Functional and Rheological Properties of Piñuela (Bromelia karatas) in Two Ripening Stages.

2.1 Sample preparation

The fruit of Piñuela in Ambalema-Tolima-Colombia municipality (4°49’11’’ N, 74°48’30’’ W) in the central-western Colombian zone was collected. The juice, after washing, peeling and shredding the fruit, was obtained. Then, it was filtered and centrifuged at 1,118 g for 15 min (Hettich centrifuge EBA20). The supernatant was recover and used immediately. To determine the rheological behaviour of the samples, juices filtrates were concentrated by evaporation (70 °C, until 5.3 to 65.0 °Brix). Then, they were lyophilized and stored (4 °C). The samples with distilled water for analysis were reconstituted.

2.2 Physicochemical properties of juice

Titratable acidity [10], pH (Schott Instruments, Handylab pH 11, Germany) and soluble solids, e. g. °Brix (Atago PAL- α model, USA) were determined in the juice. Quantification of reduction and total carbohydrates were carried out according to the methodology proposed by Loewus [11] and Norris and Ribbons [12] using a spectrophotometer (Thermo Fisher Scientific Inc., Helios gamma, Waltham, USA) with quartz cell one centimeter wide. Finally, the pectin content was determined by the gravimetric method [13].

2.3 Total phenolic content and total flavonoids

The total phenolic content was measured using the Folin Ciocalteau (FC) method, proposed by Dastmalchi et al. [14]. The total flavonoid content was assessed using two complementary colorimetric methods: quantification of flavones and flavonols with aluminium chloride [15], and evaluation of flavanones and flavanonols by 2,4- dinitrophenylhydrazine reagent [16].

2.4 Antioxidant activity

2.5 Polyphenol oxidase activity (PPO)

The PPO activity in juices was measured using the method described by Falguera [18] with some modifications. The increase of the absorbance was measured at 420 nm for 60 min with 10 min intervals using catechol as substrate (prepared in citrate buffer at 50 mM, pH 4). 1 mL of juice was mixed with the catechol solution (2.5 mL). One PPO unit was defined as the amount of enzyme responsible for the increase of one unit of absorbance at 420 nm in 10 min.

To find the best activity conditions of the PPO enzyme, pH scan was performed using catechol in citrate buffer (pH = 4.0, 5.0, 6.0) and phosphate buffer (pH = 7.0 and 8.0); then, with the pH of highest activity, temperature scan (10–60 °C) was performed with 10 °C intervals.

2.6 Rheological behaviour

Viscosity was assessed at 20 °C using a Haake RS-80 RheoStress Rheometer (Gebrüder Haake GmbH, Karlsruhe, Germany) with a Z40-DIN concentric-cylinder sensor system (radii ratio 1.0847) [19]. For temperature control, a Thermo Haake C25P bath (Gebrüder Haake GmbH) was used, using a glycol–water solution (50 % w⁄w) as coolant fluid, which allows an interval variation of ± 0.2 °C. The samples were sheared at a constant shear rate of 400 s^–1 for 3 min, after which a downward ramp to 0 s^–1 and another upward ramp until 400 s^–1 were accomplished. The average shear stress values of these two ramps were used to calculate the juice viscosity and to build the corresponding rheogram, which gives information about the flow-behavior of the different samples [20]; eqs (2) to (7).

Where, σ shear stress (Pa), σ₀ yield stress (Pa), γ˙ shear rate (s^–1), η viscosity (Pa•s), K consistency index (Pa•sⁿ), n flow behavior index (dimensionless).

2.7 Statistical analysis

The tests were performed with three replicates, reported as the average of three determinations, standard deviation (SD). On data, a check for homogeneity of variances was carried out, whereas one-way analysis of variance (ANOVA) and multiple comparisons Fisher’s LSD post hoc tests were then applied to identify noted differences among ripening stages. A P value of less than 0.05 was considered statistically significant. In order to determine the level of association between the assessed parameters, a principal components analysis (PCA) using the statistical program InfoStat/Professional® version 1.2 was used.

