Variation in Physical-Chemical Parameters and Phenolic Compounds in Fruits of Four Calafate Clones

Abstract

Calafate (Berberis microphylla G. Forst) is an evergreen shrub with blue berries that grows naturally in Patagonia, in South America. It has beneficial nutraceutical characteristics for human health. The objective of the research was to evaluate the effect of different harvest dates of calafate clones in the south-central zone of Chile on the polyphenolic content, antioxidant capacity, quality parameters and fruit yield. To meet this objective, during three consecutive years, four wild calafate clones located in the town of San Ignacio, Chile, were selected. Where a harvest period was established from 110 to 140 days after full bloom (DAFBs), each of the harvests carried out were used for the following measurements: antioxidant capacity, determination of anthocyanin content, concentration of polyphenols, phenolic compounds, soluble solids, total titratable acidity, pH, fruit yield and quality. Among the main results, it can be highlighted that clone 2 was the one that obtained the highest concentration of soluble solids, with 38.0 °Brix at 140 DAFBs. Together, it was the one that obtained the highest content of total polyphenols and concentration of anthocyanins, with 1121 g GAE kg⁻¹ fw and 714 g cy-3-glu 100 g⁻¹ fw, respectively.

1. Introduction

Within the native species of berries in Patagonia in South America (Chile and Argentina) that stand out for their high content of polyphenolic compounds and antioxidant activity, there is a wild species called “calafate” (Berberis microphylla G. Forst), a member of the Berberidaceae family, which grows naturally as an evergreen shrub and can reach a maximum height of 4 m [1]. In Chile, we can find it from the Maule Region to Tierra del Fuego, growing mainly on the margins of forests and also in thickets that form in the Magellanic Steppe [2]. It has been shown that the calafate fruit is a great source of anthocyanins that correspond to water-soluble pigments responsible for the blue coloration of the fruit. Highlighting its pharmacological properties, calafate has anti-inflammatory, antioxidant and chemoprotective effects [3]. It has been shown that an ethanolic extract of calafate root has hypoglycemic effects, capturing glucose in liver cells resistant and not resistant to insulin (hepG2) through activation of monophosphate-activated protein kinase (AMPK) [4]. It is important to point out that calafate is collected from bushes that grow in the wild, and is not only sought for fresh consumption but also for the production of various products, such as candies, jellies, pulps for the production of ice cream, alcoholic and non-alcoholic beverages, and also cosmetic products, thus generating a high demand for calafate fruit [5]. Additionally, there is a high unsatisfied demand from the pharmaceutical-homeopathic industry, since there are no commercial crops evaluated in production [1,6]. Given this, it is necessary to establish agronomic parameters and the selection of clones that may have great potential as a natural and healthy source of antioxidants for future intensive crops with high commercial value.

Currently, there is a great diversity of native fruit species, which have been developed commercially and which have outstanding levels of phenolic compounds and antioxidant capacity, such as: blackberries (Rubus spp.), blueberries (Vaccinium corymbosum L.), raspberries (Rubus idaeus L.) and strawberries (Fragaria x ananassa Duchesne ex Rozier), among others, for which values between 0.44 and 1.45 mg L⁻¹ of gallic acid have been observed for the total content of polyphenols [7]. However, in various studies, it has been shown that calafate has an antioxidant capacity 10 times greater than that of other fruit species, such as apples, oranges and pears, and 4 times greater than that of blueberries [8,9,10,11,12].

