Biological material

The fruits of blackberries and wild raspberries were collected in the municipalities of Uruapan and Nahuatzen in the state of Michoacán, Mexico. The collection sites were referenced by geographic coordinates for each species, which were identified by molecular biology in the Molecular Biology Laboratory of the Technological Institute of Tlajomulco, Jalisco, Mexico.

The species were recognized as Rubus adenotrichus (1 815 masl, 19° 26’ 48’’ north latitude, 102° 4’ 38’’ west longitude), Rubus pringlei (2 769 masl, 19° 40’ 30’’ north latitude; 101° 50’ 8’’ west longitude), Rubus glaucus Beth (2 769 masl, 19 ° 40' 29 '' north latitude, 101° 50’ 9’’ west longitude) and the commercial variety Tupy (1 210 masl; 19° 39’ 16’’ north latitude; 101 91’ 50’’ west longitude). The fruits were selected in maturity of consumption according to their coloration. At the time of collection, the size of the fruit was recorded by means of a Vernier (standard analogue Truper), number of drupillas and weight in grams with a digital scale (Sartorius, model BL210S).

Physicochemical proximal composition

The physicochemical parameters were evaluated in fresh fruit, through the application of official analysis standards for: percentage of humidity (^{AOAC 934,06/2007}), titratable acidity expressed in citric acid (^{AOAC 942,15/2007}), pH by potentiometry (^{AOAC 981, 12/2002}), total soluble solids (°Brix) with a 0-30 scale refractometer (Atago, master-BX/S28M model); likewise, the reducing sugars were evaluated according to the 3,5-dinitrosalicylic acid (DNS) method (^{Amid et al., 2014}), which consisted in the preparation of the DNS reagent by dissolving 75 g of sodium tartrate and potassium tetrahydrate in distilled water , adding 50 mL of 2 M sodium hydroxide and 75 mL of hot distilled water, finally 0.25 g of 3,5-dinitrosalicylic acid was added, the reaction was carried out with 100 μL of the extract, adding 1 mL of the reagent DNS and incubating for 10 min. The absorbance was determined at 570 nm and the results were expressed in grams of fructose per 100 g of sample.

Quantification of polyphenols, flavonoids, total anthocyanins and antioxidant capacity

The extraction was made by maceration from 1 g of fresh fruit with 9 mL of 80% ethanol, for 48 h at 4 °C in the absence of light. The mixture was centrifuged at 13 000 rpm for 10 min, the supernatant was recovered and used for the quantification of total polyphenols (PT), total flavonoids (FT), total anthocyanins (ANT) and antioxidant capacity (CA).

The PT were quantified by what was reported by ^{Zielinsli and Kozolwaska, (2000)}. 50 μL of sample was taken, mixed with 200 μL of distilled water and 250 μL of Folin-Ciocalteu reagent at 1 N, after three minutes, 500 μL of 7.5% Na₂CO₃ was added. It was incubated for 15 min and the absorbance was recorded at 760 nm. The results were expressed in gallic acid equivalents per hundred grams of fresh weight (mg EAG/100 g PF). A calibration curve was made with gallic acid at different concentrations 0.01-0.50 mg mL^-1 (ten data points R²= 0.998).

The FT were evaluated using the information reported by ^{Chang et al. (2002)}, mixing 100 μL of the extract with 200 μL of 1 M potassium acetate, 200 μL of 10% aluminum nitrate and 1 mL of 80% ethanol, then incubated for 40 min and absorbance was read at 415 nm. The results were reported in quercetin equivalents per hundred grams of fresh weight (mg EQ/100 g PF). The calibration curve was calculated at different quercetin concentrations that included ten points between 0.001-0.01 mg mL^-1 (R²= 0.998).

For ANT, it was based on the differential pH method, according to ^{Wrolstad et al. (2005)}. Two buffer solutions were prepared, one with pH 1 of potassium chloride at 0.025 M and the second with pH 4.5 of sodium acetate at 0.4 M. In both solutions the pH was adjusted with concentrated HCl. 20 μL of the extract was taken and it was completed to 1.5 mL with the respective solution. The absorbance of each solution was recorded at 520 and 700 nm. The concentration of total anthocyanins was determined by the following equation:

Where: MW= molecular weight of cyanidin-3-glucoside (449.2 g mol^-1); DF= dilution factor (75) and Σ= molar extinction coefficient (26 900) and the value of A was obtained with the equation: A= (Abs 520-Abs 700) pH 1 (Abs 520-Abs 700) pH 4.5. The results were expressed in mg of total anthocyanins per hundred grams of fresh weight (mg/100 g PF).

