Exploration of the Potential Bioactive Molecules of Tamarillo (Cyphomandra betacea): Antioxidant Properties and Prebiotic Index
, Lourdes Pérez-Chabela 3, Zaida Nelly Juárez 4, Gabriel Abraham Cardoso-Ugarte 5 and Beatriz Pérez-Armendáriz 1,*Round 1
Reviewer 1 Report
The manuscript entitled "Exploration of the potential bioactive molecules of tamarillo (Cyphomandra betacea): antioxidant properties and prebiotic index" is an interesting one, but in order to be published it needs a major revision.
My recommendations are the following:
I recommend a clear presentation of the purpose of this study in the abstract.
The introduction is very general. I suggest improving it with details on the properties of tamarillo that are already present in the literature. I recommend focusing on the bioactive properties of tamarillo, not necessarily on functional foods, because we are not talking about them here. Tamarillo is a potential ingredient in functional foods. (We cannot demonstrate without clinical or cellular studies). The same recommendation for the conclusion.
Describe the storage conditions of tamarillo after harvest, until their analysis.
Only lyophilization is described in the Samples preparation section. The description of the drying conditions is missing (he wrote that you also did conventional drying in Samples preparation. The results are presented only for lyophilized samples. Please check.). How were the epicarp separated from the pulp?
Statistical analysis of results is missing. The standard deviation is not enough.
Why are no results presented for pulp, Table 1: Phenolic compounds, Antioxidant capacity and Antioxidant activity?
The results and discussions are very succinctly presented. I recommend improving discussions for Bromatological analysis, Antioxidant properties. Only a few references are listed, without presenting the results of other studies.
Author Response
Response to Reviewer 1 Comments
Point 1: I recommend a clear presentation of the purpose of this study in the abstract.
Response 1: Abstract: Tamarillo is an alternative for the consumption of food with high added value through various technological methodologies with nutritional quality and low cost, generating an economic impact on society. The objective of this research was to evaluate the potential of tamarillo red variety, as a source of bioactive compounds, to generate scientific information on the importance of its chemical composition, antioxidant and prebiotic properties. Different analyses were carried out: spectroscopic methods (IR, UV, NMR) of pulp flour and epicarp flour, antioxidant properties, prebiotic activity, and bromatological analysis. The spectral obtained by FTIR, UV, and NMR allowed the identification of chemical structures associated with the inulin-like functional groups. Pulp flour presented positive prebiotic activity scores. Total phenolic compounds content in epicarp flour was 206.23 mg/100g dry weight, with an acceptable antioxidant property (ABTS+ = 6.27 TEAC and DPPH= 131.26 of IC50 ascorbic acid). The results of tamarillo as a source of bioactive molecules with important physiological properties as antioxidant and putative prebiotic is a good alternative looking to formulate functional foods
Point 2: The introduction is very general. I suggest improving it with details on the properties of tamarillo that are already present in the literature. I recommend focusing on the bioactive properties of tamarillo, not necessarily on functional foods, because we are not talking about them here. Tamarillo is a potential ingredient in functional foods. (We cannot demonstrate without clinical or cellular studies).
Response 2: Mexico possesses biodiversity or biological diversity [1] and has a wide variety of exotic fruits, which are traditionally consumed locally, being an excellent opportunity for innovation and food security. However, given the diversity of exotic Mexican and native species, some of these fruits have not been studied in terms of their physiological properties and chemical composition, C. betacea is a high value-added product with a high antioxidant potential and was cultivated for the first time in other countries [2]. In Mexico, C. betacea is known as tamarillo and can be found in the wild or in backyards. It is collected annually in the northeastern Sierra of the State of Puebla, on the border with the State of Veracruz. C. betacea is given different names, such as tree tomato in Colombia and Ecuador; chile tomato in Spain; tamarillo in New Zealand, Canada, Brazil and Malaysia; and tree tomato or chilto in Argentina and Chile. Currently, only three countries, Australia, Colombia and New Zealand, commercially cultivate this fruit [3].With the aim of finding sources of functional foods, many investigations have shown that C. betacea possesses various bioactive compounds and belongs to the Solanaceae family, it is characterized by its content of antioxidants, such as polyphenols, anthocyanins, carotenoids and vitamins A, C, B and E [4, 5]. These compounds present important physiological effects, related to their natural pigments [6, 7]. Additionally, the prebiotic activity of C. betacea was reported [8], the chemical characteristics of the pulp and mucilage covering the fruit seed, identifying hydrocolloids as arabinogalactan protein associated with pectin, as well as hemicellulosic polysaccharides, producing short-chain fatty acids (SCFA), acetate and propionate as a result of in vitro fermentation of mucilage and pulp hydrocolloids, respectively. The association of these bioactive compounds as prebiotic can be produced secondary metabolites associate with the health [9]. Therefore, the objective of this research was to evaluate the potential of tamarillo red variety, as a source of bioactive compounds, to generate scientific information on the importance of its chemical composition, antioxidant and prebiotic properties.