According to studies carried out on wild plants, calafate could be defined as a super berry, since it can significantly exceed the concentrations of phenolic compounds and anthocyanins of most cultivated berries, as well as other species native to Patagonia in South America, such as the murtilla (Ugni molinae) and the maqui (Aristotelia chilensis (Mol.) Stuntz.) [13]. One of the factors that influences the variations in the polyphenolic content and concentration of antioxidants is the evolution of these during the process of the ripening of fruit until the optimal moment of harvest, not clearly specifying the point that determines the optimization of both the polyphenolic and anthocyanin content, as well as the production and quality of the calafate fruit [14]. Within multiple studies, various levels of quantification of polyphenols and anthocyanins have been reported, both among species of the same genus and in other berries and fruit species [15]. This is corroborated in other native species of berries, such as maqui, where variations in the development of phenolic compounds and antioxidant activity have been reported during the fruit ripening process [16]. On the other hand, Rodarte-Castrejón et al. [11] determined, for blueberries, that the optimization of production together with the polyphenolic concentration is achieved when the fruit reaches 100% blue coverage. Contradictory results are reported in the research by Fredes et al. and Spinoza et al. [16,17], who point out that for the maqui, the optimization of the concentration of polyphenols and antioxidant capacity is achieved in stage three of maturation (dark blue color). On the other hand, in the case of blackberries, in addition to depending on the cultivar, they present a variety of colors during their maturation, which is due to the expression of anthocyanins depending on the pH of the culture medium. Showing its maximum color in acid media with red-orange tones, on the contrary, when cultivated in neutral media, blackberries have an intense red-violet color, and in alkaline media, red, purple and blue colors are manifested. Therefore, the optimal harvest time for blackberry cannot be assigned transversally to the different culture media, but rather, it must be associated with the pH of the soil in the place of establishment [18].

It is important to point out that there are no investigations on the dates or times of harvests that optimize the production, polyphenolic concentration and antioxidant capacity of the calafate fruit, which are necessary to establish a base line of the time of harvest for this species, in order to optimize: the content of phenolic compounds, the antioxidant capacity, the nutritional characteristics, productivity and quality of the fruit [16,19]. For this, it is hypothesized that the evolution of nutritional and phenolic parameters increase after fruit ripening (100% blue coloration) without significantly reducing its quality and yield parameters. It is for this reason that this research aimed to evaluate the effect of the different harvest dates of the calafate fruit in the south-central zone of Chile on the polyphenolic content, antioxidant capacity and quality and yield parameters of the fruit in order to establish a baseline for future research projects on calafate domestication in the central zone of Chile and subsequent implementation of commercial orchards. To respond to the objective set forth in this study, different selected clones from the central zone of Chile were used, and the physical-chemical, productive and quality parameters of the fruit were analyzed.

2. Materials and Methods

2.1. Plant Material, Environment and Extraction

During three consecutive seasons, surveys were carried out in the Ñuble region (south-central zone of Chile) to identify wild clones of Berberis mycrophylla G. Forst, as part of the project for the identification and domestication of calafate as a commercial alternative, financed by the Adventist University of Chile. From said prospecting, 4 calafate clones (36.85° S, 71.95° W) similar in size and productive ages (>10 years old) were selected and identified. Their taxonomic identification was carried out in the systemic botany laboratory in the Universidad de Concepción, Campus Chillán, Chile. The environmental characteristics of the locality under study were obtained from the Huemul meteorological station of the Agricultural Research Institute, Chile [20]. The data obtained corresponded to: accumulated rainfall, maximum and minimum air temperatures and accumulation of growing degree days (GDDs). The GDDs corresponded to the accumulation of the mean temperature above a base temperature (Tb, T > 10 °C) [21].

During the second season, at the beginning of flowering, the populations were individually identified and marked. Poor quality units with damage to twigs, buds, leaves and flowers were discarded. The calafate fruit samples were collected in isolation from 3 individuals of each group of clones, with only ripe fruit being collected, as determined by 100% dark blue coverage. Samples were collected at 10-day intervals, from 110 days after full bloom (DAFBs) to 140 DAFBs. The fruits were transferred into a container at 10 °C from the time of harvest, to be stored at −80 °C on the same day. It is important to point out that for each group of clones, three individuals were harvested in isolation (three replicates) for each of the established harvest dates. Next, in each of the repetitions, 100 random fruits were considered to quantify: equatorial diameter, soluble solids (SSs), average fruit weight, carbohydrates (%) and pH and acidity (%) [22]. The same sample previously selected was used for the chemical analyses of the fruit in each of the repetitions and harvest dates.

2.2. Determination of the Capacity and Nutraceutical Content of the Fruit

The DDPH antioxidant capacity was determined through the decolorization of the 1.1-Diphenyl-2-picrylhydrazyl free radical, according to methodology of Pinto-Morales et al. [23]. The antioxidant capacity was expressed in μmol Trolox equivalent (TE) 100 g⁻¹ fresh weight.

Total anthocyanins were determined by a differential pH technique, according to the methodology described by Pinto-Morales et al. [23], and data are expressed as mg of cyanidin 100 g⁻¹ of fresh weight.

