Scientia Horticulturae 263 (2020) 109107
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Biochemical composition as a function of fruit maturity stage of bell pepper (Capsicum annum) inoculated with Bacillus amyloliquefaciens
T
Jonathan Cisternas-Jameta,b, Ricardo Salvatierra-Martínezc,d,e, Antonio Vega-Gálvezf,*,1, Alexandra Stolld,g,**,1, Elsa Uribef,g, María Gabriela Goñih,i a
Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad Santo Tomás, Chile Centro de Investigación y Modelación de Negocios CIMON, Universidad Santo Tomás, Chile c Programa de Doctorado en Biología y Ecología Aplicada, Departamento de Biología, Universidad de La Serena, La Serena, Chile d Laboratorio de Microbiología Aplicada, Centro de Estudios Avanzados en Zonas Áridas (CEAZA) Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena, Chile e Laboratorio de Patología Vegetal, Universidad de Tarapacá, General Velásquez 1375, Arica, Chile f Departamento de Ingeniería en Alimentos, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena, Chile g Instituto de Investigación Multidisciplinario en Ciencia y Tecnología, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena, Chile h Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Godoy Cruz 2290, Ciudad Autónoma de Buenos Aires, Argentina i Grupo de Investigación en Ingeniería de Alimentos, Departamento de Ingeniería Química y Alimentos, Facultad de Ingeniería, Universidad Nacional de Mar del Plata, Juan B. Justo 4302, Mar del Plata, Argentina b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacillus Biofertilizer Nutraceutical Antioxidants Vitamin C
The use of growth promoting bacteria in sweet pepper plants (Capsicum annuum), such as some Bacillus strains, has previously been related to increased yields and plant resistance. However, it is also important to evaluate the effect that inoculation has on the ripening process and on the nutritional composition of the fruits. In the present work, the effect of root inoculation of sweet pepper plants with Bacillus amyloliquefaciens on the composition of sweet peppers harvested at different stages of maturation is evaluated. It was possible to determine a clear effect of inoculation on the fixation of Ca and Fe, and the content of vitamin C and compounds with antioxidant capacity. Root inoculation with Bacillus amyloliquefaciens generated an increase in the concentration of calcium, iron and vitamin C of 561 mg kg−1, 182 mg kg−1 and 561 μg 100 g−1 d.m., respectively in Red II and Green I compared to the control samples. An increase in antioxidant capacity was generated, which is reflected in an increase in the ORAC test of 1618 umol TE 100 g−1 d.m. and in 587 umol TE 100 g−1 d.m. for Green I and Red I crops respectively. On the other hand, the effect of the fruit ripening process was significant, especially in relation to the development of natural pigments and phenolic compounds, with high antioxidant potential. An increased of extractable pigments of 57 color units with respect to the control sample in Red II is highlighted, which enhances the organoleptic attractiveness of the fruit. These results would allow producers to determine the time at which to harvest to maximize the nutritional contribution of sweet peppers.
1. Introduction Bell peppers are popular in all America mainly due to their characteristic flavor, associated to the presence of capsaicinoids (ÁlvarezParrilla et al., 2011). Moreover, they are also recognized as good sources of other bioactive compounds, such as vitamin A and C, phenolic compounds, carotenoids, and flavonoids (Bae et al., 2014; Álvarez-Parrilla et al., 2011; Ghasemnezhad et al., 2011). These
bioactive compounds such as ascorbic acid, phenolics, flavonoids and pigments with antioxidant activity, are related with potential health functionality against several chronic non transmissible diseases like cancer, cardiovascular, diabetes, cataracts, neurological diseases and Alzheimer’s, among many others (Deepa et al., 2007). Antioxidant compounds, in particular, have now attracted a large amount of research as they are directly related to the functional or nutraceutical properties of the vegetables.
⁎
Corresponding author at: Departamento de Ingeniería en Alimentos, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena, Chile. Corresponding author at: Laboratorio de Microbiología Aplicada, Centro de Estudios Avanzados en Zonas Áridas (CEAZA) Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena, Chile. E-mail addresses:
[email protected] (A. Vega-Gálvez),
[email protected] (A. Stoll). 1 Both contributed substantially and equally to this study. ⁎⁎
https://doi.org/10.1016/j.scienta.2019.109107 Received 7 October 2019; Received in revised form 3 December 2019; Accepted 5 December 2019 0304-4238/ © 2019 Published by Elsevier B.V.
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suggested that besides biocontrol effects, this strain of B. amyloliquefaciens also improves plant performance. In the present article, the strain B. amyloliquefaciens BBC047 used to evaluate the effect of its inoculation on the physicochemical and nutraceutical composition of bell pepper during its fruit ripening. Therefore, physicochemical and bioactive compound content and antioxidant activity of bell peppers in different maturity stages are measured to elucidate impact of the bacterial inoculation on fruit quality.
