Assessing Nutritional Traits and Phytochemical Composition of Artisan Jams Produced in Comoros Islands: Using Indigenous Fruits with High Health-Impact as an Example of Biodiversity Integration and Food Security in Rural Development

In the Comoros Islands, as in other developing countries, malnutrition and food insecurity affect a very large percentage of the population. Developing fruit-based products in order to make profit, reduce poverty and improve indigenous people diet could be very important for local population of countries as Comoros Islands. The aim of the present work was to study the chemical composition of jams and jellies produced from seven fruit species harvested in Grand Comore Island. The following parameters were studied sugars and organic acids, total phenolics, total anthocyanins and high-performance liquid chromatography (HPLC) fingerprint of the main phytochemicals. Antioxidant activity was also measured. A multivariate approach (Principal Component Analysis) was performed in order to better characterize the products and to set a potential analytical tool for jam characterisation. Results showed that the analysed products are a good source of polyphenolic constituents, as caffeic and gallic acids, catechin and quercetin and volatile compounds, as limonene and γ-terpinene: these molecules may be considered as suitable markers for these fruit-derived products as characterizing the chromatographic patterns. The characterisation of these products and their nutritional and nutraceutical traits is important as valorisation of local food production for poverty reduction and rural development. Further benefits of this approach include the maintenance of local agro-biodiversity as raw material for fruit-based products and the strengthening of food security practices.

Centrifuge model PK 120, Cologno Monzese, Italy). This operation was carried out 3 times. All the supernatants were recovered and transferred to small glass tubes and kept frozen at -20°C for further analysis [1].

Organic acids, sugars, and monoterpenes
For the extraction of organic acids, sugars, and monoterpenes three replications were considered. Five grams of fruit jam/jelly were put into a test tube and 25 mL of 95% ethanol solution were then added. After 30 min in the dark, the extracts were homogenized with an Ultra-Turrax (T25, IKA WERKE) for about 1 min and then centrifuged for 10 min at 4,000 rpm in an ALC Centrifuge PK 120 (ALC International, Cologno Monzese, Italy). This operation was carried out 2 times. All the supernatants were recovered and transferred to small glass tubes and kept frozen at -20°C for further analysis [2].

Vitamic C
A total of 10 g of fruit-derived product (three replications) was put into a 50-mL test tube and 10 mL of extraction solution (0.1 M citric acid, 2 mM EDTA disodium salt and 4 mM sodium fluoride in methanolwater, 5:95 v/v) were then added. The extracts were homogenized with an Ultra-Turrax (IKA-Werke T25) for about 1 min and then centrifuged for 10 min at 4,000 rpm at room temperature in an ALC Centrifuge PK 120. The supernatants were recovered and transferred to a 15-mL test tube through filter cloth and then acidified with 4 N HCl to decrease pH solution to a value of 2.2-2.4 pH units. Acidified samples were centrifuged for 5 min at 12,000 rpm at 4°C with an ALC Multispeed refrigerated centrifuge PK 121R (ALC International, Cologno Monzese, Italy) [3].

Spectrophotometric analysis
The total polyphenol content (TPC) was determined by the Folin-Ciocalteu method [4,5], according to which the Folin-Ciocalteu reagent (2.5 mL) and 15% Na2CO3 (10 mL) were added to the extract (0.5 g), which was previously diluted with deionised water (30 mL). After 120 min, the absorbance was measured at 765 nm. The content of total phenolics was expressed as mg of gallic acid equivalents (GAE) per 100 g of product (Pr). The standard calibration curve was plotted using gallic acid at concentrations of 0.02-0.1 mg/mL. To evaluate the effect of sugar and citric acid added to the jams/jellies, a blank containing 65% sugar and 0.02% citric acid was used, although no correction was necessary at the dilution level used.

Apparatus and chromatographic conditions
Separation and identification of compounds were performed by HPLC analysis, using an Agilent 1200 High-Performance Liquid Chromatograph coupled to an Agilent UV-Vis diode array detector (Agilent Technologies, Santa Clara, CA, USA).
The chromatographic conditions were set in order to obtain a phytochemical fingerprint containing compositional information with a good resolution and a reasonable analysis time. Different linear gradients in different slopes were used for optimizing the molecule separation because some compounds were similar in structure with each other in the same chemical class. Formic and phosphoric acid was added for enhancing the resolution and eliminating peak tailing because most of the compounds were also weakly acidic. Detection was performed with an UV -Vis Diode Array Detector by scanning from 190 to 400 nm: selected wavelengths were suitable to achieve more specific peaks as well as a smooth baseline after a full-scan on the chromatogram.

Identification and quantification of bioactive compounds in the extracts
Identification of compounds was carried out by comparing retention times and spectroscopic data with those of authentic standards in the same chromatographic conditions. Total bioactive compound content (TBCC) was determined as the sum of selected markers having a positive role in human health as reported in the "multi-marker approach" by Mok and Chau [10]. Five polyphenolic classes were considered: benzoic acids (ellagic acid, gallic acid), catechins ((+)catechin, (-)epicatechin), cinnamic acids (caffeic acid, chlorogenic acid, coumaric acid, ferulic acid), flavonols (hyperoside, isoquercitrin, quercetin, quercitrin, rutin), and tannins (castalagin, vescalagin). Vitamin C (as the sum of ascorbic and dehydroascorbic acids), monoterpenes (limonene, phellandrene, sabinene, -terpinene, and terpinolene), organic acids (citric acid, malic acid, oxalic acid, quinic acid, succinic acid, and tartaric acid), and sugars (fructose, glucose, and sucrose) were also considered to obtain an analytical fingerprint. The total sugar content was calculated as the sum of fructose, glucose, and sucrose identified and quantified in the final products. Values include the initial total fruit sugar content and the sucrose added during the jam preparation. All the results were expressed as mg/100 g of Pr (product), except sugars (expressed as g/100 g of Pr).
External standard calibration method was used for quantitative determinations by plotting the peak area (y) of the compound versus the sample concentration (x): for each analytical standard three manual injections (20 L) at each concentration were performed. The limit of detection (LOD) and the limit of quantification (LOQ) of the used chromatographic methods was defined as the lowest amount of analyte that gives a reproducible peak with a signal to-noise ratio (S/N) of 3 and 10 respectively. All the samples were analyzed in triplicate, and the results were reported as means ± standard deviation (SD) in order to assess the repeatability of the used methods. Accuracy was checked by spiking samples with a solution containing each bioactive compound in a concentration up to a maximum of 1.0 mg· mL −1 .

Statistical analysis
In order to establish the statistical differences between means the data were treated by one-