Point 3: The same recommendation for the conclusion
Response 3: This research identifies for the first time the structures associated with prebiotics with similarity in inulin. In addition, the amount of antioxidants present in Mexican tamarillo is similar to those cultivated in other regions. The presence of bioactive components in tamarillo and its role as a prebiotic, allows showing this fruit with potential for industrial applications in the area of health foods.
Point 4: Describe the storage conditions of tamarillo after harvest, until their analysis.
Response 4: Fruit was purchased at the local market in the community of Santiago Yaonahuac, Puebla. 50 kg of fruit were stored refrigerated at a temperature of 5 to 8 °C. C. betacea was blanched at a temperature below boiling point for 5 min to facilitate peeling and separate the peel (epicarp) from the pulp. The pulp was then reduced to a size of 3x2 cm2. The batches were divided into pulp and peel, using two methods for drying the samples; i) Lyophilization, in a freeze-drying system (FreeZone®, lyophilizer, Labconco-7343000, Kansas City, MO, USA) in which the equipment conditions were followed towards obtaining high efficiency in the process. Drying time was set according to the characteristics of the samples, with a collector temperature of -50 °C and vacuum conditions of 0.22-0.05 mbar. After drying, the particle size of the samples was reduced, and two different flours were obtained, freeze-dried pulp flour (FPF) and freeze-dried epicarp flour (FEF). ii) Convection dehydration, in a computerized convection oven, model HCU (San-Son®, Estado de México, México). The processing conditions for pulp were temperature of 65 °C for 10 hours, and for the shell, dehydration was performed at 65 °C for 4 hours; the dehydrated samples obtained were reduced in particle size. The dry pulp flour obtained by convection (DPFC) and the dehydrated epicarp flour obtained by convection (DEFC) were crushed in a food processor (KRUPS GX4100 Electric Spice Herbs and Coffee Grinder with Stainless Steel Blades) from coarse to fine for 20 seconds. All the flours obtained by the methods were kept in airtight jars at room temperature in a dark place until their use for each analysis.
Point 5: Only lyophilization is described in the Samples preparation section. The description of the drying conditions is missing (he wrote that you also did conventional drying in Samples preparation. The results are presented only for lyophilized samples. Please check.).
Response 5: The fruit was purchased at the local market in the community of Santiago Yaonahuac, Puebla. 50 kg of fruit were stored refrigerated at a temperature of 5 to 8 °C. The next process was to blanch C. betacea at a temperature below boiling point for 5 min to inactivate oxidation and facilitate the separation of the peel (epicarp) from the pulp. The pulp was then reduced to a size of 3x2 cm. The batches were divided into pulp and peel, using two methods for drying the samples; i) Lyophilization, in a freeze-drying system (FreeZone®, lyophilizer, Labconco-7343000, Kansas City, MO, USA) in which the equipment conditions were followed towards obtaining high efficiency in the process. Drying time was set according to the characteristics of the samples, with a collector temperature of -50 °C and vacuum conditions of 0.22-0.05 mbar. After drying, the particle size of the samples was reduced, and two different flours were obtained, freeze-dried pulp flour (FPF) and freeze-dried epicarp flour (FEF). ii) Convection dehydration, in a computerized convection oven, model HCU (San-Son®, Estado de México, México). The processing conditions for pulp were temperature of 65 °C for 10 hours, and for the shell, dehydration was performed at 65 °C for 4 hours; the dehydrated samples obtained were reduced in particle size. The dry pulp flour obtained by convection (DPFC) and the dehydrated epicarp flour obtained by convection (DEFC) were crushed in a food processor (KRUPS GX4100 Electric Spice Herbs and Coffee Grinder with Stainless Steel Blades) from coarse to fine for 20 seconds. All the flours obtained by the methods were kept in airtight jars at room temperature in a dark place until their use for each analysis.
