extfieldsofstudy: Chemistry Medicine

text:
Quality Parameters of Juice Obtained from Hydroponically Grown Tomato Processed with High Hydrostatic Pressure or Heat Pasteurization

The effect of processing such as high hydrostatic pressure (HHP) (400-600 MPa/15 min) or low pasteurization temperature (LPT) (74°C/2 min) or high pasteurization temperature (HPT) (90°C/1 min) on selected quality parameters of juice obtained from hydroponically cultivated beef tomatoes was investigated. The total polyphenols content (TPC), total phenolic index (TPI), Trolox equivalent antioxidant capacity (ABTS) and ferric reducing antioxidant power (FRAP) were analysed in the fresh and processed juices stored for 0, 7 and 14 days. What is more, colour parameters (L∗, a∗, b∗, ∆E), the activity of polyphenol oxidase (PPO) and peroxidase (POD) and microbial stability were also analyzed following the juices storage. Among all the tested samples, the juice exposed to 600 MPa for 15 min showed superior quality. Samples treated with 600 MPa for 15 min and stored for 0, 7 and 14 days had high TPC, TPI, ABTS, FRAP and a∗ values. As demonstrated, these tested samples at the end of the storage period retained 90% and 95% of their polyphenol content and antioxidant capacity, respectively. As in the case of pasteurization, juice processing at 600 MPa for 15 min clearly reduced the activity of food-spoiling enzymes (PPO, POD) as well as the microbial count. The obtained results showed that TPC was significantly and positively correlated with TPI, ABTS and FRAP parameters.

Introduction
Tomato polyphenols are known to contribute significantly to the nutritional quality of tomato-based products [1]. The activity of plant enzymes such as polyphenol oxidase (PPO) and peroxidase (POD) results in the degradation of polyphenols, leading to browning discolouration particularly in fresh or lightly processed products [2]. Almost 60% of the annual production of tomatoes is highly processed into ketchups, juices, puree, or other preserves. Processing involving temperature treatment distinctly enhances the enzymatic and microbiological stability of tomato-based products. On the other hand, it may lead to substantial changes in the quantity and quality of thermolabile phytochemicals [3]. Tomato pulp subjected to pasteurization demonstrated 15% and 20% decreases in rutin and chlorogenic acid contents, respectively. However, the content of naringenin chalcone was completely lost after the canning process and the pasteuriza-tion (93°C) of the tomato pulp [4]. L-ascorbic acid is likely one of the more reactive compounds present in tomato; thus, it is particularly vulnerable to thermal processing [5]. Thermal treatment not only results in the loss of labile bioactive compounds but also may affect product properties such as flavour, colour, or texture. As indicated, thermally treated tomato juices showed undesirable changes in colour parameters [6,7]. The distinct changes in L * , a * , b * or ΔE values observed in the cited above reports for heat-treated samples resulted not only from the formation of Maillard reaction products but also from decomposition of thermolabile phytochemicals, which occurred upon processing.
Compared to heat treatment, HHP provides better retention of antioxidants from the food matrix already at ambient temperature. What is more, the polyphenols of fruits or vegetables as well as their antioxidant properties can be preserved more effectively by high hydrostatic pressure (HHP) than by thermal treatment [8]. Considering above, HHP processing can be an alternative for heat treatment in the context of food quality and nutritional value. Tomato juice (pH 3.9) exposed to 600 MPa for 1 min demonstrated a higher total phenolic content (20%) and antioxidant activity (24%) than thermally treated tomato juice [9]. A tomato sample treated at 600 MPa for 15 min showed higher (by almost 9%) total phenolic content and higher (by 38%) antiradical power as compared to the thermally processed puree [10]. What is more, a number of attempts have been made to use HHP instead of high-temperature treatments to inactivate food-spoiling microorganisms [11,12] and unwanted food enzymes [13]. The results obtained by Terefe et al. [2] indicated that the optimal conditions to inactivate POD in strawberries were 600 MPa at 60°C for 10 min, but no substantial PPO inactivation was noted when these conditions were applied. Marszałek et al. [14] showed that pressurization (500 MPa) of strawberry puree at 50°C after 15 min was required to achieve 72% and 50% PPO and POD inactivation, respectively.
As indicated, some critical aspects regarding plant variety selection and the conditions of the HHP process must be considered when applying HHP to obtain novel products with high nutritional quality [15].
The effects of HHP processing on the quality of tomato juice have been extensively studied in the literature [1,9,15]. However, the quality parameters of tomato-based products obtained from fruit grown in a hydroponic system remain insufficiently investigated. Currently, hydroponic systems are considered to be one of the most profitable and popular systems in crop production [16]. On the other hand, hydroponic crops may significantly differ in terms of their quality parameters, especially in terms of the content of antioxidants or the activity of food spoiling enzymes, compared to crops obtained using conventional systems. Therefore, this study evaluated the effect of HHP treatment (400-600 MPa/15 min) on the phenolic content, antioxidant capacity, colour parameters, and PPO and POD activity as well as the microbial stability of juice obtained from hydroponically cultivated beef tomatoes. All the aforementioned evaluations were also performed on juices subjected to a low pasteurization temperature (LPT, 74°C/2 min) or a high pasteurization temperature (HPT, 90°C/1 min). The quality parameters of the tomato juices treated with HHP, LPT, and HPT were also examined upon sample storage at 6 ± 2°C for 0, 7, and 14 days.