Total polyphenols were determined by colorimetry using the method of Folin–Ciocalteu according to the methodology described by López et al. [24]. The results are expressed in mg of gallic acid 100 g⁻¹. All analyses and their procedures, including those for DDPH, anthocyanins and total polyphenols, were performed in the food chemistry laboratory at the Universidad of Concepcion, Chillán, Chile.

2.3. Yield, Physical-Chemical Parameters and Fruit Quality

For each harvest, the total weight (g) of fruits per plant were counted, of which 100 fruits were randomly selected for each treatment and each repetition, and for this a Precisa precision balance was used (Precisa instruments AG, Dietikon, Switzerland). To measure the equatorial diameter (D, mm), a digital foot meter was used, with ± 0.03 mm precision (Electronic Digital Calipter, Atraco, Inc., Washington, DC, USA). These same fruits were used to measure soluble solids (°Brix) using a refractometer (PCE–0.32 holding instruments., Germany). The harvest of fruits was carried out when the fruit reached 100% dark blue coverage [23].

The pH was determined with an Inolab PH7110 digital pH meter (Xylem Analytics Germany GmbH, Weilheim, Germany). The titratable acidity was determined by manual titration, where a 0.1 N NaOH solution was used. The results are expressed as the percentage of acidity (g of citric acid 100 g⁻¹) (Equation (1)).

$$\mathrm{acidity} (\%)=\frac{\mathrm{vol} \mathrm{de} \mathrm{NaOH} \times \mathrm{concentration} \mathrm{NaOH} \times 0.064 \left(\mathrm{meq} \mathrm{citric} \mathrm{acid}\right)\times 100}{\mathrm{Weight} \left(\mathrm{g} \mathrm{sample}\right)}$$

(1)

2.4. Experimental Design and Statistical Analysis

The statistical analysis was carried out using general and mixed linear models, using the INFOSTAT (Infostat, Cordoba, Argentina) version 2018 software [25]. Differences between means were determined using Fischer’s comparison test (p < 0.05). The experimental design used was a completely randomized design, with three replications, with 3 sub-samples from each experimental unit.

3. Results

3.1. Environmental Descriptions

In Figure 1a, it can be seen that the precipitation was mainly distributed between 20 and 140 DAFBs, with the highest rainfall being between 20 and 80 DAFBs, close to 200 mm. A total accumulated precipitation of 321 mm was reached in the study season, from full bloom to the end of harvest (140 DAFBs). Additionally, in Figure 1a, the accumulation of degree days during the development of calafate fruit can be observed, highlighting that the number of GDDs necessary from full bloom to the start of harvest (110 DAFB) was 215 GDDs. Furthermore, it can be observed that to complete the harvest (140 DAFB), 418 GDDs were necessary (Figure 1a). The maximum temperatures (Tmax) recorded fluctuated between 8 and 36 °C (Figure 1b), and the average Tmax of the fruit development season was 20 °C. It should be noted that the minimum temperatures (Tmin) recorded fluctuated between −3 and 11 °C, with an average Tmin close to 6 °C (Figure 1b).

3.2. Fruit Quality and Weight Parameters

Figure 2a shows the total fresh weight of ten calafate fruits for each of the four clones and harvests carried out, highlighting that for the harvest carried out at 110 DAFBs, clone 4 obtained the highest average weight of the fruits, being significantly higher than that of the rest of the clones for that harvest date, with an average of 2.7 g. The average weights of the 10 fruits of clones 1, 2 and 3 were 1.4, 2.3 and 1.6 g, respectively (p > 0.05). At 120 DAFBs, the weights of the fruits of clone 4 were also significantly higher (2.3 g; p < 0.05) than those of clones 1, 2 and 3 at 1.1, 1.8 and 1.7 g, respectively, with clones 2 and 3 having higher weights than clone 1 (p > 0.05). Regarding the harvest date 130 DAFBs, there were no significant differences in the weight of the 10 fruits of the different clones, with clones 2, 3 and 4 (p > 0.05), with values of 1.5, 1.5 and 1.5 g having higher weights than clone 1 at 1.1 g (p < 0.05).