Point 6: How were the epicarp separated from the pulp?
Response 6: C. betacea was blanched at a temperature below boiling point for 5 min to facilitate peeling and separate the peel (epicarp) from the pulp.
Point 7: Describe the storage conditions of tamarillo after harvest, until their analysis.
Response 7: All the flours obtained by the methods were kept in airtight jars at room temperature in a dark place until their use for each analysis.
Point 8: Statistical analysis of results is missing. The standard deviation is not enough.
Response 8:
Table 2. Prebiotic activity index for both tamarillo pulp and epicarp flour with different lactic acid bacteria.
|
Substrate/Flour |
L. casei
|
L. plantarum
|
L. paracasei
|
P |
|
DPFC |
0.08±0.00 c |
1.49± 0.01a |
0.33±0.01b |
0.0001 |
|
DEFC |
-0.35±0.02 b |
1.30± 0.01a |
-0.02±0.01c |
0.0001 |
|
P |
0.0001 |
0.5482 |
0.0002 |
|
|
|
||||
a,b Means with same letter in same column are not significantly (P>0.05) different.
DPFC= dehydrated pulp flour by convection, DEFC= dehydrated epicarp flour by convection.
Point 9: Why are no results presented for pulp, Table 1: Phenolic compounds, Antioxidant capacity and Antioxidant activity?
Response 9: Ready There was an error in attaching the data, it was only determined in freeze-dried pulp simple.
Point 10: The results and discussions are very succinctly presented. I recommend improving discussions for Bromatological analysis, Antioxidant properties. Only a few references are listed, without presenting the results of other studies.
Response 10: The proximal composition of the pulp and epicarp of C. betacea, shown in Table 1, is composed of approximately 86 % moisture in pulp and 68 % in epicarp. Morillas [16] Reported 87.72 % moisture in tamarillo, becoming perishable food, as shown by recent studies claim that the moisture content in fruits and vegetables hover around 75 to 90 % Brazil and Siddiqui [17]; in proteins 0 % is in pulp and epicarp; 61.3 % of total carbohydrates in pulp and 20.5 % in epicarp, in minerals it was 1.1 % and 2.1 % respectively; finally, 4.04 % was obtained for pulp and 8.58 % in epicarp of total dietary fiber. Prohens [4] showed in C. betacea, protein values ranging between 1.5 and 2.5 %. Tamarillo contains a great variety of minerals, with a greater presence of potassium, calcium, copper, iron, manganese and magnesium, Vasco C, Avila J, Acosta-Quezada [5, 18], noted that the high potassium content is similar to that of plantain; Acosta-Quezada [18], reported total sugars of 28.1 % and 52.0 % on a dry basis. Regarding total dietary fiber Lister [19], reported that a single serving of C. betacea of approximately 60 g can contribute to the recommended daily intake (RDI) of minerals with values in the range of 5.0-7.2 % for the red and yellow varieties. Regarding total dietary fiber Morillas [16], reported values similar to those obtained in this research, with a value of 13.37 %, while Mutalib [20] reported percentages of 4.10 and 6, respectively. C. betacea contributes 11 % of the (RDI). According to Vergara-Valencia et al. [21], C. betacea can be considered a nutritious fruit due to its balance in dietary fiber. In addition, these properties of solubility and viscosity have profound effects on the functionality of dietary fiber, during food processing, and in the gastrointestinal tract Mudgil [22], another property is the adsorption of fat is part of the soluble dietary fiber for the purposes of stabilization of emulsions in the processing of high-fat foods, as well as to observe physiological effects in humans Yaich [23]. The difference in results is due to various factors such as, for example, the analyzed crop, maturity index, geographical and environmental conditions.