3.1 Physicochemical parameters and activity of polyphenol oxidase

The results of the physicochemical characterization of piñuela fruit juices are shown in Table 1. These values reveal significant changes in the piñuela that may be associated with fruit ripening process. These changes are: (a) decrease of acidic organic substances in cells reflected in changes of pH from unripe to ripe state; (b) the greater buffering capacity of intracellular components, evidenced by the higher free acidity (titratable) of the ripe fruit; (c) loss of texture and increased permeability of the fruit due to the fact that the content of soluble solids is three times higher in the ripe state than in unripe state. Similarly, it is possible to notice that the change from unripe to ripe causes the oxidative degradation of reserve materials such as starch, resulting in the formation of reducing and non-reducing simple sugars, with the consequent increase of sweetness and astringency in the plant product.

The piñuela maturation process could also be accompanied with an increase of the soluble pectin fraction because of the rupture of the interpolymer cross-links [21–23] and with an increase in the activity of the Polyphenol Oxidase (PPO). However, the statistical analysis did not show significant difference (p = 0.4084) between unripe and ripe state (20 °C and natural fruit pH).

It is important to recall that the fruit ripening requires significant amounts of energy to perform inherent physiological processes in that state [23], resulting in the increase of respiratory mechanisms. Energy generation through respiration in turn produces free radicals, but those are counteracted by the active cell antioxidant defence mechanisms that include phenolic compounds production. Therefore, the relationship between enzyme activity and phenolic compounds is clearly understood, as these specific compounds are the specific substrates of the enzyme (PPO) [24]. Table 1 reveals that the enzyme activity is ten times higher in ripe than in unripe fruit.

A principal component analysis applied to the above referenced variables, revealed that PPO activity in piñuela has a close correlation with pH (r = 0.94), titratable acidity (r = 0.97), flavones/flavonols system (r = 0.97) and total carbohydrates (r = 0.94) but in a lesser extent with soluble solids (r = 0.86) and total phenols (r = 0.81). Studies on apple [25], banana [26] and avocado [27] have shown the influence of environmental conditions in the enzymatic browning process, which is closely linked to the PPO functionality.

A PPO activity scanning in piñuela juices over time (Figure 1), clearly shows that the maximal enzyme activity is reached after 60 min in the ripe fruit, undergoing dramatic changes in enzyme activity before and after that time (more in ripe than in unripe fruit). Later, the activity decreases in both states, perhaps due to exhaustion of the substrate as other authors have found [28, 29]. Meanwhile, it can be seen in Figure 2 that changes between pH 5 and 8 on ripe fruit are not significant; however, for unripe state there is a steady increase as the pH rises to a maximum activity peak at pH 8.

Figure 1:

Polyphenol oxidase activity of piñuela juices in natural conditions at 20°C.

Figure 2:

Polyphenol oxidase activity of piñuela juices in natural conditions at 20 °C and pH between 4 and 8.

The results obtained in this study demonstrate that the enzyme, either in unripe or ripe fruit, works at its full capacity at 20 °C; being more critical in unripe, rather than in ripe state (Figure 3). Assuming that at 20 °C the maximum enzyme activity (100 %) is achieved, it appears that at a temperature of 30 °C, an inhibition of more than 89 % of the enzymatic activity in the ripe state can be reached, while in the unripe state inhibition is around 93 % [30].

Figure 3:

Polyphenol oxidase activity in piñuela juices at a temperature range from 10 to 60 °C.

Temperature is a parameter of careful observation in all processing operations and food preservation. In the control of heat treatments, it is essential in reducing and inactivating the enzyme activity [25, 31–33].

Results showed that the PPO of B. karatas appears to be activated between 20 and 80 min as maximum, it is relatively tolerant to acid pH and temperatures oscillating amongst 10 and 20 °C. From the results also, pH and temperature greatly influence when the fruits are immature, and decline as they hit peak ripeness.

3.2 Antioxidant capacity

In a previous study in GIPRONUT (Research Group of Natural Products), phenolic compounds such as coumarins, flavonoids and phenylpropanoids were identified [34]. Thus, in this work it was found that total phenolic compounds increased 40 % during fruit development, which could be interpreted as an indicator parameter of the physiological processes. That occurs during fruit ripening or as evidence that these compounds are required for protection of the nutrients against the attack of pathogens and predators [35–37], perhaps to protect the cells against oxidative stress caused by the ripening process or because of both processes.