While genetics of the fruit plays an important role in the composition, several other factors must be considered, like e.g. cultivar, agronomic practices, harvest date and environmental conditions during growing and postharvest and storage handling (Patthamakanokporn et al., 2008). The levels of bioactive compounds present in bell pepper vary throughout growth and maturation, which is important to the color and flavor of the pepper but also to their nutraceutical composition (Estrada et al., 2002). For example, green peppers are rich in chlorophyll, contributing to the characteristic color and, also providing antioxidant activity (Álvarez-Parrilla et al., 2011). On the other hand, red bell pepper has high levels of carotenoids (Deepa et al., 2007). Changes in the phytochemical composition of peppers occurring during maturation affect the antioxidant activity and may affect their consumption (Patthamakanokporn et al., 2008). Bae et al. (2014) examined the impact of maturity stage in greenhouse grown peppers, and they found that mature peppers generally had higher ascorbic acid and total phenolic content than immature peppers. However, harvest date is often decided according to market demand and not by the optimum composition of the fruits. Nevertheless, a better understanding of the changes on bioactive compounds content during maturation could lead to a enhance product, with potential use as a functional ingredient. Little is known regarding the impact of agricultural practices on the content of bioactive compounds, including fertilization or use of biostimulants like plant growth promoting rhizobacteria (PGPR). As the agronomical benefits of PGPR are known and are not in discussion in the present article, the next stage should bring into focus the effects that could potentially increase the value of the product as a functional ingredient due to the inclusion of bioactive compound in the diet. Soil microbiota plays an important role in maintaining the soil ecological balance and the sustainability of the natural ecosystem or the agroecosystem (Jaizme-Vega et al., 2004). Several studies highlight the agronomical benefits of using PGPR, where crop yield was increased, as well as disease protection and crop improvement are well acknowledged (Hafeez et al., 2006; Choudhary and Johri, 2009; Bacilio et al., 2014; Babu et al., 2015). The use of PGPR in modern agriculture allows to reduce chemical fertilizers and fungicides (Adesemoye et al., 2009) with the consequent decrease of environmental impact of agriculture. In PGPR interaction with the host plant, several mechanisms are known, such as: production of phytohormones, mobilization of phosphorous, siderophore production, inhibition of plant ethylene synthesis, antibiotic production and induction of plant systemic resistance (Cezón et al., 2003; Ali et al., 2014). PGPR action is associated to their ability to successfully colonize the roots of the plant (Cezón et al., 2003). However, the impact of harvest time on the phytochemical compound content is also of interest for inoculated bell pepper and should be evaluated. Among the most employed PGPR, the genus Bacillus is one of the most comprehensively investigated (Babu et al., 2015). There is evidence of the positive effect on plant development or the establishment of seedlings in different crops and fruits: banana (Jaizme-Vega et al., 2004), arugula (Eruca vesicaria) (Ali et al., 2014), tea plant (Chakraborty et al., 2006), tomato (Lucas-García et al., 2003; Li et al., 2017), wheat (Hafeez et al., 2006). B. amyloliquefaciens in particular has been successfully employed as biofertilizer in different crops, as chili, bell pepper or tomato (Herman et al., 2008; Adesemoye et al., 2009; Gowtham et al., 2018). B. amyloliquefaciens is also capable to reduce significantly the incidence of fungal disease. Nonetheless, despite its use in agriculture, their impact on fruit quality parameters and nutritional value is poorly studied. In a previous study, Salvatierra-Martinez et al. (2018) performed a physiological and molecular characterization of an autochthonous strain of Bacillus amyloliquefaciens (BBC047) and studied its antifungal activity against Botritis cinerea (in vitro and in tomato). Root and foliar/ root inoculation were able to reduce the severity of B. cinerea infection, but only root inoculation led to growth promotion in the tomato plants (enhanced vertical growth and increased biomass). These results
2. Materials and methods 2.1. Growing conditions of the Bacillus amyloliquefaciens BBC047 The B. amyloliquefaciens BBC047 strain was provided by CEAZA (Centro de Estudios Avanzados en Zonas Áridas, La Serena, Chile). In a previous study, the growth promotion capacity of this Bacillus strain and its antifungal activity were tested (Salvatierra-Martinez et al., 2018). B. amyloliquefaciens was cultivated in LB media (30 °C, 150 rpm, 36 h) and a standardized concentration of 107 cfu/mL was used for inoculation. 2.2. Inoculation of Bacillus amyloliquefaciens BBC047 Bell pepper (Capsicum annum, var. Lamuyo, Correntín) was inoculated with B. amyloliquefaciens in the seedbed before transplant, while no inoculated seedlings were used as control. A random block array was used with 20 plants per block and 80 plants per treatment. Three inoculations were made in the seedbed, at 7 day intervals, before transplant of the seedlings to the greenhouse. In each of the inoculations, 2 mL of bacterial culture (107 CFU/mL) were added to each seedling while in the trays (240 cells, polystyrene, 25 mL of substrate per cell). 2.3. Experimental conditions during growing The experiment was carried out in a greenhouse for commercial production of sweet pepper, provided by Allfresh Company (Coquimbo, Chile). During the growing period the average temperature was 18.2 ± 4.5 °C and the relative humidity was 52.7 ± 3.4 %. Regular irrigation and fertilization was employed throughout the cultivation, according to standard practices for bell pepper. 2.4. Harvest of the bell peppers and postharvest processing of the samples The Bell peppers were harvested in three different stages of ripening differentiated according to the color of the fruits. The first harvest Green, were green fruit and marketable in size (90 days after transplantation). In the second Red I, fruits with 80 % red color were harvested (125 days after transplantation). In the third Red II, we obtained green fruits and 100 % red fruits (150 days after transplantation), respectively. All harvested fruits were transported to the research laboratory in less than two hours. In the laboratory, fruits were washed, disinfected with sodium hypochlorite (25 ppm, 30 min), rinsed with distillated water and cut into 0.5 cm3 pieces. Afterwards, samples of cut bell pepper (1000 g) were homogenized in a blender (Oster, Model BRLYo7-Z00-051, Mexico) for each treatment. Homogenized pulp was refrigerated at −80 °C until further processing. Frozen bell pepper pulp samples were freeze-dried (3 days, −40/30 °C, 200 mTorr) in a freeze-dryer (Virtis, Model Advantage Plus XL-70 Gardiner, NY, USA). Dried pulp samples were stored in polyethylene bags at 4 °C until analysis 2.5. Physicochemical composition Physicochemical characterization of dried bell pepper was carried out according to AOAC methodologies, as referenced by Vega-Galvez 2
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dichromate (0.3 g L−1) in 1.8 M sulfuric acid. The extractable pigments value is obtained by Eqs. (5) and (6), where Ag corresponds to the absorbance of the standard, If is the correction factor of the cell and the instrument and A (460 nm) is the absorbance of the sample.