At 140 DAFBs, the weight of the fruit was similar between clones 1, 2 and 3 (p > 0.05). It should be noted that values of fruit weight were not registered for clone 4, since the fruit was collected in its totality at 130 DAFBs. We can point out that as the harvest date progressed, the weight of the calafate fruits decreased by up to 100%, as happened to clone 1 at 140 DAFBs (Figure 2a). Additionally, in Figure 2a, it can be observed that, when analyzing the behavior of each clone individually for the weight of the fruit for the different harvest dates, the trend was similar in all clones evaluated, showing a decrease in the weight of the fruit from 110 DAFBs to 140 DAFBs. It was clone 3 that obtained lower weight loss at 25%. Clones 1, 2 and 4 had a greater average weight decrease than clone 3, with 57, 56 and 45% decreases in fruit weight (Figure 1a), respectively. It should be noted that, despite having a high % of weight loss, clone 4 obtained the highest average weight of the 10 fruits for all harvests.

Regarding the equatorial diameter of the fruit (Df), in Figure 2b, it can be seen that at 110 DAFBs, clones 2 and 4 had significantly larger diameters with a Df of 6.3 and 6.5 mm, respectively, than clones 1 and 3, with a Df of 5.3 and 5.3 mm, respectively. At 120 DAFBs, the Df of clone 4 was significantly higher, at 6.7 mm, compared to those of clones 2 and 3, which were equal to each other (p > 0.05), at 5.9 and 5.5 mm Dfs, respectively (Figure 2b). It was clone 1 that obtained the lowest Df at 120 DAFBs, with 4.9 mm. With respect to 130 and 140 DAFBs, no significant differences were observed between the equatorial diameters of the fruit among the different clones, with average Df values close to 5.0 mm being observed for the different harvest dates (Figure 2b). It should be noted that, as with the average weight of the fruit, clone 4 was the one which obtained the highest average equatorial diameter, at 6.1 mm, compared to 5.0, 5.6 and 5.4 mm for clones 1, 2 and 3, respectively.

Figure 2c shows us the concentration of soluble solids (SSs), where it can be observed that as the fruit ripened from 110 DAFBs, the concentration of soluble solids in the fruit increased up to 140 DAFBs. Clone 2 was the one that obtained the highest concentration of soluble solids towards the end of the study, reaching 38.0 °Brix (p < 0.05). Regarding 110 DAFBs, clones 1 and 2 reached 15 °Brix (p > 0.05), with both achieving a higher value than clones 3 and 4, whose values were 1.6 and 2.7 °Brix (p < 0.05). At 120 DAFBs, the highest concentrations of soluble solids were recorded for clones 1, 2 and 3, with values close to 23 °Brix (p > 0.05), with clone 4 registering the lowest value at 17 °Brix (p < 0.05). At 130 DAFBs, clone 2 had a significantly higher value than the rest of the clones with a value of 37.5 °Brix (p < 0.05), followed by clones 3, 1 and 4, whose registered values were 26.8, 22.3 and 18.2 °Brix (p < 0.05). At 140 DAFBs, clone 2 was the one for which the highest concentration of soluble solids was recorded, at 38.0 °Brix (p < 0.05), followed by clones 3 and 1, whose values were 19.7 and 16.0 °Brix (p < 0.05) (Figure 2c). It should be noted that no data were recorded for clone 4 at 140 DAFBs, since the fruit was fully harvested at 130 DAFB.

Figure 3a shows the proportion of total sugar (AT) in the calafate fruit for the different harvest dates. It can be observed that at 110 DAFBs, the AT values were similar to each other for the different clones evaluated (p > 0.05). This behavior registered variations at 120 DAFBs, at which point the fruit of clone 3 was significantly higher in AT (Figure 3a) at 4.4% compared to clones 1 < 2 = 4, with average values of 2.4, 3.3 and 3.4% total sugar (p < 0.05). This trend was reversed for 130 and 140 DAFBs, with clone 2 having the highest concentration of AT in the fruit at 11.6 and 2.7% for 130 and 140 DAFBs, respectively. It is important to point out that, on average, for the different harvest dates, clone 2 was the one that obtained the highest average concentration of TA, with an average of 4.9% (Figure 3a).

On the other hand, Figure 3b shows the pH of the fruit for the different harvest dates, not observing significant differences between clones 1, 2, 3 and 4 at 110 and 140 DAFBs, whose values fluctuated between 3.2 and 3.7 (Figure 3b). It should be noted that clone 4 was the one for which recorded the maximum pH value of 3.7 (p < 0.05) at 120 DAFBs was recorded, and clone 2 was the one for which the maximum pH value of 3.6 (p < 0.05) at 130 DAFBs (Figure 3b) was recorded.