Sample Preparation.
The mature tomatoes of the Beef variety grown in a hydroponic system were purchased from the Łegajny greenhouse complex, Łegajny, Poland. The juice was produced from fresh fruits using a juicer (domestic appliance, BOSCH CNCJ04, Germany) equipped with a ceramic blade. The seed and skin were automatically separated upon juice processing. The fresh juice was directly subjected to high hydrostatic pressure treatment or thermal pasteurization.
2.3. High Hydrostatic Pressure (HHP) Process. The juice samples were enclosed in Teflon tubes (50 mL), deaerated, tightly sealed, and subjected to HHP using a high-pressure system (Unipress U-303, Warsaw, Poland). The Teflon tubes were put into a high-pressure chamber with a capacity of approximately 100 mL filled with a pressure-transmitting medium (water-propylene glycol (propane-1,2-diol), 1 : 1, v/v), which also minimized adiabatic heating. The samples were pressure-treated at 400 or 600 MPa for 15 min. The compression and decompression rates were 8 MPa/s and 10 MPa/s, respectively. The sample temperature reached 32 ± 1:5°C at a pressure of 400 MPa and 38 ± 1:5°C at a pressure of 600 MPa. Data acquisition software (OMEGASOFT, OMB DAQ-54, OMEGA Inc., Hungary) was used to collect all the parameters of the HHP treatment. The analyses were carried out in triplicate for each treatment.
A thermocouple positioned at the juice cold point was used to control the temperature. The time for the juice to reach the required temperature was less than 5 min. Once the pasteurization temperature was reached, the juice samples were immediately cooled to room temperature by immersing the bottle in a water/ice mixture. The analyses were carried out in triplicate for each treatment.

Storage Condition.
Nonprocessed tomato juice was used as a reference sample. The untreated and HHP-, LPT-, and HPT-treated juices were stored at 6 ± 2°C in the dark and analysed after 0, 7, and 14 days of storage.
2.6. Extraction Procedure. The juices were lyophilized prior to the extraction of phenolic compounds (Labcono 195, England), and the extraction procedure was performed using a water : methanol mixture (20 : 80, v/v). The juice powder and the extraction mixture (1 : 20, w/w) were treated at 70°C for 15 min under continuous shaking using a water bath (Julabo SW 22, Germany). The process was repeated three times. The extracts obtained were combined, filtered through filter paper, and concentrated under reduced pressure on a rotary evaporator (Buchi Rotavapor R-200, Switzerland) with a warm water bath at 54 ± 2°C and lyophilized. The freezedried extracts were stored at -24°C until analysis. International Journal of Food Science and Ciocalteu's phenol reagent [17]. After colour development, the absorbance was measured at 725 nm (Beckman DU® 7500 spectrophotometer, California, USA) after sample incubation (TH-24 block heater, Meditherm, Poland) at 20°C for 20 min. The results were expressed as mg of gallic acid (GAE)/g of juice dry matter (dm). The linearity range of the calibration curve was from 0.062 to 0.50 mg/mL (R 2 = 0:999).
2.8. Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC). For the RP-HPLC fingerprint analysis of individual phenolic compounds present in the tomato juice extract, a Shimadzu system (Shimadzu Corp., Kyoto, Japan) consisting of two LC-10AD pumps, an SCTL 10A system controller, an SPD-M 10 A photodiode array detector, and a prepacked LUNA C 18 column (4 × 259 mm, 5 μm, Phenomenex) was used. A flow rate of 1 mL/min, injection volume of 20 μL, a gradient elution of acetonitrile-water-acetic acid (5 : 93 : 2, v/v/v) (solvent A) and acetonitrile-water-acetic acid (40 : 58 : 2, v/v/v) (solvent B), and a 0-50 min solvent B from 0% to 100% were applied [18]. The tomato juice extract was dissolved using a water : methanol mixture (20 : 80, v/v) and filtered through a 0.45 μm filter (Chromafil Xtra PET-45/25, Macherey-Nagel). The separation of compounds was monitored at 260 and 320 nm. The identification was performed based on the retention times and the UV spectra of the standards and the samples.
2.9. Trolox Equivalent Antioxidant Capacity (ABTS ⋅+ ). The method described by Re et al. [19] was used to determine the TEAC of tomato juice extracts.
To perform the measurements, the ABTS ⋅+ solution was diluted with a water : methanol mixture (20 : 80, v/v) to an absorbance level of 0:70 ± 0:02 at 734 nm. For the spectrophotometric assay, 1.48 mL of the ABTS ⋅+ solution and 20 μL of the respective extract, Trolox or blank (80% methanol), were mixed, and absorbance was measured at 734 nm (Beckman DU® 7500 spectrophotometer, California, USA) directly after sample incubation (TH-24 block heater, Meditherm, Poland) at 30°C for 6 min. The results were expressed as μmol Trolox equivalents/g of juice dm. The linearity range of the calibration curve was from 0.0 to 2.0 mM (R 2 = 0:999).