et al. (2009). Water content determination was performed in a vacuum oven (Gallenkamp, OVL570, Leicester, UK) according to AOAC N° 934.06. Crude protein content was performed in a Kjeldhal automatic system (VelpScientifica, UDK 20 and UDK 129, Usmate, Italy) using a 6.25 conversion factor. Lipid content was measured by a Soxhlet extractor (Gerhardt, Köningswinter, Germany). Ash content was determined according to AOAC N° 923.03 in a muffle (Labtech, LEF-103S, Kyonggi-Do, Korea) at 550 °C. Water activity was measured in an automatic aw-meter (Aqualab, 4TE, Pullman, USA). Total carbohydrates were determined by difference. All measurements were performed in triplicate.
If =
Extractable pigments =
2.6. Reducing sugars
The minerals Cu, Fe, Ca, and K were measured according to Briones et al. (2011) in the crude ash residue obtained from the dried samples, using an atomic absorption spectrophotometer (Shimadzu Instruments Inc., SpectrAA-220, Kyoto, Japan) after digestion with a H2SO4:HNO3:HClO4 mixture (3:2:1). Mineral content was expressed as mg/g of dry mass. All analyses were performed in triplicate.
Chlc (μg / mL) = (−1.67·A664 ) − (7.60·A647 ) − (24.52·A630 )
(3)
TC (μg / mL) =
(1000·A 470 ) − (1.82·Chla) − (85.02·Chlb) 198
(6)
Vitamin C was measured as ascorbic acid following Islam et al. (1993). 1 g of the dried samples was mixed with 0.2 g of oxalic acid (Fluka, Germany). Then, 30 mL of deionized water is added and the mixture is homogenized in an Ultraturrax (IKA, T18, Staufen, Germany) at 9000 rpm for 2 min. After, 20 mL of diethyl ether was added and a second homogenization was performed for 30 s. The final mixture was centrifuged (4472g, 15 min, 4 °C). Then, a 5 mL aliquot of the supernatant was transferred to a flask containing 5 mL of 4 % m/v solution of KI (Sigma Aldrich, Saint Loius, USA), 2 mL of a 10 % v/v solution of CH3COOH (MerkKGaA, Darmstadt, Germany) and 0.15 mL of 1 % m/v starch solution. Titration of the mix was carried out with NBS (N-Bromosuccinimide, C4H4BrNO2, MerkLGaA, Darmstadt, Germany) as a titration agent (0.2 g of NBS in 1 L of deionized water). A standard ascorbic acid solution was prepared (51.12 mg of ascorbic acid and 2 g of oxalic acid in 250 mL of deionized water). All determinations were performed in triplicate and results are expressed as mg of Vitamin C per 100 g of dried mass (mg VitC/100 g d.m.).
Photosynthetic pigments, such as Chlorophyll a, b and c and Total Carotenoids were measured according to Lichtenthaler (1987). A 0.20 g of dried bell pepper sample (Green) was use to obtaining the extract. Chlorophyll a, b and c were calculated according to the Eqs. (1)–(3), while Total Carotenoids (including xanthophylls and β-carotene) were calculated by Eq. (4) (Jeffrey and Humphrey, 1975; Lichtenhaler, 1987), where A664, A647, A630 and A470 represent the absorbances at the corresponding wave length obtained for the extracts.
(2)
Mass sample (g )
2.11. Vitamin C
2.8. Photosynthetic pigments
Chlb (μg / mL) = (−5.43·A664 ) − (21.03·A647 ) − (2.66·A630 )
A (460nm)16.4 If
Methanolic extracts were obtained from dried bell pepper samples according to Vasco et al. (2008) with modifications. Samples of green bell pepper (0.3 g) were placed in Falcon tubes with 20 mL of the methanol:water mix (50:50) in an orbital shaker (BOECO, OS-20, Hamburg, Germany) with continues agitation (250 rpm, 20 °C) for 1 h, then centrifuged (4472g, 4 °C) in a refrigerated centrifuge (Eppendorff, 5804 R, Hamburg, Germany) and the supernatant was separated. Three more extractions were performed followed by another centrifugation. Finally, all the extracts were concentrated in a rotary evaporator (IKA, RV 10, Staufen, Germany) at 40 °C. Dry extract was reconstituted in methanol to 10 mL and kept refrigerated.
2.7. Mineral content
(1)
(5)
2.10. Methanolic extract obtained for antioxidant activity and total phenolic determination
The first stage of the reducing sugar determination was the extraction procedure, described by Sricharoen et al. (2016), using 0.1 g of dried sample. Then, reducing sugar content was determined in the extract by the methodology proposed by Miller (1959) in a UV-VIS spectrophotometer (Spectronic, 20 Genesis, Rochester, USA) at 540 nm. Fructose (MerkKGaG, Darmstadt, Germany) in range of 0.5–3 g L was used for the calibration curve, resulting in the following equation y = 0.738 x – 0.0675 (R2 = 0.995). All determinations were performed in triplicate and results are expressed as g of Fructose per 100 g of dried mass.
