioxidant effects [1,2]. However, coffee production generates huge quantities of by-products that have a negative effect on the environment if they are not treated [3].

Cascara and silverskin are the primary by-products of coffee cherries pulping and green coffee roasting, respectively [3]. In this work, the CGA profile of these two coffee by-products were studied and compared with those of coffee beans (green and roasted), aiming a possible valorization in a circular economy context.

The samples were all of Coffee arabica from Colombia. A solid-liquid extraction with 40 ml of ethanol: water (1:1) was performed for 30 min, using 0.4 g (roasted and green coffee) or 1.2 g (silverskin and cascara) of sample. The caffeoylquinic acids (CQA) and feruloylquinic acids (FQA) were analyzed by RP-HPLC-DAD [4].

The results show that these coffee by-products have a significantly lower CGA content than green or roasted beans. In all samples, the predominant CGA was the 5-CQA. Cascara contained the highest concentration of 5-CQA among the by-products (1.1 mg/g dw), while green beans presented the highest amount (49.57 mg/g dw). In fact, roasted beans and silverskin are roasted at high temperatures, rendering CQAs susceptible to degradation, transesterification, isomerization, and conversion into lactones [5]. FQA are present in smaller amounts, with the highest concentration found in green beans (6.78 mg/g dw).

To conclude, while cascara and silverskin contain less CGA than coffee beans, they can still be considered a source of these high-value compounds. CGA in by-products could be recovered and used to improve the functionality of foods as well as in the pharmaceutical industry.

  • Open access
  • 45 Reads
Study of the antihypertensive peptides derived from alpha-lactalbumin hydrolysate after simulation of digestion

Alpha-lactalbumin is a whey protein which is a cheese making industrial residue of high biological value. The antihypertensive capacity of three peptides obtained from the simulated gastrointestinal digestion of alpha-lactalbumin hydrolysates was studied.

Alpha-lactalbumin hydrolysis was performed using Alcalase enzyme and subsequently subjected to a simulated digestion process by using pepsin and pancreatin enzymes to mimic digestion conditions. The peptides were identified from a RP-HPLC fractionation of the digest and subsequent identification by mass spectrometry analysis. Three peptides from alpha-lactalbumin sequence were obtained: IWCKDDQNPH (P1), KFLDDDLTDDIM (P2) and DKFLDDDLTDDIM (P3). The in vitro antihypertensive activity of the peptides was determined by studying the inhibition of angiotensin converting enzyme, with P1 being the only peptide with antihypertensive activity detected by this methodology (IC50=3.91±0.2 mg/mL).

In order to correlate structural (molecular dynamics simulations) and physicochemical properties (charges, hydrophobicity, solvent accessible areas (ASA, ASA+, ASA-, ASAH, ASAP)) with potential mechanisms of antihypertensive capacity in silico methods were performed. The peptides P1, P2 and P3 had a negative global charge and were hydrophilic. After molecular modelling, the peptide structures were submitted to a refinement based on an energy minimization and further molecular dynamics simulation to assess their global size and conformational space. After 50 nanoseconds simulation, the global structures, solvated and immersed in an ionic water solution similar to that of blood, were studied in their ASA´s. Some secondary structure (alpha-helix) was observed in the P1 peptide but in general, all peptides showed an extended folding. Surfaces were charge code colored and in a visual inspection it could be conjectured that all of them exposed the charge, mainly negative charge, to the solvent surface, in agreement with the GRAVY index which was also evaluated.

In conclusion, the structure and amino acid composition of peptide 1 assessed by in silico studies agrees with the antihypertensive activity obtained by the in vitro study.

  • Open access
  • 48 Reads
A simplified SPE-GC-FID method for detection of adulteration of olive oil with sunflower oil

The unique distribution between free and esterified minor compounds represents a promising tool for the evaluation of olive oil authenticity. For this purpose, we developed and in-house validated a simple SPE-GC-FID method aimed at assessing free and esterified hydroxylated minor compounds patterns in olive and sunflower oils. The methodology showed suitable repeatability relative standard deviation, which was lower than 7.5%, in all cases. The method, which is intentionally based on simple instrumentations, allows quantification of hydroxylated minor compounds in a single chromatographic run while reducing sample preparation, employment of toxic solvents, and analysis time compared to already existing procedure. Finally, the analytical approach has been used for the analysis of pure oil samples, comprising 15 authentic extra-virgin olive oils selected from different European countries, revealing different hydroxylated minor compounds profiles and proportion of free and esterified forms in olive and sunflower oils. The herein proposed SPE-GC-FID method has been then applied to the quantification of free and esterified hydroxylated minor compounds as well as their ratio in pure extra virgin olive oil mixed in different amounts to mimic olive oil adulteration with refined sunflower oil at different levels of 2, 5, 10, 15, and 20% (w/w).

This work is developed in the context of the project OLEUM 'Advanced solutions for assuring authenticity and quality of olive oil at global scale', funded by the European Commission within the Horizon 2020 Programme (2014-2020, GA no. 635690). The information expressed in this abstract reflects the authors' views; the EC is not liable for the information contained therein.

  • Open access
  • 62 Reads
Extraction of Chamomile (Matricaria recutita L.) essential oil using the green technology of Microwave free-solvent extraction

The essentials oils are important in food industry, mainly for flavors and bioactive compounds. In the last years, new techniques for the extraction of essential oil has been developed aiming to improve yields, among them, the green technology of microwave solvent-free extraction (ME). This study aimed to assess the benefits of this method on the Chamomile (Matricaria recutita L.) essential oil quality and yield compared to the extraction by steam distillation (SD). The oil obtained by ME and SD presented a blue color, a solubility in 70% ethanol (v/v) of 4, a relative density of 0.929-0.925g/ml, a refractive index of 1.5013-1.4790, and an acidity index of 6.23 and 3.43 respectively. The yields were significantly different between extraction methods, being the highest 0.5ml (0.083% v/w) for ME and 0.2ml (0.03%) for SD. The GC-MS analysis of chamomile essential oils obtained showed a markedly difference in its principal components such as Chamazulene, α-Bisabolol, α-Bisabolol oxide A and α-Bisabolol oxide B. The ME had a 97% and 20% more content of Chamazulene and α-Bisabolol respectively, whilst SD an 88% and 12% more content of α-Bisabolol oxide A and B respectively. Considering that Chamazulene content is strongly related to price and α-Bisabolol has been associated to the anti-inflammatory properties of Chamomile, the results suggest that ME is an outstanding alternative for essential oil extraction due to much higher yield and quality compared to the steam distillation.