Ferric
Reducing Antioxidant Power (FRAP). The FRAP assay was carried out according to the procedure described by Benzie and Strain [20]. A Fe 3+ -TPTZ complex was generated at pH 3.6 (300 mM acetate buffer) by mixing 10 mM TPTZ in 40 mM HCl and 20 mM ferric chloride (1 : 1, v/v). Then, 75 μL of tomato juice extract or blank (80% methanol) and water (225 μL) were added to the complex solution (2.25 mL). The absorbance was read at 593 nm (Beckman DU® 7500 spectrophotometer, California, USA) after sample incubation (TH-24 block heater, Meditherm, Poland) at 37°C for 30 min. Trolox was used to prepare a calibration curve (from 0.0 to 1.0 mM; R 2 = 0:999). The results were expressed as μmol Trolox equivalents/g of juice dm.
2.11. Colour Measurements. The colour of fresh, untreated, HHP-, LPT-, and HPT-processed tomato juices at 0, 7, and 14 days of storage was analysed using the colour space Color-Flex (HunterLab, USA). The following colour parameters were determined: L * (represents lightness, where L * = 0 (black) and L * = 100 (white)), a * (-a * = greenness and +a * = redness), and b * (-b * = blueness and +b * = yellowness). A standard white and black plate was used to calibrate the instrument before analysis. The obtained results were expressed in accordance with the CIELab system with reference to the standard illuminant D65 and a visual angle of 10° [21].
Additionally, in the processed and stored tomato juice, the total colour differences (ΔE) were calculated using the following equation [22]: where ΔL, Δa, and Δb are the differences in L * , a * , and b * values between the untreated (control) sample and the treated sample on a particular day of storage.
The absorbance of the mixture was measured at 420 nm (for the PPO assay) and at 485 nm (for the POD assay) at 25°C for 15 min using a UV-visible spectrophotometer (UNI-CAM Heλios Alpha & Beta, Cambridge, UK).
A blank sample was prepared in the same way by substituting the supernatant with 0.2 M phosphate buffer. The PPO and POD activity was expressed as a change in absorbance at the respective wavelength ΔOD/min/g of fresh weight (fw) of the analysed sample.
The residual activity (%) of the enzymes studied was calculated as follows: where A 0 represents the activity of the enzyme in the untreated (reference) sample and A represents the enzyme activity in the treated sample.
2.13. Microbiological Evaluation. The total aerobic mesophilic count (TAMC), total yeast count (TYC), and total mould count (TMC) were analysed in the fresh, untreated juice and 3 International Journal of Food Science in the juices subjected to processing (HHP, LPT, and HTP) upon storage at 6 ± 2°C for periods of 0, 7, and 14 days.
A sample of juice (10 mL) was diluted with 90 mL of saline peptone (SP, 1 g/L peptone, 8.5 g/L NaCl) and homogenized in a stomacher (Model 400, Seward, London, UK) at a regular speed for 2 min.
To determine the TAMC of the juice samples, the homogenates were serially diluted and plated on plate count agar (Merck, No. 105463), followed by incubation at 30 ± 1°C for 3 days. Mould and yeasts were determined by plating the homogenates in dichloran rose bengal chloramphenicol agar (Merck, No. 100466), followed by incubation at 25 ± 1°C for 5 days.
After incubation, the plates were counted, and the results were expressed as colony-forming units per 1 mL (CFU/mL). The determinations were carried out in triplicate. The detection limit was <1 CFU/mL.
2.14. Statistical Analysis. Data are presented as the mean ± standard deviation ðSDÞ and were processed statistically using Statistica 13 software (Statsoft, USA). Differences between the obtained data were investigated by one-way analysis of variance (ANOVA) and Tukey's test (p ≤ 0:05).
Pearson's correlation analysis was carried out to determine the correlation between all the studied parameters (TPC, TPI, ABTS, FRAP, L * , a * , b * , ΔE, PPO, and POD). Principal component analysis (PCA) was also carried out to analyse the original variables (TPC, TPI, ABTS, FRAP, L * , a * , b * , ΔE, PPO, and POD) and to replace them with the appropriate number of components. The PCA was based on a correlation matrix.

Results and Discussion
3.1. The Effects of Processing on the Total Phenolic Content (TPC) and the Total Phenolic Index (TPI). The total phenolic content (TPC) obtained for methanolic extracts of tomato juices subjected to HHP (400-600 MPa/15 min), LPT (74°C/2 min), and HPT (90°C/1 min) processing is presented in Table 1.
The TPC measured in the extract obtained from the untreated tomato juice constituted 7.08 mg GAE/g dm. The content of phenolics presented in our study is comparable to the data obtained for juices produced from conventionally grown tomatoes [23]. As noted by these authors, the TPC found in commercially available tomato juices ranged from Table 1: Effect of processing on the total phenolic content (TPC), total phenolic index (TPI), and antioxidant capacity (ABTS, FRAP) in untreated, HHP-treated, and pasteurized (LPT and HPT) tomato juice after 0, 7, and 14 days of storage. The results are expressed as the mean ± SD, n = 6. * Uppercase letters indicate differences between treatment types (in columns) on particular days of storage, whereas lowercase letters indicate differences between storage times for individual samples (in lines) (p ≤ 0:05).