Chla (μg / mL) = (11.85·A664 ) − (1.54·A647 ) − (0.08·A630 )
0.600 Ag
2.12. Total phenolic content (4)
Total phenolic content (TPC) was determined by the Folin-Ciocalteu methodology, as described by Zhuang et al. (2012) with small modifications. 500 μL of the methanolic extract was mixed with 500 μL of the Folin-Ciocalteu reagent (Merck, KGaA, Darmstadt, Germany) in a 15 mL Falcon tube. After 5 min, 2 mL of the Na2CO3 solution (Fluka, Germany; 20 % p/v) were added. Fifteen min later, 10 mL of distillated water were added and the mix was centrifuged (3578g, 5 min, 4 °C). Later, absorbance of the extracts was measured in a VIS-Spectrophotometer (Spectronic, 20 Genesis, NY, USA) at 725 nm. A calibration curve was constructed with Gallic Acid (Sigma Aldrich, Saint Louis, USA) with a range of 54.6–546 μg of Gallic Acid per mL, resulting in the following equation y = 0.00419 x + 0.0819 (R2 = 0.997). All determinations were performed in triplicate and results are expressed as mg of Gallic Acid Equivalents per 100 g of dried mass (mg GAE/100 g d.m.)
2.9. Extractable pigments To quantify the amount of extractable pigments present in the samples, a slightly modified methodology of Association of Official Analytical Chemists (AOAC)/ASTA Methods (Govindarajan et al., 2009) was used. Dried bell pepper samples (Red I and Red II) were passed through a sieve (US Standard Sieve Series No. 35; 500 microns, Chicago, ILL. USA), then 100 mg of powdered samples were put into a 100 mL flask with acetone, stirred and kept in darkness for 16 h. Next, these mixes were centrifuged at 4472g for ×15 min. A 2 mL sample of the supernatant was taken and read at a wavelength of 460 nm in a VISSpectrophotometer (Spectronic 20 Genesis, NY, USA). The standard is a solution of ammonium and cobalt sulfate (35 g L−1) plus potassium 3
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Table 1 Physicochemical composition of bell pepper (Capsicum annum) inoculated with Bacillus amyloliquefaciens harvested at different stages of maturity. Parameters 1
Moisture
2
Water Activity
1
Fat
1
Ash
1
Crude protein*
1
Carbohydrates
1
Reducing Sugars**
Mineral content (mg kg−1 d.m.) Cu Fe*** Ca K***
Green
Red I Ac
Red II Aa
C I C I C I C I C I C I C I
6.67 ± 0.39 7.27 ± 0.41Aa 0.349 ± 0.001Aa 0.357 ± 0.001Aa 0.98 ± 0.02Bc 1.12 ± 0.01Ab 8.12 ± 0.49Ab 8.49 ± 0.26Aa 0.26 ± 0.01B 0.38 ± 0.01A 90.64 ± 0.50Ab 90.01 ± 0.51Ac 21.39 ± 1.23a 20.79 ± 1.29a
10.52 ± 0.21 8.37 ± 0.54Ba 0.315 ± 0.001Ab 0.257 ± 0.004Bb 1.93 ± 0.02Aa 1.45 ± 0.03Ba 9.45 ± 0.61Aa 7.52 ± 0.19Bb 0.26 ± 0.01B 0.43 ± 0.04A 88.36 ± 0.58Bc 91.59 ± 0.59Ab 35.85 ± 0.76b 36.69 ± 0.75b
8.62 ± 0.54Ab 7.46 ± 0.38Ba 0.257 ± 0.001Ac 0.251 ± 0.002Bb 1.16 ± 0.06Bb 1.35 ± 0.07Aa 5.91 ± 0.28Ac 5.31 ± 0.13Bc 0.27 ± 0.01B 0.42 ± 0.02A 92.52 ± 0.64Aa 92.92 ± 0.53Aa 34.79 ± 1.71c 35.29 ± 1.27c
C I C I C I C I
131.93 ± 2.01Aa 106.35 ± 1.95Ba 376.00 ± 9.33Ba 399.17 ± 1.95Aa 955.68 ± 7.50Ba 1149.17 ± 27.32Aa 5423 ± 31.75Aa 2563 ± 27.37Bb
86.42 ± 1.26Bb 97.98 ± 1.06Ab 162.67 ± 9.04Bc 222.07 ± 11.07Ac 482.93 ± 40.91Ab 489.06 ± 37.15Ac 3127 ± 23.30Ab 3171 ± 30.38Aa
63.98 ± 1.10Bc 83.33 ± 2.33Ac 187.99 ± 12.89Bb 369.62 ± 15.19Ab 108.22 ± 5.56Bc 669.35 ± 45.44Ab 2966 ± 28.76Ac 2135 ± 33.18Bc
Ref: C: control no inoculated; I: inoculated. Values are expressed as mean ± standard deviation (n = 3). Different uppercase letters in the same column indicate significant differences (p < 0.05) according to LSD test among INOCULATION levels. Different lowercase letters in the same row indicate significant differences (p < 0.05) according to LSD (LSD) among MATURITY levels. 1 Expressed as g/100 g. 2 Dimensionless. * No significant interaction, MATURITY no significant as a factor. ** No significant interaction, INOCULATION no significant as a factor. *** No significant interaction, both MATURITY and INOCULATION were significant factors.
previous solution was diluted in 25 mL of phosphate buffer, obtaining a 100 nM solution. For the AAPH solution 976.3 mg of the reagent were added to 10 mL of buffer (0.36 M). 200 μL of the 100 nM solution was mixed with 40 μL of the extract (diluted 1:9 in phosphate buffer) and incubated (37 °C, 20 min). After that, 35 μL of the AAPH solution is added and the decay of the fluorescein is measured. A calibration curve was made using Trolox as the standard (in replacement of the extract) in a range of 6.25–200 μM, with a regression curve y = 0.000161 x – 21.9 (R2 = 0.972). All determinations were performed in triplicate and results are expressed as μmol of Trolox Equivalents per 100 g of dried mass (μmol TE/100 g d.m.).