In Figure 3c, the total acidity (%) of the fruit can be observed, showing erratic variations and no well-defined trends among the clones. However, clone 4 was the one that showed a downward trend in AT (Figure 3c) as the DAFBs advanced (Figure 3c). Regarding the recorded AT values, these fluctuated between 1.0 and 1.6% (Figure 3c). It should be noted that there was no evidence of differences in the average TA of the fruit between the different clones evaluated, with TA values fluctuating between 1.2 and 1.3%.

3.3. Evolution of Polyphenolic Content, Anthocyanin Concentration and Antioxidant Capacity of the Fruit

Figure 4 shows the concentration of total polyphenols in the fruit of different clones. In general, it can be observed that as the different harvest dates progressed, the concentration of total polyphenols (TPC) remained relatively stable in all the clones evaluated from 110 to 130 DAFBs. At 140 DAFBs, the TPC increased in all the clones evaluated. At 110 DAFBs, clone 2 had a significantly higher value than the rest of the clones evaluated (Figure 4) with 898 g GAE kg⁻¹ fw. Clones 1 and 3 had similar values (p < 0.05) at 769 and 797 g GAE kg⁻¹ fw, respectively. It was clone 4 that obtained a lower TPC at 110 DAFBs, with 705 g GAE kg⁻¹ fw. It should be noted that at 120 DAFBs, clones 1, 2 and 4 were similar to each other (p > 0.05), but had significantly higher values than clone 3, with polyphenolic contents of 779, 741, 788 and 619 g GAE kg⁻¹ fw, respectively. At 130 DAFBs, clones 1, 2, 3 and 4 were equal to each other (p > 0.05), with polyphenol concentration values of 820, 923, 963 and 882 g GAE kg⁻¹ fw, respectively (Figure 4). At 140 DAFBs, no results were obtained for clone 4, since the fruit of the plant fully ripened at 130 DAFBs; however, clone 2 was significantly superior to clones 3 and 1 (p > 0.05), with polyphenol concentration values of 1923, 1697 and 1132 g GAE kg⁻¹ fw, respectively (Figure 4). It is important to point out that the highest average value of the total content of polyphenols was obtained for clone 2, with an average value of 1121 g GAE kg⁻¹ fw, and the highest TPCs were observed for clones 1, 3 and 4 at the concentrations of 875, 1019 and 792 g GAE kg⁻¹ fw, respectively.

On the other hand, in Figure 5, it is possible to observe the variation in the concentration of total anthocyanins (TAC) during the different harvest dates for the different clones evaluated. At 110 DAFBs, it can be seen that the highest TAC corresponded to clone 4 with 394 mg cy-3-glu 100 g⁻¹ fw; this was significantly higher than for clones 1, 2 and 3, with values of 297, 346 and 316 g cy-3-glu 100 g⁻¹ fw (p > 0.05), respectively. A similar trend was observed at 120 DAFBs, where clone 4, at 601 mg cy-3-glu 100 g⁻¹ fw, also had a significantly higher value than the rest of the clones, and the TAC values observed in clone 1 = 2 > 3 were 532, 481 and 339 mg cy-3-glu 100 g⁻¹ fw. It should be noted that clone 2 at 130 DAFBs was the one that obtained the highest TAC (p < 0.05), with a value of 709 mg cy-3-glu 100 g⁻¹ fw, compared to clones 3 and 4, whose values of TAC were 646 and 609 mg cy-3-glu 100 g⁻¹ fw, respectively. It was clone 1 which had the lowest TAC value at 130 DAFBs, with a value of 449 mg cy-3-glu 100 g⁻¹ fw. Therefore, at 140 DAFBs, clone 2 was the one that obtained the highest TAC value with 1321 mg cy-3-glu 100 g⁻¹ fw, followed by clone 3, with a value of 1105 mg cy-3-glu 100 g⁻¹ fw (p < 0.05). Finally, at 140 DAFBs, clone 1 had the lowest TAC (p < 0.05) at 476 mg cy-3-glu 100 g⁻¹ fw. It is important to point out that the highest average value of TAC among all the harvests of the different clones evaluated corresponded to clone 2, with a TAC of 714 mg cy-3-glu 100 g⁻¹ fw, followed by clones 3, 4 and 1, with average TAC values of 601, 535 and 439 mg cy-3-glu 100 g⁻¹ fw, respectively.