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International Journal of Food Science approximately 2.0 to 9.4 mg GAE/g dm. On the other hand, the concentration of polyphenols in the fresh, untreated juice was almost 30% and 40% higher than that found in hydroponically grown tomatoes [24,25]. However, as demonstrated in the literature, the plant genotype and growth conditions are key determinants of the nutritional quality of the obtained product [26]. As shown in our study, pressurization did not significantly modify polyphenol content in the tested juices. In contrast, the fresh juices subjected to LPT and HPT yielded 21% and 31% reductions in TPC, respectively. Despite such a marked decrease, the polyphenol concentration in the tested juices did not differ significantly from that (4.59-8.67 mg gallic acid/g juice, dm) measured in commercially available, heat-pasteurized juices [27]. Our results are also in accordance with another reference in which a 22% decrease in TPC was observed after juice processing at 90°C for 1 min [9]. Similarly, only a slight variation (6% decrease) in TPC was found by these authors after juice treatment with HHP (600 MPa/1 min).
Among all the treated and stored (for 7 and 14 days) samples, the juice exposed to 600 MPa/15 min showed the highest TPC ( Table 1). The value obtained for the tested juice on day 14 of storage was more than 10% and 30% higher than the values noted for the LPT-and HPT-treated juices, respectively. The obtained results clearly indicated that the HPTtreated juice showed a distinct reduction in TPC (by 15%) compared to the untreated sample. In contrast to our results, Vallverdu-Queralt et al. [23] found a minor variation (2-5% drop) in TPC in pasteurized tomato juices stored for 3 months.
Considering the results obtained for individual samples (Table 1), it is evident that LPT-and HPT-treated juices showed slight, insignificant variations in TPC throughout the entire storage period. At the end of the storage period, the juices exposed to HHP processing had 21% and 10% lower TPC values than those of the fresh samples exposed to 400 MPa/15 min and 600 MPa/15 min, respectively.
A more substantial drop (by 29% and 39%) in TPC was noted in the pasteurized and HHP-treated juices, respectively, after 1 month of storage [9]. Considering the above, it is evident that not only the processing and storage conditions but also the raw material used in the production step substantially affected the obtained values.
The total phenolic index (TPI) was quantified as the sum of the individual components identified in the analysed extracts using HPLC by comparing their retention time and UV spectra against the reference standards ( Table 1).
As a result of processing, TPI decreased by 9% and 18% in the LPT-and HPT-treated juices, respectively, compared to that in the untreated sample (153.57 μg/100 g dm). However, the pressurized juices showed no substantial changes in TPI values. Similarly, after 7 days of storage, the juices exposed to HHP or LPT treatment showed no significant changes in TPI values compared to those in the untreated sample. In contrast, an almost 40% drop in TPI was noted in the HPT-treated juice during that storage period. Similarly, almost half of the polyphenol concentration in the HPT-treated juice was lost after 14 days of storage. However, the pressurized juices had 9% and 48% higher TPI values (on average) than those noted for the reference and HHP-treated juices, respectively. A similar trend was observed in the work of Dede et al. [6]. In the cited study, the heat-treated juice demonstrated a 70% loss of antioxidants during 30 days of storage, whereas only slight changes were noted in the stored and HHP-treated juices. The polyphenol quantification by HPLC showed clearly that only the HHP-treated juices maintained individual compounds at high levels during the entire storage period.
3.2. The Effects of Processing on Antioxidant Capacity. The antioxidant capacities of the hydrophilic extracts of the analysed tomato juices determined in the ABTS and FRAP tests are presented in Table 1.
The ABTS value obtained for the juices exposed to HHP or LPT did not change significantly compared to that of the untreated juice (8.44 μmol Trolox/g juice dm) ( Table 1). In contrast, the antioxidant capacity of the HPT-treated juice was approximately 26% lower than the value noted for the reference sample. Similarly, Jayathunge et al. [9] found that pressurized (600 MPa/1 min) fresh tomato juices successfully retained their capacity to scavenge ABTS radicals. These authors also found that thermally processed (90°C/2 min) juice showed a significant reduction (by approximately 15%) in ABTS values.
In the first 7 days of storage, the pressurized (400 MPa/ 15 min) juice and LPT-treated juice showed nonsignificant variation in their ABTS values compared to those of the stored reference material (Table 1). However, a 15% drop in antioxidant capacity was noted for the HPT-treated stored juices. Among all the samples tested after 7 days of storage, the juice processed at 600 MPa/15 min had the highest ABTS value. After 14 days of storage, the juices treated with HHP and LPT were characterized by similar, still relatively high ABTS values, which were on average 40% higher than the value noted in the HPT juice.
We also found no changes in the ABTS value of the LPTtreated juice after 14 days of storage compared to the fresh, pasteurized juice. Similarly, 92% of the antioxidant potential of the juice exposed to 600 MPa/15 min was retained. However, the juices treated with 400 MPa/15 min and HPT retained 85% (on average) of their ability to scavenge ABTS radicals. As found by Fernandez Garcia et al. [28], tomato puree exposed to HHP treatment (500-800 MPa/5 min) retained 60-70% of its antioxidant capacity after 21 days of storage.
The antioxidant capacity of the analysed juice extracts determined in the FRAP assay ( Table 1) showed clearly that the juice processed with 600 MPa for 15 min had the highest FRAP values of all the analysed samples. The tested juices retained 95% of their reducing power at the end of the storage period. Similarly, the HPT-treated juice also showed stable FRAP values during storage. However, its scavenging power was 6% lower than that found for the HHP-treated (600 MPa/15 min) juices. It was also found that pressurized (250 MPa/15 min) juice stored for 30 days had approximately 40% higher total antiradical capacity than the heat-pasteurized (80°C/1 min) sample [6]. Among all the processed juices, the 5 International Journal of Food Science greatest decrease (by 13%) in FRAP values was noted for the LPT juice after 14 days of storage.

The Effects of Processing on the Colour Parameters.
In this study, colour parameters such as L * (lightness), a * (redness), and b * (yellowness) were recorded for the fresh, untreated juice and for the juices subjected to HHP, LPT, and HPT processing upon storage for 0, 7, and 14 days. The obtained parameters were also used to calculate the ΔE coefficient to estimate the total colour change as a result of processing and storage ( Table 2). As shown in the literature, the ΔE coefficient clearly characterizes total colour changes in the processed products, from not noticeable (0-0.5), slightly noticeable (0.5-1.5), noticeable (1.5-3.0), and well visible (3.0-6.0) to great (6.0-12.0) [29,30].
The ΔE values obtained for the HHP-treated (400-600 MPa/15 min) juices reached an average value of 2.59, indicating noticeable total colour changes after processing (Table 2). However, almost threefold higher ΔE values were noted in the juices subjected to LPT and HPT treatment compared to those in the pressurized samples. Similarly, it was found that the tomato and carrot juices subjected to heat processing (80°C/1 min) showed a greater variation in total colour compared to the HHP-treated juices (250 MPa/ 35°C/15 min) [6]. The changes in ΔE values observed, in the above cited report, for HHP-treated tomato and carrot juices did not exceed 15. In turn, the thermally treated samples presented distinctly higher values for this parameter. According to Dede and coauthors [6], these discrepancies may be attributed to the different effects of HHP and thermal treatment on the release of carotenoids from the protein complexes and/or the breakdown of phytochemicals in the treated material.
The results presented in Table 2 indicated that the heatpasteurized juices showed approximately 13% (LPT sample) and 25% (HPT sample) decreases in a * values. They also showed an increase (by 14% and 27% on average) in the L * and b * parameters, respectively. In the study of Kelebek et al. [30], a similar drop (17%) in the a * value was noted in the hot break tomato paste. These authors explained that phenomenon as an increase in the redness of the tomato paste due to the evolution of the brown colour along with the processing time.
As expected, the untreated juice stored for 7 and 14 days demonstrated the most distinct changes in total colour. We The results are expressed as the mean ± SD, n = 3. * Three independent measurements: L * (lightness), a * (redness), and b * (yellowness). ΔE is the colour difference between the control and processed tomato juices. * * Uppercase letters indicate differences between treatment types (in columns) on particular days of storage, whereas lowercase letters indicate differences between storage times for individual samples (in lines) (p ≤ 0:05).