2.13. Antioxidant activity by DPPH assay The antioxidant activity of the green bell pepper was determined according to Lafka et al. (2011) with minor modifications. A solution of DPPH radical (2,2-diphenyl-1-picrylhydrazyl, Sigma Aldrich, Saint Louis, USA) was prepared adding 50 mg of DPPH radical in 250 mL of methanol. 3.9 mL of that solution was mixed with 0.1 mL of the methanolic extract. After 30 min (dark, 20 °C), absorbance of the mixture was measured in a VIS-Spectrophotometer (Spectronic, 20 Genesis, NY, USA) at 517 nm. A calibration curve was constructed with Trolox radical (6-hydroxy-2,5,7,8-tetramethylchromomne-2-carboxyl acid, Sigma Aldrich, Saint Louis, USA) as a standard, with a range of 0.1–1.0 mM, resulting in the following equation y = −0.611 x + 0.549 (R2 = 0.998). All determinations were performed in triplicate and results are expressed as μmol of Trolox Equivalents per 100 g of dried mass (μmol TE/100 g d.m.).
2.15. Statistical analysis Results reported in this paper are LSMEAN values (least square mean, estimators of means by the method of least squares) with their standard deviations, obtained of three independent replica. Data were analyzed using InfoStat v.2.0 (2017). For all parameters, except Chlorophyll and Carotenoids Content, the variation factors were: INOCULATION (I: plants inoculated with B. amyloliquefaciens, C: plants without inoculation), MATURITY (Green I, Red I and Red II) and the corresponding interaction. A Two-Way ANOVA was performed. When significant differences among factors were found (p < 0.05), the LSD multiple comparison test (α < 0.05) was performed. For Chlorophyll and Carotenoids Content, MATURITY was not considered as a variation source, since only Green Peppers were analyzed, therefore a One-Way ANOVA was performed.
2.14. Antioxidant activity by ORAC assay The antioxidant activity by ORAC (Oxygen Radical Absorbance Capacity) assay was performed according to Zou et al. (2015). The extracts from the freeze dried samples of bell pepper were carried out following the same methodology as for the DPPH assay until the rotary evaporation of the solvent. In this case, after the evaporation, the dry extract is reconstituted in phosphate buffer (75 mM, pH 7) until 10 mL of final volume. Two solutions were prepared using phosphate buffer as diluent. The first one was a solution of fluorescein sodium (C20H10Na2O5, Sigma Aldrich, Saint Louis, USA) and the second, of AAPH reagent (2,2′-azobis (2-methylpropianamidine) dihydrochloride, Sigma Aldrich, Saint Louis, USA). The fluorescein sodium solution was prepared in two stages; initially 5 mg of the reagent were added to 25 mL of phosphate buffer. Finally, in a further dilution, 4.7 μL of the 4
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3. Results and discusion
Green stage, both for inoculated and control samples when compared to Red I and Red II. However, when maturity progresses, Cu content was lower and an increased was found for inoculated samples when compared to control. Cu is involved in the metabolism of carbohydrates and in the photosynthetic process, which could explain its gradual decrease as the fruit ripens. In a similar work (Khan et al., 2017), the application of PGPB with a mixture of compost and Trichoderma harzianum T22 in tomatoes, was also linked to a significant increase in the Cu concentration (from 36.85 mg kg−1 to 41.70 mg kg−1). Similar results are found for Ca content (Table 1), where Green stage presented higher values than Red stages, for inoculated or control samples. Then, Ca content gradually decreases in control samples in response to plant maturity process, while inoculated samples on Red II samples are higher. Salvatierra-Martinez et al. (2018) reported that B. amyloliquefaciens BBC047 can produce auxins like Indoleacetic Acid (IAA), which are closely linked to the transport of Ca in plants (Tadesse, 1997). In a similar study (Ekinci et al., 2014) the application of Bacillus subtilis in cauliflower also generated an increase in Ca concentration. For Fe content, no significant interaction was found, with both factor being a significant source of variation (Table 1). Fe content was higher in inoculated samples, in all ripening stages, if compared with control. As for maturity stages, Green presented higher Fe content, but Red II was higher than Red I, indicating that no clear tendency was followed during ripening. According to the characterization carried out by Salvatierra-Martinez et al. (2018), this strain (BBC047) does not produce siderophores. However, BBC047 may possess other mechanisms, which lead to increase the capture of this mineral. Khan et al. (2017) detected a significant increase in Fe concentration (from 195.85 mg kg−1 to 234.30 mg kg−1) applying an amendment of compost and Trichoderma harzianum T22 on tomato plants. Similar results were found for K content, no significant interaction was observed but both factors were (Table 1). Inoculated plants presented lower or equal values of K content than control plants. Similar results were obtainad by Esitken et al. (2009) after the inoculation of different PGPRs on strawberry and by Khan et al. (2017) in their study with compost and Trichoderma harzianum T22 on tomato. K concentration decreases irregularly from Green to Red II regardless inoculation. According to Tadesse (1997), this reduction of K content along ripening could be associated with the involvement of K in color development.