Finally, Figure 6 shows the antioxidant capacity (AC) expressed as a percentage of inhibition, highlighting that at 110 and 130 DAFBs, all the clones evaluated registered a % inhibition close to 94 and 90%, respectively, with no significant differences between them for any harvest date. A similar trend was observed at 120 DAFBs for clones 2, 3 and 4 (p > 0.05), whose values corresponded to 90, 88 and 87%, respectively, with clone 1 having a significantly lower inhibition value of 82%. The aforementioned trends were not observed at 140 DAFBs, highlighting that only clone 1 was significantly superior to the rest of the clones, with an inhibition value of 85%, and clones 2 and 3 only reached an inhibition of 72 and 71%, respectively.

4. Discussion

According to the environmental results observed in the Ñuble region, in general the climatic conditions of the study area vary considerably with respect to the conditions of predominant wild development in Patagonia. This factor is relevant, since it affects the vegetative development of the calafate. In particular, the annual accumulated precipitation (PP) in the study site was 472 mm, well above the precipitation recorded in the same year in the locality of Punta Arenas, which reached 144 mm of annual accumulated precipitation [20]. This would be relevant, since according to Arena and Curvetto [5], wild calafates require an annual accumulated precipitation between 295 and 324 mm. This factor could be affecting the vegetative and productive development of the species as a result of the lower availability of the annual water resource; therefore, the domestication of this species in the Ñuble region is proposed as a viable alternative from the point of view of water resources (Figure 1a). These results could indicate that the optimal environmental conditions for the development of calafate in Patagonia could be affected by the effects of climate change [26]; furthermore, from the point of view of PP, the Ñuble region has characteristics beneficial for the development and domestication of this species.

It should be noted that the accumulation of thermal weather in the study locality reached 321 GDDs (Figure 1a) with average temperatures above 10 °C, quite the opposite of those observed at the INIA Punta Arenas meteorological station [20], where it reached 31 GDDs (base 10 °C); therefore, the clones evaluated in this study should start their vegetative development before the clones from Patagonia, as they are even able to reach higher productivity due to environmental conditions such as temperature (Figure 1b), which was less extreme throughout the development of the crop [27]. This greater accumulation of thermal time is mainly due to the average temperatures recorded in the town of San Ignacio (13.0 °C; Figure 1b), which had an average T° higher than 5 °C above the average recorded in Patagonia (8.1 °C) [28], with the mean maximum and minimum temperatures for San Ignacio being 20 and 6 °C, respectively. This is well below what was recorded in Patagonia for the same year, where temperatures of 11 and 3 °C were recorded for the maximum and minimum annual average temperature (Figure 1b), respectively.

On the other hand, regarding the average weight of the 10 fruits in this study (Figure 2a), clone 4 at 110 DAFBs was superior to the rest of the clones, with an average weight of 2.7 g, and the minimum weight was 1.1 g. These results are lower than for other species of Berberis spp., where for B. mycrophilla, the fruit weight was at least 200% lower than that of Berberis heterophylla Juss., with an average fruit weight between 0.91 and 2.26 g [29]. On the other hand, in blueberries, the highest weight obtained per fruit, according to the research carried out by Rodríguez and Morales [8], was 1.94 g, which also shows a lower average weight of the fruit of B. microphylla compared to other species of berries. In addition to the genetic variations in each species in the potential size of the fruit, there are variations in the biochemical processes of fruit maturation, such as the rates of photosynthesis and water accumulation, which could favor high differentials in the final size of the fruit among the aforementioned species [30].

Regarding the equatorial diameter of the fruits evaluated, it was observed that clone 4 was the one with the largest average diameter at a value of 6.7 mm. This result correlates with the average weight of the fruit, since higher average weights correspond to larger equatorial diameters (Figure 2a,b). According to the study carried out by Romero et al. [31], which consisted of comparing different wild berries that are distributed in different areas of Chile, calafate fruits from the Aysén region had a maximum fruit diameter of 10 mm. On the other hand, Dalzotto et al. [32], in the province of Río Negro, made various measurements of calafate, and the resulting equatorial diameter of their samples was between 7 and 11 mm. Therefore, although the average weights obtained were lower in this study, the equatorial diameters were similar to those found in the aforementioned studies. It should be noted that the size of the fruit could be expected to be higher in our study compared to fruit from the aforementioned locations, mainly due to a higher GDC. However, what is indicated in the previous paragraph may have been generated by higher total plant growth and higher total fruit yield.