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International Journal of Food Science may speculate that the enzyme (PPO, POD) activity found in the untreated juice ( Figure 1) along with the microbial instability of the untreated juice ( Figure 2) was responsible for the major observed colour changes, especially in the changes in the a * value. An interesting phenomenon was observed in the stored HHP-treated samples. The tested juices at 7 and 14 days of storage had 12% and 17% (on average) lower b * values, respectively, than the freshly pressurized juices. However, only a slight variation was noted in the a * values of the analysed samples. This suggests that the yellow colour was unstable in the stored HHP-treated juices. Compared to the HHP-treated juices, juices exposed to heat pasteurization demonstrated less advanced but still noticeable and visible changes in ΔE values after 7 and 14 days of storage, respectively. Following Marszałek and coauthors [31], we speculate that the processes combined with the storage time may disrupt the equilibrium between labile phytochemicals, leading to a transformation in their structure that, in turn, creates less or more advanced changes in the analysed colour parameters.
3.4. Activity of PPO and POD. The initial activity of PPO determined in the freshly prepared (untreated) tomato juices was lower (A 0 = 0:14 ΔOD/min/g fw) than the activity of POD (A 0 = 35:70 ΔOD/min/g fw). The initial activity of POD measured by Hernandez and Cano [32] in fresh tomato puree reached a similar value (40.41 ΔOD/min/g fw) as the data presented in our study. However, these authors obtained much higher PPO activity (0.79 ΔOD/min/g fw) than the value noted for our sample. The effects of processing on the PPO and POD residual activity in tomato juices are shown in Figures 1(a) and 1(b). While the fresh juice treated with 400 MPa/15 min showed reduced (by 25%) PPO activity (Figure 1(a)), the POD activity did not change significantly after the processing applied (Figure 1(b)). The PPO was completely inactivated when 7 International Journal of Food Science the juice was exposed to 600 MPa for 15 min or subjected to thermal pasteurization (LPT/HPT). The complete inactivation of POD was also achieved after LPT and HPT treatment, whereas the treatment at 600 MPa/15 min reduced its activity by 90%. In contrast to our results, Marszałek and coauthors [13] found that PPO was more resistant to pressure (200-  International Journal of Food Science 900 MPa) than POD. However, these authors studied the effect of processing on the initial activities of pure and isolated enzymes. Since the mechanism of enzyme inactivation under HPP was not fully explained, we could speculate that the differences in the chemical structure of PPO and POD are not the main determinants of their barostability and that the behaviour of the food matrix components and interaction between them should also be taken into account when considering the enzyme stability in the HHP-treated food system. At 7 and 14 days of storage, a significant increase (by approximately 36% and 7%, respectively) in PPO activity was found for the juice treated with 400/MPa/15 min. An almost 200% increase in the residual activity of PPO was also observed by Sulaiman and Silva [33] for strawberry puree stored under frozen conditions for 30 days. However, these authors did not explain this finding. On the other hand, this phenomenon may be related to the fact that the structure of the cellular membranes was disturbed as a result of HHP processing of tomato material [32], which subsequently could have led to a gradual PPO release during sample storage. The POD activity determined in the freshly prepared juice as well as in the HHP-treated (400 MPa/15 min) juice remained at a similar level throughout the entire storage period (Figure 1(b)).