3.1. Physicochemical composition of bell pepper 3.1.1. Physicochemical composition Physicochemical composition of bell pepper at different maturity stages is presented in Table 1, for inoculated or non-inoculated samples. A significant interaction was found between INOCULATION and MATURITY for fat, and ash content (Table 1). There is no clear effect of inoculation on the fat content, while a slight increment is observed for Red I and Red II compared to Green. These results may indicate a greater influence of maturation over inoculation. In a similar work, the Rhizombium TPV08 and PETP01 strains were applied to the pepper plant (Silva et al., 2014) and it was found that the TPV08 strain generated significant increases in the fatty acid content of the fruit and leaves of the plant, which could suggest an increase in the percentage of lipids. Although an actual trend is not manifested, data suggested that the increase in the percentage of lipids in Red stages may be due to a higher synthesis of fatty acids that will allow the development and maturation of the fruit. Even less clear is the effect on ash content, with very variable results, affected both by maturity and inoculation. The higher ash content was found for Green in Inoculated samples, and for Red I for Control samples. As ash values do not manifest a predictable trend or behavior, it could only be implied that the mineral content of the samples varies as the ripening process progresses. No significant interaction was found for crude protein content, with only INOCULATION being a significant factor (Table 1). A clear increment was observed in inoculated samples for all maturity stages, compared to CONTROL samples. The increase in the protein content could be associated to increase nitrogen reductase enzymatic activity or production of Indolacetic acid (Salvatierra-Martinez et al., 2018), which would stimulate the growth and development of the plant. In general, total carbohydrates were not affected by inoculation, except in Red I where a slight increment was found (Table 1). On the other hand, ripening of the fruit did change the total carbohydrate content in the bell pepper samples, but without a clear trend as maturity progress. Changes in total carbohydrates can be associated with enzymatic processes during the maturation process, which could imply modifications of the cell wall composition and an increase in the enzymatic activity of carbohydrates (Tan et al., 2013)
3.2. Photosynthetic pigments and extractable color 3.1.2. Reducing sugars For Reducing Sugars only MATURITY was a significant factor (Table 1). An increment was observed for Red I and Red II compared to Green fruits. All reducing sugar values, for both control and inoculated samples, increased significantly as ripening progresses. In the work developed by Ghasemnezhad et al. (2011), in which five varieties of pepper were evaluated in two stages of maturation, it was determined that the percentage of total soluble solids increased in all ripened samples with respect to the less mature peppers. The authors pointed out that while fruit ripens, the degradation of pectins and cellulose occurs, which then are used in the ripening process for the biosynthesis and accumulation of sugars. Together with the increase in the percentage of soluble solids, a significant increase in total acidity was also reported for all pepper varieties during ripening (Ghasemnezhad et al., 2011). The increase in acidity of this study is based on the increase in organic acids content, mainly related to those present in the Krebs cycle due to increased metabolism during the maturation process (Chitarra and Chitarra, 1990)
3.2.1. Chlorophyll content Chlorophyll a, b and c values are presented in Fig. 1. These values are only presented for Green, both inoculated and non-inoculated. Inoculation did not affect Chlorophyll content of green Bell peppers, with mean values of 101.9, 32.3 and 5.4 μg g−1 dm for Chlorophyll a, b and c, respectively. As no effect was observed in Chlorophyll content, inoculation with B. amyloliquefaciens will no interfered with normal photosynthesis of the plant or with the antioxidant capacity that could be associated to the pigment. 3.2.2. Carotenoids Carotenoids content results are presented in Fig. 1, also for Green peppers only. As in Chlorophyll content, no effect of inoculation was observed in Carotenoids content, with a mean value of 35555 μg g−1 dm. Howard et al. (2000) determined that carotenoids (measured as αcarotene, β-carotene and zeaxanthin) increased during the maturation process in different varieties of peppers (Capsicum annuum). It is also known that the increase in the concentration of carotenoids is a function of the ripening process due to the synthesis of pigments generated by the change in color of the fruit. However in our study, carotenoids were measured indirectly through the determination of photosynthetic pigments only in Green stage, without considering Red I and Red II (in which the color change would have been evident).
3.1.3. Mineral content Mineral content of the bell pepper samples is presented in Table 1. For inoculated and control treatments Cu and Ca content were affected both by inoculation and the different maturity stages at harvest, presenting a significant interaction. Cu content was significantly higher in 5
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3.3. Bioactive compounds content 3.3.1. Vitamin C Results of Vitamin C quantification are presented in Fig. 2. Both inoculation and maturity at harvest produced changes in Vitamin C content, with a significant interaction. Vitamin C is an antioxidant compound whose functions and benefits are widely documented (Howard, 1994; Loewus, 1999; Valpuesta and Botella, 2004). Among them, its importance in physiological processes and its antioxidant role in climacteric fruits stand out (Gnayfeed et al., 2001). Inoculation clearly increased Vitamin C content in Green bell peppers (a 434 % increase for inoculated samples respect to control). However, in all other maturity stages, Vitamin C content in inoculated samples was the same or lower than in control samples. The increment of vitamin C content in Green samples, may suggest that ascorbic acid is fulfilling its antioxidant role within the fruit while in the process of development and ripening, which could be associated to several oxidative stress reactions. This hypothesis is based in that Vitamin C is not less produced by control plants but it is being used (Goñi et al., 2010). When considering ripening stages at harvest, vitamin C showed an increasing trend, from Green to Red I, with a slight reduction in Red II. These results are in agreement with a study evaluating the bioactive compounds in Paprika (pungent spice red pepper), by Gnayfeed et al. (2001). Authors noted that ascorbic acid (together with α-tocopherol) accumulates progressively during the maturation process of fruit, reaching its maximum amount just when the pepper was in its ripe-red state. Then, when reaching the state of over-ripe, the amount of ascorbic acid decreases due to the loss of water and the beginning of the process of senescence of the fruit. This increase could be associated to the concentration of reducing sugars, which also reach their maximum in Red I. As a result, the increase in ascorbic acid could be a response for the synthesis from reducing sugars such as D-fructose-6-P or D-glucose1-P (Valpuesta and Botella, 2004). Ghasemnezhad et al. (2011) evaluate the amount of biocomponents present in pepper at two different harvest dates. Authors affirm that ripening process increases total acidity due to the number of metabolic reactions related to the Krebs cycle and to the synthesis of several pigments and enzymes.