3.2 Antioxidant properties
The yield of the ethanolic extracts of C. betacea FPF was 6.25. The result of the determination of phenolic compounds of the FPF ethanolic extract of C. betacea was 206.23 mg EAG/100 g dry weight. The antioxidant capacity of the FPF extract of C. betacea, by ABTS, shows a concentration obtained of 6.27 µmol/Trolox to inhibit 50% of free radicals. As for the percentage of antioxidant activity of the ethanolic extract of FPF in the DPPH method, the % AA was 91.74 with the concentration of 250.00 µg/m; it showed an IC50 of 131.26 µg/mL, being lower than the control (IC50 of 780.60 µg/mL) as shown in Table 1. Different authors have reported the presence of phenolic compounds in C. betacea; Orqueda et al. [24], reported 223.80 mg/100g of flavonoids in the pulp; Espin et al. [25] identified phenolic acids in the dried pulp (421.6 mg/100g), in cultures of C. betacea in New Zealand. In the epidermis (peel) and pulp, they identified polyphenols ranging from 54.67 to 278.03 mg/100g, mainly phenolic acids. The recommendation for flavonoid intake is between 250-400 mg/day, considering the seasonality of food sources Peluso and Palmery [26]. The results of this research agree with those reported Vasco et al. [5] with values of 4.2 to 10.3 µmol/Trolox of antioxidant activity and higher than those reported Hurtado et al. [27], of 1.90 µmol/Trolox/g. Therefore, C. betacea showed its free radical inhibition capacity and antioxidant activity due to the presence of natural phytochemicals with antioxidant potential. Ordoñez et al. [28], attributed this antioxidant activity to the presence of flavonoids, polyphenols, and vitamins in the fruit. The values obtained were lower than those reported Mutalib et al. [20], with an IC50 of 800 µg/mL in C. betacea pulp. The difference in ABTS and DPPH values of antioxidant compounds present in ethanolic extracts of FPF is due to the type of extractable compounds, hydrophilic and lipophilic Rufino et al. [29]. Saura Calixto [30], showed that about 50% of the total dietary antioxidants, mainly polyphenols cross the small intestine bound to dietary fiber, thus attributing the transport of dietary antioxidants through the gastrointestinal tract, releasing the fiber matrix in the colon by the action of the bacterial microbiota, producing metabolites and an antioxidant environment.
The presence of polyphenols in C. betacea was reported [25], identifying rosmarinic acid as a compound with important biological, antioxidant, anticancer and diabetes control properties. These results are similar to those reported [31], who demonstrated the presence of flavonoids in the red variety of C. betacea. Other compounds were identified [7] as rutin, caffeic acid and chlorogenic acid. According to Wan S. [3], C. betacea is a potential functional food due to its biological properties, antioxidant, anti-inflammatory, antiviral, antibacterial, antidepressant and anticancer effects, in addition to its natural pigments, which are often associated with the prevention of chronic diseases [32].
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript studied the physical and chemical characteristics of freeze-dried pulp and peel of tamarillo. The samples showed important physiological properties such as antioxidant and putative prebiotic. The results indicate that the fruit might serve as potential food processing ingredients.
Line 84 The author mentioned two methods, lyophilization and convection oven drying, however, only freezing dried samples were shown in the paper. I think it is more meaningful to study the retention of active substances by processing technologies.
2.6. Antioxidant properties ABTS and DPPH methods are the commone methods to determin the antioxidant activity. I think it is not necessary to be specifically described as antioxidant activity and antioxidant capacity.
Section of Materials and Methods
The concentration of the reagents should be In front of the reagent.
Section of Resluts. The results should be presented more clearly. Combine the results and discussion might be a good choice.
3.4 Spectroscopic Methods Some paragraphs just have one sentence. Some useful structural information of bioactive molecules relating to physiological properties can be shown here.
Line 108 What is the abbreviation of FPF and FEF.
Line 211 free radical scavenging capacity could be better.
Author Response
Response to Reviewer 2 Comments
Point 1: Line 84 The author mentioned two methods, lyophilization and convection oven drying, however, only freezing dried samples were shown in the paper. I think it is more meaningful to study the retention of active substances by processing technologies.