Microbiological Evaluation.
The results of the total aerobic microbial count (TAMC), total yeast count (TYC), and total mould count (TMC) obtained for the analysed juices are presented in Figures 2(a)-2(c).
For the fresh reference juice, 10 3 total bacteria were initially noted (Figure 2(a)). On days 7 and 14 of storage, the untreated juice contained 10 8 and 10 9 microorganisms/mL, respectively. A rapid increase in the yeast population (by nine orders of magnitude) was noted in untreated tomato juice at 14 days of storage (Figure 2(b)). In contrast, the growth of mould in the juice during the analysed storage period was reduced to 10 1 microorganisms/mL (Figure 2(c)). This phenomenon could be related to the fact that yeast and mould have special nutritional requirements; thus, a competition effect might occur between them during the tested storage period. Moreover, the toxicity of yeast towards mould, due  to the specific metabolites excreted by yeast, might also affect the mould population in the analysed juices [34].
The results clearly showed that the HHP (400-600 MPa/15 min), LPT, and HPT processes were equally effective in inhibiting the growth of microorganisms to a level below the limit of detection (<1 CFU/mL) during storage (Figures 2(a)-2(c)). Our results are in accordance with data presented by Plaza and coauthors [35], who also found a significant decrease (by 4 logarithmic units) in the total microbial count after tomato puree was exposed to 400 MPa for 15 min. As indicated by these authors, the processing parameters were sufficient to completely inactivate the yeast and mould populations. Similarly, the pressurization of tomato juice at 600 MPa for 1 min or its thermal processing at 95°C for 20 min also effectively reduced the total viable count below the detection limit < 1 CFU/mL [9].
3.6. Principal Component Analysis (PCA). PCA was carried out based on all analysed samples and variables (total phenolic content (TPC), total phenolic index (TPI), antioxidant capacity (ABTS, FRAP), colour parameters (L, a, b, and ΔE ), and enzyme activity (PPO/POD)) to characterize the structure and regularity in the relationships between variables and outcomes.
The first factor (PC1, 42.80%) was marked by high loadings on ABTS (-0.8385), TPI (-0.8122), and TPC (-0.7964), and the second factor (PC2, 25.68%) by high loadings on a * (-0.7540) and POD (0.6235). The factors with opposite signs exerted opposite effects (Figure 3). In the analysis, TPC, ABTS, and TPI were located close to one another due to the presence of significant and positive correlations. On the other hand, these parameters appeared to be weakly correlated with a * . The location of the a * vector confirms its significant and negative impact only on PC2. The impact of a * on PC1 is rather minor (-0.3193). Similarly, the TPI vector has high negative loadings on PC1, but it has a negligible impact on PC2 (-0.0929). Thus, TPI is significantly correlated only with ABTS and TPC parameters. The correlation coefficients (Table 3) confirmed the relationships among the tested parameters. Our results are in accordance with other literature data [15,23]. In the cited references, phenolic concentrations were positively and significantly correlated with the scavenging capacity of tomato fruit.
All the analysed samples were additionally described in the coordinate system of the first two principal components. The score plot generated by PCA classified and detected five different groups in the data structure ( Figure 4). The nature of the differences between the obtained clusters resulted more from the processing than from the juice storage conditions. Regardless of the storage period, the samples treated with HPT formed cluster I. These samples had low TPC, TPI, ABTS, and FRAP values but high values of L * and b * . A similar trend was observed in the LPT-treated and stored samples, which formed cluster II. Samples treated with 600 MPa for 15 min and stored for 0, 7, and 14 days were in cluster III and had high TPC, TPI, ABTS, FRAP, and a * values. Cluster IV included the juices treated with 400 MPa/ 15 min (fresh and stored) and the fresh, untreated sample. The composition of that cluster indicates the negligible impact of processing at 400 MPa/15 min on the quality

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International Journal of Food Science parameters of the juice. As shown in Figure 4, the untreated juice stored for 7 and 14 days formed cluster V and had high PPO, POD, and ΔE values.

Conclusions
Compared to the heat-pasteurized juices stored for 14 days, the juice exposed to 600 MPa for 15 min showed superior quality. Samples treated with 600 MPa for 15 min and stored for 0, 7, and 14 days had high TPC, TPI, ABTS, FRAP, and a * values. As demonstrated, these tested samples at the end of the storage period retained 90% and 95% of their polyphenol content and antioxidant capacity, respectively. As in the case of pasteurization, juice processing at 600 MPa for 15 min clearly reduced the activity of food-spoiling enzymes (PPO, POD) as well as the microbial count. The obtained results showed that TPC was significantly and positively correlated with TPI, ABTS, and FRAP parameters. The pressurized and stored juices demonstrated high stability in their a * values, while variations in yellow colour were observed. As indicated by the results presented, HHP (600 MPa/ 15 min) without the use of temperature allowed to preserved polyphenol content and antioxidant capacity of the tomato juice. The preservation of a high nutritional value in the HHP-treated juice with a simultaneous complete inactivation of undesirable microflora and enzymes allows to demonstrate the superiority of the HHP process over the heat treatment.

Data Availability
Data supporting the results were preserved at the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, in Olsztyn, Poland, and they are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Plant Growth of Beetroots ( Beta vulgaris L.) with Nitrogen Supply at Suboptimal Elevations in a Tropical Region

fertilization respectively. High root yields were reported in other experiments in a ABSTRACT The present study was designed to study the possibility of beetroots to be cultivated at suboptimal elevations in the tropics. Four pot experiments were conducted in the field each at 1700, 850, 520 and 320 m asl (above sea level) in the region of Malang, East Java. A randomized block design with four replicates was used to impose the treatment of nitrogen (N) fertilizer consisting of 0, 0.4, 0.8 and 1.2 g N/plant (~ 0, 100, 200 and 300 kg N/ha). In the experiments at the elevation of 1700 m and 320 m asl, the treatment of 0 and 80 g chicken manure per plant (~ 0 and 20 t/ha) was involved. The treatment of 0 and 0.6 g P2O5 and 0 and 0.8 g K2O per plant was involved in the experiment at 850 m and 520 m asl respectively. Plant growth (total dry weight, leaf area and leaf number) was observed on day 20, 40, 60 and 90 after sowing by destructive plant samplings. In the present paper, data were reorganized to analyze the effect of elevation and N fertilizer on plant growth with the elevation as the main factor and N fertilizer as the sub factor.