Table 2 shows that flavonoids are a representative group within phenolic compounds in piñuela, especially those of flavone and flavonol type (0.7 mg/100 g in the unripe tissue and 20 mg/100 g in ripe tissue). It can also be noted that during the transition from unripe to ripe, the percentage of flavanones decreases (relative to the total flavonoids) by 9 %, while flavones increase by 9.4 %, which could be explained by the fact that flavanones are considered precursors in flavones biosynthesis [38]. Based on the above, there were significant differences (P < 0.0001) in flavones/flavonols content between the two stages of maturity, occurring the opposite with flavanone/flavanonol type (P = 0.9373).

Likewise, Table 2 reveals the results from in vitro antioxidant activity exhibited by fruit juices. The antioxidant activity of ripe fruit expressed as superoxide inhibition percentage was significantly higher (P < 0.0001) than the juice from unripe fruits; however, it did not exceed the control activity (ascorbic acid) at any conditions, which is a renowned exogenous antioxidant used in industry [39]. The activity of the Superoxide Dismutase (SOD) enzyme (first line cellular defence against oxidative stress), showed a high capacity to stabilize the superoxide anion generated by xanthine oxidase during the reaction [40] in both maturation states without statistical difference (P = 0.2651).

In order to find the changes in antioxidant activity through maturation, a separate principal component analysis for each state was performed. It was demonstrated that in ripe stage the superoxide anion inhibition appears to be greatly associated to flavanones/flavanonols system (r = 0.99); two types of flavonoids that also act as vascular pigments contributing with the whitish colour of piñuela fruits [41]. For unripe fruit, the same superoxide anion inhibitory activity but with total phenols (r = 0.96) was observed.

In turn, independently whether the fruits are ripe (r = 0.78) or unripe (r = 0.80), the SOD activity displayed moderate association with flavanones/flavanonols system for ripe (r = 0.90) and unripe fruit (r = 0.81) and somewhat less with the flavones/flavonols system. These results indicate that the phenolic compounds (including flavonoids) contribute significantly to the antioxidant activity of piñuela fruits. Besides, the SOD enzyme activity maintains a direct relation with phenolic compounds, meaning that the enzyme manifests its protective action only when soluble antioxidants have been reduced to low levels, and vice-versa. The change in the behaviour of the antioxidant activity is associated with fruit development. The flavonoids, in addition to their antioxidant functions, act as messengers inhibiting protective enzymes [42] as well as in the possible exerted regulatory properties on the development and communication pathways between plant and adequate environment for their colour and aroma; considering the increase in flavonoids content as a clear indicator of fruit maturity [43]. These metabolites can be produced as a result of environmental stresses and can act as plant protection product in different ways; variations in these metabolites could also account for structural changes such as aroma, colour, taste and other properties that are evident while the ripening process takes place, process that also includes the integrity protection of the fruit against adverse conditions.

3.3 Rheological behaviour

Among the different rheological models, Helschel-Bulkley was found to be the best model that fits the piñuela juices flow behaviour always with high coefficients of determination (R² > 0.99). For juices that contain less soluble solids, the model that best fits the experimental data was Newton equation. For flow, the rheograms were accomplished with a downward ramp to 0.12 s⁻¹ and another upward ramp until 400 s⁻¹. To verify whether the samples show thixotropy, they were sheared at a constant γ˙ of 400 s^–1 for 3 min. It was not observed any variation in σ. Therefore, the samples do not show thixotropy, accordingly, the ramps are matching. On the other hand, the equipment used does not allow to work with γ˙ lower than 0.12 s^–1.

The results (Table 3 and Table 4) show that juices in both ripening stages exhibited values less than one for shear σ0 and n in all temperatures tested, except in, as already stated, for the juices with less soluble solids content (n = 1). These are typical characteristics of flow behaviour for pulps and fruit juices that are not clarified and have moderate amounts of pectin, showing a time-dependent behaviour and following in most cases the behaviour of pseudoplastic materials with yield stress [44–46].

It is important to note that the consistency index is strongly influenced by the content of soluble solids, which is directly reflected in the sharp increase of the consistency of K and σ0 when the content of total soluble solids and the apparent viscosity increase, as shown by the results (Figure 1). Some other authors have described that behaviour [44–48].

However, the flow n, which in most cases tends to increase with T [44, 45, 49], did not follow an increasing trend in all tested concentrations. Data reported by other authors does not follow a clear trend, either [50].