The CA was performed by the DPPH radical method (1,1-diphenyl-2-picrylhydrazyl), according to the reports by ^{Brand et al. (1995)}. For this, 150 mM DPPH was dissolved in 80% methanol. An aliquot of 100 μL of the extract of each sample was taken and 900 μL of DPPH solution was added. The mixtures were incubated for 30 min and their absorbance was determined at 515 nm. The results were expressed as micromolar equivalent of trolox per gram of fresh weight (μmol ET/g PF). The calibration curve was calculated at different concentrations of trolox that included ten points between 1 to 1 500 μM mL^-1 (R²= 0.998).

Statistical analysis

The statistical analyzes were carried out with the statistical package Statgraphics Centurion Version XV.II. The mean values and their standard deviations were reported. One-way analysis of variance was performed, multiple means were compared using the Tukey test (p≤ 0.05).

Physicochemical proximal composition

The results of the physicochemical analyzes carried out on the wild species of the genus Rubus, show variations in the length of the fruit ranging from 1.7 to 2.23 cm (Table 2). On the other hand, it was recorded from 34 to 89 in the number of drupillas, being the species R. pringlei the least amount and Rubus adenotrichus who reported the largest number of durpillas. Regarding the relationship between length and number of drupillas, it was observed that the greater the length of the fruit, the lower the number of drupillas. The unit weight varied between 1.1 and 3.1 g, with R. pringlei being the lowest weight and R. glaucus Beth with the statistically highest value.

Average with different letters indicate significant differences between rows (p≤ 0.05).

Regarding the moisture content, it is highlight R. glaucus Beth, showing significant difference with respect to the other wild species. Although it is known that the high humidity in fruits and vegetables is a disadvantage because, as there is greater availability of free water, the shelf life is reduced, in the same way the concentration of sugars and therefore the sweetness of the product is reduced. As the wild to have lower percentages of moisture is expected a longer shelf life than the commercial variety Tupy.

The content of total soluble solids in the four species presented significant statistical differences; however, the highest values were observed for the wild species R. adenotrichus and R. pringlei with 12.4 ±0.19 and 10.7 ±0.10 °Brix, in contrast the Tupy variety reported 9.8 ±0.09 °Brix, while Rubus glaucus Beth obtained the lowest amount of soluble solids (7.6 ±0.1 °Brix) compared to the commercial variety.

The high content of total soluble solids is reflected in the sweetness of the fruit, characteristic qualified in fruits incorporated in the elaboration of wines and liquors (Coronel, 2008). Regarding the percentage of titratable acidity values of 0.8-1.1 were found, being the wild species those who showed statistically lower percentages of acidity, with respect to the commercial variety.

In the fruits, as the maturation progresses, the organic acids are breathed or converted into sugars, decreasing their content (Seymour et al., 1993), so that their flavor is not only affected by the sugar content, but also by the presence of volatile compounds and organic acids, being citric, malic and tartaric acid the most abundant in fruits of blackberries and raspberries, constituting citric acid between 30-95% with respect to total organic acids (^{Petkovsek et al., 2012}).

Some organic acids such as citric acid, have been widely incorporated in the food industry, because it acts as a regulator of acidity, provides firmness, enhances flavor, acts as a preservative and antioxidants, in addition to providing stability to lipids and antioxidants primary as phenolic acids (gallic and ferulic acid) and are capable of inactivating metal ions that act as pro-oxidants, such as iron and copper through the formation of chelates (^{Quitmann et al., 2014}).

The ratio of total soluble solids and titratable acidity of R. adenotrichus reported 14.76 ± 0.41, was included as the highest value in wild fruits of Rubus hirsutus Thunb with ratios ranging from 31.31-11.23. Although the relationship obtained in the Rubus glaucus and pringlei species were statistically lower, with respect to R. adenotrichus, they are within the reported ^{Lewers et al. (2010)} in 21 commercial varieties of blackberries and raspberries (4.8-11.9). ^{Lewers et al. (2010)} make reference to a more pleasant flavor for strawberry with a relation around 10, in turn this could be achieved with high to moderate levels of total soluble solids and low titratable acidity, as in the case of the wild species of the present work.