Figure 3a shows the total content of soluble solids (SSs) in the different clones evaluated, where the highest concentration of SSs was observed at 130 DAFBs, with clone 2 reaching the highest average SS concentration, with a value of 28.4 °Brix. Furthermore, as the DAFBs advanced, the color and the SS content advanced hand in hand; this is due to the fact that during the ripening of the fruit, there is a decrease in the content of organic acids due to respiration and an increase in sugar coming from the synthesis and sap transfer [33]. These parameters are also affected by environmental factors, mainly temperature, solar radiation, rain, shading and nitrogen levels in the soil, all of which can alter the final evolution of the fruit [34]. Similar results were observed by Arenas and Curvetto [5] and Cáceres et al. [35], whose studies showed that the concentrations were 24.8 and 26.8 °Brix, respectively. The case of other wild berries found in Chile is different, such as the murtilla (Ugni molinae), which has an average value of 13.1 °Brix, and the white strawberry at 11.6 °Brix [31]. With regard to the concentration of total sugar, clone 2 surpassed the others with an average of 4.9% [36]. In blueberries, the percentage of sugar content ranges between 10 and 14% [37]. In maqui, the values of total sugar concentration are around 41.2 g 100 g⁻¹ fw [38]. The content of sugars is influenced by nutrients, plant growth regulators and physical factors that affect transport, metabolism, accumulation and the relationship between them. Additionally, maintaining a higher content of sugar in the pulp is a base indicator of maturity and taste quality; it has been described that these contribute to the quality of the fruit, in relation to weight, firmness, color and flavor, in calafate [1]. It should be noted that both the concentration of soluble solids and total sugars increase once the fruit is harvested as a product of dehydration; this is due to the degradation of polysaccharides in cell membranes, which contributes to the increase in sugars [39], but as the fruit ripens, and as a result of its respiration, they begin to lose weight as a consequence of the consumption of these sugars, which was observed in our SS and AT results (Figure 2c and Figure 3a) [40].

The study by Arena [41] demonstrated that the pH can be a parameter with variable results due to the adaptability of the plant to changes such as light intensity and the level of fertilization, where the results were 2.78 and 2.82 for medium and high light intensity. Cáceres observed [35] that in a commercial orchard trial, the average pH was 3.42, maintaining a close similarity with the results of this research, where the average pH of wild clones was 3.4.

It is important to highlight that regarding the total acidity, no significant differences were found between the clones, with the averages of these being 1.2 and 1.3%, and very similar values were found for the fruit of Untusha (Berberis lobbiana), which, in the mature state, revealed around 1.56% of total titratable acidity [42], and in a study carried out in calafate by Sztarker, it had the same value for total acidity as the Untusha fruit with values between 1.52% and 1.56% [43]. In blueberries, the total acidity is lower compared to the fruits mentioned above with a value of 0.89% to 1.2% [44]. On the other hand, the camarosa variety of strawberry also has a lower total acidity value compared to the Berberideceae family, but very is similar to the blueberry, with 0.8% total acidity [9].

In this investigation, there was evidence that the highest average polyphenolic content was for clone 2, at 1121 mg GAE 100 g⁻¹ fw. These results coincide with previous studies, where Pinto-Morales et al. [23] observed that in the commercial cultivation of calafate under different styles of organic fertilization management, the average value of polyphenolic content was 1100 mg GAE 100 g⁻¹ fw. On the other hand, Speisky et al. [7] reported the polyphenolic content of 27 species in Chile, and indicated that the average content for calafate was 1201 mg GAE 100 g⁻¹ fw, which agrees with our results (Figure 4), but found lower polyphenolic contents for murta, at 863 mg GAE 100 g⁻¹ fw, blackberry at 671 mg GAE 100 g⁻¹ fw, and blueberry, at 529 mg GAE 100 g⁻¹ fw. On the other hand, in studies where three types of wild fruits of the genus Rubus sp., which have nutraceutical benefits such as calafate, were compared, average values of polyphenolic content of 285 to 592 mg of GAE 100 g⁻¹ fw were observed [10], showing that the calafate fruit has higher concentrations of total polyphenols than other species.