Response 1: The fruit was purchased at the local market in the community of Santiago Yaonahuac, Puebla.50 kg of fruit were stored refrigerated at a temperature of 5 to 8 °C. The next process was to blanch C. betacea at a temperature below boiling point for 5 min to inactivate oxidation and facilitate the separation of the peel (epicarp) from the pulp. The pulp was then reduced to a size of 3x2 cm. The batches were divided into pulp and peel, using two methods for drying the samples; i) Lyophilization, in a freeze-drying system (FreeZone®, lyophilizer, Labconco-7343000, Kansas City, MO, USA) in which the equipment conditions were followed towards obtaining high efficiency in the process. Drying time was set according to the characteristics of the samples, with a collector temperature of -50 °C and vacuum conditions of 0.22-0.05 mbar. After drying, the particle size of the samples was reduced, and two different flours were obtained, freeze-dried pulp flour (FPF) and freeze-dried epicarp flour (FEF). ii) Convection dehydration, in a computerized convection oven, model HCU (San-Son®, Estado de México, México). The processing conditions for pulp were temperature of 65 °C for 10 hours, and for the shell, dehydration was performed at 65 °C for 4 hours; the dehydrated samples obtained were reduced in particle size. The dry pulp flour obtained by convection (DPFC) and the dehydrated epicarp flour obtained by convection (DEFC) were crushed in a food processor (KRUPS GX4100 Electric Spice Herbs and Coffee Grinder with Stainless Steel Blades) from coarse to fine for 20 seconds. All the flours obtained by the methods were kept in airtight jars at room temperature in a dark place until their use for each analysis.
Point 2: 2.6. Antioxidant properties ABTS and DPPH methods are the commone methods to determin the antioxidant activity. I think it is not necessary to be specifically described as antioxidant activity and antioxidant capacity.
Response 2: 2.6. Antioxidant properties
Total polyphenol content was determined by the method proposed Singleton and Rossi [12]. Folin-Ciocalteu's reagent was added to 1 mL of ethanolic extract of FPF and mixed with 8 mL of 0.7 M Na2CO3. The mixture was allowed to stand for 2 h at room temperature in the dark. The absorbance was measured at 765 nm and extrapolated with a catechol standard curve (0 to 100 mg/mL). Subsequently, in the ABTS and DPPH assays, the radical scavenging potential was evaluated only in the FPF sample was investigated according to the method described Re et al [13]. The diammonium salt of 2, 2'-azino-bis(3-ethyl-6-benzothiazoneline) (ABTS) (Sigma Aldrich, St. Louis, USA) and potassium persulfate were occupied 2.45 mM and incubated at room temperature in the dark for 12 h. After addition of 1.0 mL of diluted ABTS solution (A734nm = 0. 700 ± 0.020) to 10 µL of diluted samples or Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchrom-2-carboxylic acid) standards in ethanol, the absorbance reading was taken exactly 1 min after initial mixing and up to 10 min. The percent inhibition absorbance at 734 nm was calculated and plotted as a function of antioxidant and Trolox concentration for the reference data of the standard. Subsequently, the method described Kasote et al [14] was followed. For this, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was used at concentrations of 62.5-500 mg/mL, and ascorbic acid was used as a positive control.
Point 3: Section of Materials and Methods
The concentration of the reagents should be In front of the reagent.
Response 3 : Ready
Point 4: Section of Results. The results should be presented more clearly. Combine the results and discussion might be a good choice.