A ratio greater than 12.5 is considered a characteristic appreciated in a sweet taste for the consumer. On the other hand, ^{Vergara et al. (2016)} make reference that the industry of the United States of America prefers good-colored blackberries with a high content of soluble solids and acidity, as well as looking for low numbers of seeds and pH, while, for consumers, the qualities, as sweetness, acidity, astringency, color, firmness and absence of seeds are important for both processed and fresh fruits.

These characteristics make it possible to consider wild species as candidates for incorporation into agronomic management programs that allow increasing the size of the fruit, as well as cross-species processes, since it has benefited the obtaining of large fruits with a moderate number of seeds (^{Clark et al., 2011}).

Content of polyphenols, flavonoids, anthocyanins and antioxidant capacity

The registered values of total polyphenols for the materials studied, reported data ranging from 285.06 ±8.49 to 592.61 ±7.03 mg EAG/100 g PF. The wild species showed the highest values, being statistically different with respect to the commercial variety. The content of polyphenols found in this work exceeded that reported by ^{Wang and Lin. (2000)}, in commercial cultivars of blackberries (204- 248 mg EAG/100 g PF) and raspberries (208-267 mg EAG/100 g PF) and agree with that reported by Ramune et al. (2012) for 19 improved varieties of raspberries (278.6- 714.7 mg EAG /100 g PF). Found in the wild species Rubus hirsutus Thunb 108. 23-269. 9 mg EAG/100 g PF.

Regarding the flavonoid content (Figure 1), it ranged from 93.13 ±1.7 to 36.4 ± .4 mg EQ/100 g PF, with the lowest number of wild species. On the other hand, in the content of anthocyanins stood out the wild species with respect to the commercial variety, whose quantities fluctuated between 18.43 ±1.05 to 4.32 ±0.24 mg L^-1, being R. adenotrichus and R. glaucus who reported the greatest amount of anthocyanins, without showing significant difference between them. The values of anthocyanins found in this study surpass that reported for Rubus chamaemorus (^{Koponen et al., 2007}) and like that shown in Rubus adentrichus (^{Martínez et al., 2011}).

Figure 1 Concentration of flavonoids, total anthocyanins and antioxidant capacity (CA) in fruits of three wild species of the genus Rubus and the commercial Tupy variety (± standard deviation). Average with different letters indicate significant differences between rows (p≤ 0.05)  

Although there have been diverse studies that quantify polyphenolic compounds such as flavonoids and anthocyanins in Rubus species, it is known that the synthesis of polyphenols is given by factors such as the conditions of cultivation, species and phenolic stage of the plant. Likewise, its obtaining can be conditioned by the process and the type of solvent used (^{Valencia et al., 2013}). Therefore, the species analyzed in the present work may be suitable candidates for obtaining improved cultivars with high polyphenol content.

On the other hand, greater antioxidant potential was observed in wild species (65.7 ±2.31-25.15 ±2.65 μM ET/g PF) compared to the Tupy variety (15.35 ±1.01 μM ET/g PF), this agrees with that found in Rubus fructicosus and Rubus ideaous (25.3-35.5 μM ET/g PF) by ^{Paredes et al. (2010)}, as mentioned by ^{Cuevas et al. (2010)}, who reported that wild species of the genus Rubus have higher polyphenolic content and antioxidant potential than the commercial Tupy variety. These results demonstrate the importance of Mexican wild species as sources of polyphenols and their possible incorporation into antioxidant production programs with an industrial objective.

The antioxidant capacity of polyphenols is mainly related to structural characteristics, such as the presence of double bonds and O-diphenyl, hydroxyl or methoxy groups (^{Balasundram et al., 2006}). The absence or substitution of some of these structural characteristics reduces or inhibits the antioxidant capacity. ^{Skrovankova et al. (2015)} mentioned that the antioxidant capacity of the blackberries is influenced by the concentration of the extract. However, ^{Johnson et al. (2012)}, reported a close relationship between antioxidant activity and polyphenol content.

On the other hand, ^{Hassimotto et al. (2008)}, showed that the elimination of radicals is influenced by the total of specific compounds such as anthocyanins. In counterpart, ^{Silva et al. (2007)} showed that the relationship between the capacity and the number of polyphenols could be given by the technique implemented for the antioxidant evaluation of the compound.