During the maturation of the calafate fruit, the highest concentration of total anthocyanins was reached at 140 DAFBs in clone 2 with 1321 g cy-3-glu 100 g⁻¹ fw. Comparable data were found in the study by Arena and Curvetto [5] carried out in the city of Ushuaia, where at 126 DAFBs, the concentration of anthocyanins reached a value of 800 mg cy-3-glu 100 g⁻¹ fw. Additionally, in our study, at 120 DAFBs, the highest concentration corresponded to clone 4 at 601 g cy-3-glu 100 g⁻¹ fw. The variability between the evaluated clones could be explained by what was suggested by Quin et al. [45], who point out that those clones with higher anthocyanins values could be more sensitive to climate changes. Radice et al. [27], in the city of Moreno, Argentina, worked with plants brought from Patagonia in chemical studies, and the concentration of anthocyanins was 118 mg cy-3-glu 100 g⁻¹ fw, which was significant different to values found in this investigation [46]. These differences between the two environments could be due to the lower temperatures of the city of Moreno, Argentina. It should be noted that this research was not designed to track the seasonal variation in the chemical properties of B. microphylla fruit, since the evaluations were only carried out from the beginning of ripening and in three subsequent events (four points in time). As noted above, conclusions could only be based on this time period. Additionally, as evidenced in Figure 4, Figure 5 and Figure 6, the four harvest moments evaluated showed significant differences in the chemical profile and biological activity of the calafate extract [10,47]. It is important to point out that there are studies that indicate that most genes related to anthocyanin synthesis are regulated by environmental factors, which would explain why, under the environmental conditions of this study of high radiation and temperatures, they would be regulating the expression of key genes in the synthesis of anthocyanins, as it is pointed out in studies of photoinduced anthocynin synthesis in Vitis vinifera L., where there was less expression of genes promoting anthocyanin synthesis with low temperatures, and with high temperatures and low radiation [48,49].

In relation to the antioxidant capacity, the highest value was obtained by clone 1 with an average 85% inhibition, surpassing the other clones in all the harvests carried out. These concentrations found go hand in hand with the high presence of polyphenols present in calafate, significantly surpassing other fruits, such as blueberries, where the antioxidant capacity expressed as the percentage of inhibition was 13.89%, 19.82% in elderberry (Sambucus nigra), 25.6% in maqui and 34% in cassis (Ribes nigrum) in the research carried out by Busso et al. [50]. Arenas et al. [51], in 2017, carried out a study where they analyzed the behavior of calafate in different light irradiances where the antioxidant capacity obtained ranged from 56% to 66.8% under the high-light treatment, finding lower values than in this study (Figure 6). It is evidenced in other studies that the antioxidant activity depends on the type and total content of polyphenols [23,45,48]. Like other species, calafate has a high proportion of total anthocyanins (Figure 5), which explains why the calafate fruits with the highest antioxidant activity (Figure 6) corresponded to the fruits with the highest concentration of total anthocyanins (Figure 6).

5. Conclusions

According to the results obtained in this study, for the different wild calafate clones evaluated in the commune of San Ignacio, Ñuble Region, Chile, the duration of the period from flowering to harvest fluctuated between 110 and 140 DAFBs. To complete this period of vegetative development, the accumulation of thermal time between 215 and 418 GDCs was necessary.

Clone 4 was the one that obtained the highest fruit size and weight at 110 DAFBs (beginning of harvest), but its highest content of total polyphenols and antioxidant capacity was achieved at 120 DAFBs, without a significant decrease in the size and weight of the fruit.

Among the different clones evaluated, clone 2 was the one that obtained the highest concentration of soluble solids, at 140 DAFBs. Furthermore, it was the one which obtained the highest content of total polyphenols and concentration of anthocyanins, with 1121 mg GAE kg⁻¹ fw and 714 g cy-3-glu 100 g⁻¹ fw, respectively. However, the antioxidant capacity dropped by more than 25% at 140 DDDFs. Therefore, we may conclude that the harvest date that best optimizes the antioxidant capacity, together with the polyphenolic content and concentration of anthocyanins, is at 120 DAFB.