Response 4: 3. Results and Discussion
3.1 Bromatological Analysis
The proximal composition of the pulp and epicarp of C. betacea, shown in Table 1, is composed of approximately 86 % moisture in pulp and 68 % in epicarp. Morillas [16] Reported 87.72 % moisture in tamarillo, becoming perishable food, as shown by recent studies claim that the moisture content in fruits and vegetables hover around 75 to 90 % Brazil and Siddiqui [17]; in proteins 0 % is in pulp and epicarp; 61.3 % of total carbohydrates in pulp and 20.5 % in epicarp, in minerals it was 1.1 % and 2.1 % respectively; finally, 4.04 % was obtained for pulp and 8.58 % in epicarp of total dietary fiber. Prohens [4] showed in C. betacea, protein values ranging between 1.5 and 2.5 %. Tamarillo contains a great variety of minerals, with a greater presence of potassium, calcium, copper, iron, manganese and magnesium, Vasco C, Avila J, Acosta-Quezada [5, 18], noted that the high potassium content is similar to that of plantain; Acosta-Quezada [18], reported total sugars of 28.1 % and 52.0 % on a dry basis. Regarding total dietary fiber Lister [19], reported that a single serving of C. betacea of approximately 60 g can contribute to the recommended daily intake (RDI) of minerals with values in the range of 5.0-7.2 % for the red and yellow varieties. Regarding total dietary fiber Morillas [16], reported values similar to those obtained in this research, with a value of 13.37 %, while Mutalib [20] reported percentages of 4.10 and 6, respectively. C. betacea contributes 11 % of the (RDI). According to Vergara-Valencia et al. [21], C. betacea can be considered a nutritious fruit due to its balance in dietary fiber. In addition, these properties of solubility and viscosity have profound effects on the functionality of dietary fiber, during food processing, and in the gastrointestinal tract Mudgil [22], another property is the adsorption of fat is part of the soluble dietary fiber for the purposes of stabilization of emulsions in the processing of high-fat foods, as well as to observe physiological effects in humans Yaich [23].
The difference in results is due to various factors such as, for example, the analyzed crop, maturity index, geographical and environmental conditions.
3.2 Antioxidant properties
The yield of the ethanolic extracts of C. betacea FPF was 6.25. The result of the determination of phenolic compounds of the FPF ethanolic extract of C. betacea was 206.23 mg EAG/100 g dry weight. The antioxidant capacity of the FPF extract of C. betacea, by ABTS, shows a concentration obtained of 6.27 µmol/Trolox to inhibit 50% of free radicals. As for the percentage of antioxidant activity of the ethanolic extract of FPF in the DPPH method, the % AA was 91.74 with the concentration of 250.00 µg/m; it showed an IC50 of 131.26 µg/mL, being lower than the control (IC50 of 780.60 µg/mL) as shown in Table 1. Different authors have reported the presence of phenolic compounds in C. betacea; Orqueda et al. [24], reported 223.80 mg/100g of flavonoids in the pulp; Espin et al. [25] identified phenolic acids in the dried pulp (421.6 mg/100g), in cultures of C. betacea in New Zealand. In the epidermis (peel) and pulp, they identified polyphenols ranging from 54.67 to 278.03 mg/100g, mainly phenolic acids. The recommendation for flavonoid intake is between 250-400 mg/day, considering the seasonality of food sources Peluso and Palmery [26]. The results of this research agree with those reported Vasco et al. [5] with values of 4.2 to 10.3 µmol/Trolox of antioxidant activity and higher than those reported Hurtado et al. [27], of 1.90 µmol/Trolox/g. Therefore, C. betacea showed its free radical inhibition capacity and antioxidant activity due to the presence of natural phytochemicals with antioxidant potential. Ordoñez et al. [28], attributed this antioxidant activity to the presence of flavonoids, polyphenols, and vitamins in the fruit. The values obtained were lower than those reported Mutalib et al. [20], with an IC50 of 800 µg/mL in C. betacea pulp. The difference in ABTS and DPPH values of antioxidant compounds present in ethanolic extracts of FPF is due to the type of extractable compounds, hydrophilic and lipophilic Rufino et al. [29]. Saura Calixto [30], showed that about 50% of the total dietary antioxidants, mainly polyphenols cross the small intestine bound to dietary fiber, thus attributing the transport of dietary antioxidants through the gastrointestinal tract, releasing the fiber matrix in the colon by the action of the bacterial microbiota, producing metabolites and an antioxidant environment.