INTRODUCTION
The cultivation of beetroots (Beta vulgaris L.) has attracted increasing attention in recent years in Indonesia in order to obtain fresh root yield (taproots). Air temperature is the main limiting factor of beetroots, a subtropical species by the origin and known as a cool-weather crop, to grow in the tropics. For optimum growth, this crop requires temperatures in the range of 12-19°C (Nottingham, 2004;Starke Ayres, 2014) or 15-20°C (ARC, 2013). A reduction in yield quantity and quality (flavor and texture) was observed to occur in addition to growth retardation especially during prolonged high temperature (ARC, 2013). As a consequence, from the biological and economic point of view, this crop is only likely to be cultivated on areas of high elevations in the tropics. Based on the results of a previous study on air temperatures during the day at several elevations in the region of Malang, East Java (Sitompul, Sitawati, & Sugito, 2013), the optimal elevation for beetroot cultivation in this region is in the range of > 1800 -≤ 2900 m asl.
The results of an experiment, conducted in the region of Malang at an elevation of 1700 m asl, led to a conclusion that beetroots can be cultivated in the tropics on highlands with good growth and reasonable yields (Sitompul & Zulfati, 2019). The highest yield of 150.5 g RFW (root fresh weight) per plant was obtained from the plants fertilized with 0.6 g N/plant (~ 150 kg N/ha). This root yield, with 250,000 plants/ha (20 cm x 20 cm spacing), is equivalent to 37.6 t/ha. In other experiment designed to study watering intervals (once 3-7 days) in the objective of water use efficiency, the highest yield (RFW) of 87.5 g per plant (~ 21.9 t/ha) was observed in plants with the watering interval of once three days (Sitompul, Roviq, & Riedo, 2019). The root yields of both experiments are higher than the root yield of beetroots (cv. Early Wonder) from an experiment at Urutai-GO, Brazil (17˚29'10" S, 48˚12'38" W) at the elevation of about 800 m asl with an average annual temperature of 230C (da Silva Curvêlo, Diniz, de Azevedo Pereira, & Ferreira, 2018). The RFW of this latter experiment was 16.25 t/ha and 15.5 t/ha with conventional fertilization and organic fertilizer respectively. High root yields were reported in other experiments in a Copyright © 2020 Universitas Brawijaya tropical region of Brazil (Serra Talhada-PE, 7º57'15" LS) at an elevation of 429 m asl (dos Santos et al., 2017). The RFW of 46.48 t/ha was reported from an experiment conducted during autumn (March-June) with the application of 15.6 t/ha of green manure (Calotropis procera), A lower root yield (41.48 t/ha) was found from an experiment carried out during spring-summer (October-January) with the same application of green manure.
The cultivation of beetroots on a large scale in the tropics is hampered by limited areas of high elevations suitable for beetroots. At suboptimal elevations, beetroots may suffer from heat stress which is the main environmental stress limiting the productivity of crops worldwide (Hasanuzzaman, Nahar, Alam, Roychowdhury, & Fujita, 2013). This could be attributed to the disturbance of many biochemical reactions sensitive to high temperatures and oxidative stress due to ROS (reactive oxygen species) formation in large quantities (Hasanuzzaman, Hossain, da Silva, & Fujita, 2012;Hasanuzzaman, Nahar, Alam, Roychowdhury, & Fujita, 2013). The results of studies in Brazil as mentioned previously suggest that beetroots adapted well to suboptimal elevations down to 429 m asl. In the experiment at Serra Talhada-PE mentioned above, the annual average temperature was reported to be 24.7°C, and the maximum and minimum of monthly average temperature varied successively in the range of > 31 -< 36°C and ≥ 20 -≤ 23°C (dos Santos et al., 2017). A review showed that the negative effect of heat stress on plants, in general could be alleviated with various means including the supply of nitrogen (N), phosphorus (P) and potassium (K) nutrients (Hasanuzzaman, Nahar, Alam, Roychowdhury, & Fujita, 2013).
The present study was designed to study the adaption of beetroots to suboptimal elevations in a tropical region with the growth performance of plants used as an indicator of adaptation. The potency of nitrogen nutrients to increase the adaptability of beetroots to suboptimal elevations was also explored. A very high rate of N fertilizer (300 kg N/ha) was involved in the present study as the results of the previous experiment showed a linear increase in root yield with an increase in N fertilizer up to 150 kg N/ha (Sitompul & Zulfati, 2019). This is also supported by the results of a pot experiment in a greenhouse with a day/night temperature maintained at 22/15°C in Bloemfontein, South Africa (Rantao, 2013). This latter experiment showed an increase in root yields to 14.47 t/ha with 200 kg N/ha compared with 2.99 t/ha without N fertilizer. Better performance of beetroot growth was also reported in plant height, leaf number, leaf area, leaf fresh weight, total fresh weight and the diameter and volume of tuberous roots.

Study Sites
The study consisted of four experiments carried out in the field during the dry season (June-September) in 2019 within the region of Malang (7° 57'59" S, 112°37'57" E). Experiment 1 (as a control), 3 and 4 was undertaken at the Experimental Station of the Universitas Brawijaya in Cangar at 1700 m asl, Jatimulyo (520 m asl) and in Jatikerto (320 m asl), respectively. Experiment 2 was conducted at a farmer's field in Batu at 850 m asl. The average air temperature at Cangar during the day, based on measurements at 06.00 AM, 13.00 PM and 18.00 PM for five successive days in February and May 2016, was 20°C corresponding with the results of the previous study (Sitompul, Sitawati, & Sugito, 2013). The air temperature during the day at the site of experiments 2, 3 and 4, based on the previous study, was estimated to be 26.0, 28.3 and 29.6°C respectively.

Sowing and Treatments
Seeds of beetroot (cv. Ayumi 04), purchased from a local agricultural shop, and were grown in plastic bags (20 cm in diameter and 30 cm in height) in June 2019. In each pot (plastic bag), filled with topsoil (Andisol, sandy clay loam, pH 6.4) mixed with compost (3:1), five seeds were sown to obtain more uniform seedlings and a healthy plant per pot selected on 20 das (days after sowing). Each pot was supplied with 0.6 g P 2 O 5 (SP36) at sowing and 0.8 K 2 O (KCl) on 20 das for experiments not involving phosphorus (P) or potassium (K) fertilizer as a treatment.
The imposition of treatments in the field was arranged according to a randomized block design with four replicates. The treatment of nitrogen (N) consisting of 0, 0.4, 0.8 and 1.2 g N/plant (urea) was involved in all experiments and applied on 5 das. Other treatments were organic manure (chicken manure): 0 and 80 g/plant (~ 20 t/ha) in experiment 1 and 4 applied at sowing, phosphorus (SP36): 0 and 0.6 g P 2 O 5 /plant (~ 150 kg P 2 O 5 /ha) in experiment 2 applied at sowing, and potassium (KCl): 0 and 0.8 g K 2 O/plant (~ 200 kg P 2 O 5 /ha) in experiment 3 applied Copyright © 2020 Universitas Brawijaya on 15 das. Plants were free from weeds controlled manually, and a fungicide (benlate 0.2%) was used as necessary to control fungi (Peronospora schachtii).