In Figure 4, it is important to warn about the influence of temperature on the reduction of shear stress, which is taken by industry as an advantage to reduce energy consumption in transport processes since the reduction of the apparent viscosity also reduces the power required in the process of pumping and agitation. A fluid is able to flow due to the cohesive forces in their molecules and generally it continuously deforms when it is subjected to shear stress [47, 48].

Figure 4:

Rheograms for ripe piñuela juices of 65 °Brix at different temperatures.

3.3.1 Effect of temperature and soluble solids content

The Arrhenius equation is widely used to describe the behaviour of the apparent viscosity at different temperatures [19, 45].

(6)η=K0expEaRT

Where η is the viscosity, K₀ is a constant, E_a is the activation energy of flow, R is the gas constant and T is the absolute temperature in Kelvin.

Table 5 shows the parameters of the Arrhenius equation for a 100 s⁻¹ strain rate. The E_a values are within the normal range as in other pulps and fruit juices; finding activation energy values between 9.92 and 17.80 kJ/mol [19, 45, 48, 50]. However, an increase in soluble solids content did not correspond to higher activation energy for unripe fruit samples, as expected [51]. This could be explained, perhaps because of the molecular interactions or irreversible breakdown in the matrix structure of pulps that could also influence the activation energy [48].

Table 5:

Parameters for Arrhenius equation for piñuela juices to different concentrations and maturation states.

	SS	K0	Ea	R2
	(ºBrix)	(mPa·s)	(kJ/mol)
Ripe	65	0.777	17.30	0.9798
	44.3	5.279	9.92	0.9487
	20.9	0.061	14.31	0.9820
	17.9	0.050	11.94	0.9993
Unripe	12.5	1.19	11.94	0.9906
	8.3	0.377	13.59	0.9969
	5.3	0.003	17.80	0.9823

K₀ is a constant, E_a is the activation energy of flow, SS is soluble solids and R² is coefficient of determination

The effect of the solids content was described by a potential model (5), which fits very well to the experimental data (R² > 0.98). In the case of unripe piñuela juice, the pulp amount and its yield were very low (33.34 ± 1.32 %), for this reason the sample was insufficient and the data corresponding to the three tested solid concentrations were not sufficient to fit the data for this model. Table 6 shows the parameters of the power lay equation for concentrated juices in ripe state, in which a decrease in the frequency factor with the apparent viscosity of the samples is evident. In the Figure 5 and Figure 6 can be seen the influence of temperature and the effect of soluble solids on the viscosity of ripe piñuela juice, respectively. Being evident the influence of temperature in the solubility of solids in the juice from ripe fruit. Most of the solids have positive dissolution heats, and therefore an increase in temperature that favours the solubility, dissolution rate and influences the fluid viscosity. According to Le Chatelier’s law, an endothermic process is favoured by temperature increase but no from those exothermic processes that exhibit negative dissolution heats.

Figure 5:

Effect of temperature on the viscosity of ripe piñuela juices at different soluble solids content.

Figure 6:

Effect of soluble solids content on the viscosity of piñuela ripe juice as function of temperature.

Table 6:

Exponential equation parameters of soluble solids effect on ripe bromelia karatas of juice ripe piñuela viscosity at different temperatures.

T	η0	b	R2
(ºC)	(mPa·s)	(ºBrix-1)
10	2.040 × 10-4	3.7771	0.9856
20	2.195 × 10-4	3.6859	0.9865
30	2.007 × 10-4	3.6548	0.9850
40	1.994 × 10-4	3.6182	0.9801
50	1.341 × 10-4	3.6937	0.9812

(7)η=η0Cb

Where η is the viscosity, ηo is the viscosity when the soluble solids content is 0°Brix, b is a constant and C is the concentration expressed in °Brix.

Studying the effect of factors such as temperature and soluble solids on the viscosity of a product is needed in order to determine the rates of heat transfer, energy consumption (with increasing concentration) and to control the temperature and flow rates so it is possible to ensure continuous product flow [52]. In general, the temperature and solids content affect some of the physical properties of the juices such as density, viscosity, refractive index, boiling point and specific heat. This is explicable because the concentration of dissolved solids in the primary juice (glucose and fructose) directly affects those properties mentioned above [53].

Then, studying the variations experienced by the viscosity and density with concentration and temperature provides guidelines for designing and optimizing resources in processes of food products derived from fruits (pumping, evaporation, membrane filtration, etc.).