The presence of polyphenols in C. betacea was reported [25], identifying rosmarinic acid as a compound with important biological, antioxidant, anticancer and diabetes control properties. These results are similar to those reported [31], who demonstrated the presence of flavonoids in the red variety of C. betacea. Other compounds were identified [7] as rutin, caffeic acid and chlorogenic acid. According to Wan S. [3], C. betacea is a potential functional food due to its biological properties, antioxidant, anti-inflammatory, antiviral, antibacterial, antidepressant and anticancer effects, in addition to its natural pigments, which are often associated with the prevention of chronic diseases [32].
3.3. Spectroscopic methods
The FTIR spectra for both pulp flour and epicarp flour samples, where the main observable differences were reflected in chemical composition around 1800-1600 cm-1. In Figure 2, the FTIR spectra of the tamarillo pulp and epicarp samples the same typical bands can be observed but with different magnitude, because of the different composition among pulp and epicarp. Changes in composition are reflected in several peaks assigned to different wavelength ranges for the contribution of specific regions: 3350 cm−1 for O–H stretching modes of water absorbing, −C−H stretching in fatty acids (2900 cm−1), −C=O of stretch of methyl esterified carbonyl (1745 cm−1), asymmetric stretch of carboxylate anion −COO- (1630 cm−1), symmetric stretch of carboxylate anion (1432 cm−1), and C=O and C−C stretching of acids (1010 cm−1), respectively [33]. The noticeable changes between 1300 and 800 cm-1 correspond to the typical fingerprint region similar to citrus pectin [7], since the presence of high methoxyl pectin in tamarillo pulp and low methoxyl in mucilage have been reported [34]. The results showed a similarity with the characteristic peaks of inulin of 3270-2929 cm-1 and 1025-985 cm-1 [35].
In the UV spectroscopy, the presence of carbohydrates was identified in both tamarillo samples for pulp and epicarp, with absorption bands at 212 and 275 nm (values of 0.089 and 0.375 abs) for pulp and absorption bands at 210 and 328 nm (values of 0.145 and 0.803 abs) for epicarp (Figure 3). The results of UV showed that the peaks obtained are associated with the presence of monosaccharides as reported by Kaijanen et al. [36], where the maximum absorption peaks for xylose are from 245 nm to 255 nm and the UV spectrum for glucose, the absorbance is close to a maximum of 270 nm and significantly low at 270 nm. Nonetheless, in tamarillo pulp, the presence of common monosaccharides from different polysaccharides are similarly fractionated depending on the extraction method, since in water extraction procedure mannose- and xylose-containing polysaccharides, major constituents of hemicelluloses, presented lower extractability [33]. This is that despite of the extraction method the UV analysis confirm the presence of fermentable sugars, as mono-, oligo- or polysaccharides in tamarillo pulp, with an observable difference in pulp flour with more peaks, since pulp is the sweeter part of the fruit, than in epicarp flour, although other important components in epicarp as pigments are present. Novel delphinidin 3-O-a-L-rhamnopyranosyl-(1®6)-β-Dglucopyranoside-3’-O-β-D-glucopyranoside as a minor constituent has been reported [37], and hence tamarillo as a tropical fruit could be considered as good source of natural pigments with potential antioxidant activity.
In the spectroscopy 1H RMN, the FDPF and FDEF samples showed signals between 3 and 4 ppm, which correspond to the various carbons of fructose (sugars), and signals at 5.0 ppm that correspond to an anomeric H, characteristic of sugars (Figure 4). In the NMR spectra the presence of fructose units and glucose units, as well as anomeric carbon at 5.44 ppm, corresponding to the α1-β1 proton of the D-glucopyranosyl unit, which is located at the beginning of the inulin chain [38]. According to do Nascimento, 2013 and 2017 [39, 40] reported in C. betacea the presence of a galactose arabinose glucuronoxylan in the pulp, composed of major monosaccharides of glucose, arabinose, galactose, xylose, uronic acids, showing an antinociceptive effect in inflammatory pain models. These results are associated with dietary fiber as the presence of pectins, being the main components of the soluble fraction of fiber in the pulp. Kou, [41] showed that the presence of phenolic compounds in C. betacea have high antioxidant potential and demonstrated inhibition of LDL oxidation in vitro and ROS production in PC12 cells.