Fig. 1. Pigments content of bell pepper (Capsicum annum) inoculated with Bacillus amyloliquefaciens harvested at different stages of maturity. *Different uppercase letters indicate significant differences (p < 0.05) according to ANOVA test with two INOCULATION levels (for Chlorophyll content and Carotenoids). **Significant interaction in the Two Way ANOVA (p < 0.05). Different uppercase letters in the same MATURITY level indicate significant differences (p < 0.05) according to LSD (LSD) among INOCULATION levels. Different lowercase letters in the same INOCULATION level indicate significant differences (p < 0.05) according to LSD (LSD) among MATURITY levels (for Extractable Pigments).
3.3.2. Total phenolic content The Total Phenolic Content (TPC) was measured for bell peppers inoculated with B. amyloliquefaciens in different ripening stages and results are presented in Fig. 2. A significant interaction was also observed, with effect of both factors. Inoculation only showed a slight increase in total phenolic content in Green (20 % increase compared to control), while no differences were found in Red I (only 6 % of increase in control samples) and higher values in Red II for control compared to inoculated samples (60 % increase). Accordingly, the application of Trichoderma harzianum T22 in grape crops (Pascale et al., 2017) managed to significantly increase Total Phenolic Content, suggesting that the increase was generated by the strain as the result of the synthesis of plant hormones (auxins for example). In this study, B. amyloliquefaciens inoculation might be able to produce IAA (indoleacetic acid) with similar effects Respect to maturity, Red I had the higher value of Phenolic content for all stages, both inoculated and non-inoculated. A decrease in total phenolic content was observed in Red II, respect to Red I with continuing ripening. Similar results were presented by Conforti et al. (2006) where variation of biochemical composition of the fruits were found in different ripening stages, with lower content on ripened samples. In contrast, in the work presented by Ghasemnezhad et al. (2011) different pepper varieties showed higher Total Phenolic Content in early harvest samples compared to fully rip ones. This phenomenon has also been documented in another study by Ekinci et al. (2014). These authors suggested that when there is synthesis of amino acids
3.2.3. Extractable color The amount of Extractable pigments in each of the Red stages of maturity is presented in Fig. 1. A significant interaction was observed for this parameter. Results for Extractable color showed that Red II were higher than those for Red I crop, regardless of inoculation, which is in agreement with a greater synthesis of pigments as ripening develops in more advanced maturity stages. On the other hand, inoculation with B. amyloliquefaciens had a different effect in the amount of Extractable pigments on each development stage: it increased for Red II but it was lower in Red I. In Red I the control presented higher values of Extractable color than inoculated samples (117.6 and 87.6, respectively), while in Red II inoculation increased significantly the content of extractable color (244.7 and 187.6, respectively). The increment in the Extractable color value can be directly associated to the increase in pigment content. Fox et al. (2005), stated that carotenoids (expressed as mg of capsantin kg−1 of dry mass) increased from 79.6 mg to 121.9 mg for 30 % red and 100 % red, respectively.
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Fig. 2. Bioactive compounds content and antioxidant capacity of bell pepper (Capsicum annum) inoculated with Bacillus amyloliquefaciens harvested at different stages of maturity. *Significant interaction in Two Way ANOVA (p < 0.05) with INOCULATION and MATURITY as factors.
The antioxidant activity measured by ORAC methodology presented a different trend than by DPPH, also with a significant interaction among factors. Inoculation with B. amyloliquefaciens was able to increase antioxidant capacity by ORAC on Green bell peppers (33 % increase) and Red I (11 % increase), but no significant differences were found in Red II stage (mean value of 4230 μmol TE 100 g−1 dm). Considering the effect of maturity, less prominent differences were found, being Green only slightly higher than Red I or Red II. For ORAC antioxidant methodology no clear effect of maturity was detected. In general, antioxidant activity should follow the same trend as bioactive compounds that are responsible for such antioxidant activity. That is not the case for our results, where DPPH, ORAC, vitamin C and total phenolic content presented significant differences in behavior, both for ripening or inoculation. However, many other factors could interfere and make difficult that association. Similar results were presented by Fox et al. (2005), who working with different varieties of bell peppers at different stages of ripening, also found ORAC antioxidant activity to be extremely variable, and their results did not comply with the increased expected with ripening. This is why more than one methodology should be employ to evaluate antioxidant activity in complex systems like vegetables, and biochemical characterization of the food matrix is extremely relevant to make conclusions on the effect of different practices. Some changes in DPPH antioxidant capacity, especially those related to break color (Red I), could be directly associated to an increase in total phenolic content and Vitamin C content, but a different trend was observed for ORAC.
(such as phenylalanine, tyrosine), these could enter the metabolic pathway of the shikimic acid, acting as precursors of phenolic acids, such as gallic, ferulic and p-cumaric acids. In a previous work (Howard et al., 2000) changes in phytochemical composition and antioxidant activity of Capsicum annuum Yellow Bell as a consequence of the ripening were evaluated. Total Phenolic Content was significantly higher for the immature crop, while decreasing in the mature crop. However, in another variety Capsicum annuum Cascabella, total phenolic content was lower in the immature crop and higher in the mature crop. Similar behavior occurred with the Inferno and Mesilla varieties. These results suggested that variation in the total phenolic content also responds to the genotype of each pepper variety, which make difficult to extrapolate results among different kinds of pepper. 3.4. Antioxidant activity The antioxidant activity of bell pepper was measured in different maturity stages, on inoculated and control samples. Results are presented in Fig. 2 for both DPPH and ORAC methodologies. It is common to measure antioxidant activity by more than one methodology so different mechanisms could be evaluated and different trends were observed in the present study for each methodology. The antioxidant activity measured by DPPH presented an increment in value from Green to Red I, with a reduction in the last stage (Red II), regardless of the inoculation of the seeds. When considering inoculation, only Green presented a slight increment in antioxidant activity if compared to control samples (22 % increment). In all other ripening stages, control samples presented higher antioxidant activity than inoculated. The increase in antioxidant activity as a function of ripening was also described by Howard et al. (2000) were most of the species tested showed a significant increase in antioxidant activity in mature crop compared to less mature ones. These results were associated to the increased in the amount of several bioactive compounds, such as ascorbic acid, flavonoids or phenolic acids. Moreover, a similar trend can be observed between vitamin C content and DPPH antioxidant activity. The higher value of total phenolic content also corresponds to the higher value of antioxidant activity by DPPH.