3.4 Prebiotic activity
In general, tamarillo pulp flour presented a higher prebiotic activity than tamarillo epicarp flour. L. plantarum showed significantly (P<0.01) higher prebiotic activity for both pulp and epicarp flours as carbon sources, obtaining values of 1.49 and 1.30, respectively. Pulp flour presented positive values with all the lactic acid bacteria (Table 2). The prebiotic activity depends on the probiotic lactic acid bacteria performance in presence of a pathogenic strain, in order to be the dominant flora. In this research, under the employed experimental conditions, L. plantarum presented the higher scores 8 above 1), but in general the employed strains presented a higher prebiotic activity score employing tamarillo pulp flour than tamarillo epicarp flour. Although Diaz-Vela et al. [42] reported positive prebiotic activity values for L. rhamnosus GG with pineapple peel flour and cactus pear flour (0.19 and 0.21, respectively), it seems that tamarillo epicarp or peel presented lower fermentable carbohydrates that pulp. Nonetheless, it has been reported that tamarillo hydrocolloids were resistant to digestive enzymes and gastrointestinal conditions, indicating that can be available for fermentation by gut microbiota, producing as well short chain fatty acids [34]. Pectic polysaccharides as the found in tamarillo pulp and epicarp possess important biological activity [43]. In this view, tamarillo consumption as a source of bioactive molecules with important physiological properties as antioxidant and putative prebiotic is a good alternative looking for functional foods, since foods formulated with this fruit will present health benefits, as has been already reported as hyperlipidemia [44] or metabolic syndrome [31].
Point 5: 3.4 Spectroscopic Methods Some paragraphs just have one sentence. Some useful structural information of bioactive molecules relating to physiological properties can be shown here.
Response 5: According to do Nascimento, 2013 and 2017 [38, 39] reported in C. betacea the presence of a galactose arabinose glucuronoxylan in the pulp, composed of major monosaccharides of glucose, arabinose, galactose, xylose, uronic acids, showing an antinociceptive effect in inflammatory pain models. These results are associated with dietary fiber as the presence of pectins, being the main components of the soluble fraction of fiber in the pulp. Kou, [40] showed that the presence of phenolic compounds in C. betacea have high antioxidant potential and demonstrated inhibition of LDL oxidation in vitro and ROS production in PC12 cells.
Point 6 Line 108 What is the abbreviation of FPF and FEF.
Response 6: The flours were obtained from the Tamarillo fruit by the freeze-drying method, freeze-dried pulp flour (FPF) and freeze-dried epicarp flour (FEF).
Point 7 Line 211 free radical scavenging capacity could be better.
Response 7: Different authors have reported the presence of phenolic compounds in C. betacea; Orqueda et al. [24], reported 223.80 mg/100g of flavonoids in the pulp; Espin et al. [25] identified phenolic acids in the dried pulp (421.6 mg/100g), in cultures of C. betacea in New Zealand. In the epidermis (peel) and pulp, they identified polyphenols ranging from 54.67 to 278.03 mg/100g, mainly phenolic acids. The recommendation for flavonoid intake is between 250-400 mg/day, considering the seasonality of food sources Peluso and Palmery [26]. The results of this research agree with those reported Vasco et al. [5] with values of 4.2 to 10.3 µmol/Trolox of antioxidant activity and higher than those reported Hurtado et al. [27], of 1.90 µmol/Trolox/g. Therefore, C. betacea showed its free radical inhibition capacity and antioxidant activity due to the presence of natural phytochemicals with antioxidant potential. Ordoñez et al. [28], attributed this antioxidant activity to the presence of flavonoids, polyphenols, and vitamins in the fruit. The values obtained were lower than those reported Mutalib et al. [20], with an IC50 of 800 µg/mL in C. betacea pulp. The difference in ABTS and DPPH values of antioxidant compounds present in ethanolic extracts of FPF is due to the type of extractable compounds, hydrophilic and lipophilic Rufino et al. [29]. Saura Calixto [30], showed that about 50% of the total dietary antioxidants, mainly polyphenols cross the small intestine bound to dietary fiber, thus attributing the transport of dietary antioxidants through the gastrointestinal tract, releasing the fiber matrix in the colon by the action of the bacterial microbiota, producing metabolites and an antioxidant environment.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors responded to all my comments