4. Conclusion Root inoculation of the pepper plant with Bacillus amyloliquefaciens BBC047 increases the content of bioactive compounds, such as vitamin C and total phenols only at early stages of maturity (Green). In later stages of maturation there is no longer an effect of inoculation but of the metabolic process of maturity. On the other hand, the effect of inoculation on the fixation of Ca and Fe is clear, with increased values with respect to the control. The color changes that accompany the maturation process are considered important from the point of view of consumer acceptance, but here they also related to the pigment content 7
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and its relation to nutritional intake. These indicators can easily be used to define harvest stages and thus determine the potential destinations of a given lot. The physiological changes resulting from the ripening process are significant in the fruits of bell pepper, especially due to changes in the metabolism of carbohydrates. As a conclusion, it is possible to affirm that root inoculation of pepper plants with Bacillus amyloliquefaciens BBC047, in addition to its plant growth promotion effect, could also modulate the maturation process, which would allow the producers to better control and select harvest time, since ripening could be scheduled in advance.
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CRediT authorship contribution statement Jonathan Cisternas-Jamet: Conceptualization, Methodology, Writing - original draft. Ricardo Salvatierra-Martínez: Investigation, Methodology, Validation. Antonio Vega-Gálvez: Funding acquisition, Supervision. Alexandra Stoll: Conceptualization, Writing - review & editing, Supervision. Elsa Uribe: Supervision, Resources. María Gabriela Goñi: Conceptualization, Formal analysis, Visualization, Writing - review & editing. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments This work was financed by Projects FIC 2015 30403034 and FIC 2013 30137771-0. We would also like to mention the collaboration of Stefanie Maldonado, Seedling Company SA. The autor SalvatierraMartínez gratefully acknowledges the Comisión Nacional de Investigación Científica y Tecnológica of Chile (CONICYT) for a PhD scholarship (21140504). References Adesemoye, A.O., Torbert, H.A., Kloepper, J.W., 2009. Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. Microbiol. Ecol. 58, 921–929. https://doi.org/10.1007/s00248-009-9531-y. Ali, S., Hameed, S., Imran, A., Iqbal, M., Lazarovits, G., 2014. Genetic, physiological and biochemical characterization of Bacillus sp. strain RMB7 exhibiting plant growth promoting and broad spectrum antifungal activities. Microb. Cell Fact. 13, 144. https://doi.org/10.1186/s12934-014-0144-x. Álvarez-Parrilla, E., De La Rosa, L.A., Amarowicz, R., Shahidi, F., 2011. Antioxidant activity of fresh and processed jalapeño and serrano peppers. J. Agric. Food Chem. 59, 163–173. https://doi.org/10.1021/jf103434u. Babu, A.N., Jogaiah, S., Ito, S., Nagaraj, A.K., Phan Tran, L., 2015. Improvement of growth, fruit weight and early blight disease protection of tomato plants by rhizosphere bacteria is correlated with their beneficial traits and induced biosynthesis of antioxidant tperoxidase and polyphenol oxidase. Plant Sci. 231, 62–73. https://doi. org/10.1016/j.plantsci.2014.11.006. Bacilio, M., Moreno, M., Bashan, Y., 2014. Mitigation of negative effects of progressive soil salinity gradients by application of humic acids and inoculation with Pseudomonas stutzeri in a salt-tolerant and a salt-susceptible pepper. Appl. Soil Ecol. 107, 394–404. https://doi.org/10.1016/j.apsoil.2016.04.012. Bae, H., Jayaprakasha, J., Crosby, K., Yoo, K.S., Leskovar, D.I., Jifon, J., Patil, B.S., 2014. Ascorbic acid, capsaicinoid, and flavonoid aglycone concentrations as a function of fruit maturity stage in greenhouse-grown peppers. J. Food Compos. Anal. 33, 195–202. https://doi.org/10.1016/j.jfca.2013.11.009. Briones-Labarca, V., Venegas-Cubillos, G., Ortiz-Portilla, S., Chacana-Ojeda, M., Maureira, H., 2011. Effects of high hydrostatic pressure (HHP) on bioaccessibility, as well as antioxidant activity, mineral and starch contents in Granny Smith apple. Food Chem. 128, 520–529. https://doi.org/10.1016/j.foodchem.2011.03.074. Cezón, R., Gutierrez Mañero, F.J., Probanza, A., Ramos, B., Lucas-García, J.A., 2003. Effects of two plant growth-promoting rhizobacteria on the germination and growth of pepper seedlings (Capsicum Annum) cv. Roxy. Arch. Agron. Soil Sci. 49, 593–603. https://doi.org/10.1080/03650340310001620123. Chakraborty, U., Chakraborty, B., Basnet, M., 2006. Plant growth promotion and induction of resistance in Camellia sinensis by Bacillus megaterium. J. Basic Microbiol. 46 (3), 186–195. https://doi.org/10.1002/jobm.200510050. Chitarra, M.I.F., Chitarra, A.A., 1990. Pos-colheita de frutas e hortalicas. Fisiologia e manuseio 293. Choudhary, D.K., Johri, B.N., 2009. Interactions of Bacillus spp. and plants – with special
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