Plant Growth Observation
Plant growth in leaf area and total plant dry weight was observed with destructive samples on days 20, 40, 60 and 90 (harvest) after leaf number was recorded. Sample plants were cleaned from adherent soil as necessary and each plant was placed in a plastic bag to prevent water loss during the period between sampling and measurement. Fresh leaves were weighed to obtain fresh weight (FW) followed by the measurement of leaf area with leaf area meter (Licor LI 3100). The fresh weight of roots (taproots) was observed on 60 and 90 das. Sub-samples from the representative part of leaves, stems, and roots were dried in an oven at 80°C for 48 hours for the estimation of total and root dry weight.

Data Analysis
Statistical analyses of data including analysis of variance (ANOVA) were executed in the MS excel manually. Least significant difference (LSD for n ≤ 6) or Duncan multiple range tests (DMRT for n > 6) at P ≤ 0.05 was used to compare mean values between treatments. The present paper is focused on the growth adaptability of beetroots on suboptimal elevations with the supply of nitrogen fertilizer. For this purpose, data of all experiments were reorganized so that a Split Plot Design could be used to analyze the effect of site elevation as the main factor and nitrogen as a sub factor.
The growth pattern of plants with time was analyzed with a Richards's model (France & Thornley, 1984)  Where: W is the growth parameter (biomass or leaf area) at a particular time t (day), W 0 and W m is W at t = 0 and the end of growing period respectively, and n, k, W 0 and W m are constants. A significant difference in the growth patterns, analyzed with the Richards model, between plants at the optimal elevation (1700 m asl) and plants at other elevations was analyzed by Kolmogorov-Smirnov (KS) test (Zaiontz, 2014) at P ≤ 0.05. The application of an exponential model available in the MS excel was also explored to analyze the growth pattern of plants.

Total Dry Weight
The plant growth of beetroots, the accumulation of total dry weight (TDW) with time, was significantly influenced by the site elevation on 20, 60 and 90 das (days after sowing), but not by nitrogen (N) fertilizer on all days of observation. A significant interaction between site elevation and N fertilizer was observed on 40, 60 and 90 das prompting an interest to analyze the pattern of beetroot growth with time for all treatment combinations. It appears that the growth of beetroots up to 90 das showed a similar pattern that increased slowly at the beginning up to 40 das, then rapidly thereafter (Fig. 1).
The use of an exponential model to analyze the pattern of increases in the average TDW of plants for each treatment with time resulted in high coefficients of determination (R 2 ≥ 0.824). The coefficients of determination were not, however, significant for R 2 < 0.903 due to a limited sample number (n = 4). A much better description of TDW development with time was found with the use of a Richards model with R 2 ≥ 0.994 (P < 0.003) as drawn for all treatments (Fig. 1). The R values can be seen in the relationship between R2 values of the Richards model and those of the exponential equations ( Fig.  2A). No significant difference in the growth pattern was found between plants at the optimal elevation and others based on the KS test. A linear relationship between the observed TDW and the estimated TDW with the Richards model for all treatments is highly significant (Fig. 2B).
It is clear, however, that the biomass production (TDW) of plants was lower at suboptimal elevations than at the optimal elevation (1700 m asl) particularly at later stages of growth when N nutrient was supplied sufficiently. The interaction of site elevation and N fertilizer on TDW at harvest (90 das) showed, in general, a decrease in the TDW of N-fertilized plants with a decrease in the site elevation with a rate of decrease depending on the rate of N fertilizer (Fig. 3A). A significant, linear relationship between TDW and the site elevation (P ≤ 0.05) was found in the plants with 1.2 g N/plant that showed a reduction of 39.8 g/plant in TDW with a decrease of 1000 m in the site elevation. Similar trends, but not significant, were also found in plants with 0.8 g N/plant (N2, P > 0.10) and plants with 0.4 g N/plant (N1, P > 0.14).
Copyright © 2020 Universitas Brawijaya Fig. 1. The progress with time in total dry weight (TDW) of beetroots grown at different site elevations (E) with different rates of N fertilizer. E1, E2, E3 and E4 is 1700, 850, 520 and 320 m asl respectively; A. N0 and N1 is 0 and 0.4 g N/plant, respectively; B. N2 and N3 is 0.8 and 1.2 g N/plant, respectively. The graph for each treatment combination was generated by a Richards model. The R 2 of all graphs is highly significant (P ≤ 0.003).

A B
Copyright © 2020 Universitas Brawijaya Fig. 2. Richards R 2 was plotted as a function of exponential R 2 . R 2 = Coefficient determination, and Richards and exponential refers to Richards and exponential equation respectively. The dashed line (horizontal) and dotted line (vertical) is the border between not significant and significant R 2 of exponential equations at P = 0.05 and of Richards equation at P = 0.01 respectively  When the TDW of N1, N2 and N3 treatment was averaged for each elevation (n = 16), a good relationship (R 2 = 0.719; P < 0.05) was found between the TDW and site elevation with the linear model and exponential model (Fig. 3B). The linear relationship suggests in general that a decrease of 1000 m in the elevation led to a decrease of about 30% in the TDW compared with that at the optimal elevation. The TDW of plants without N fertilizer showed no significant difference between site elevations and was much lower than that with the supply of N nutrient at high elevations (> 800 m) particularly at 1700 m asl (Fig. 3A).

Leaf Area
Leaf area (LA) per plant was mainly influenced by the site elevation as observed significantly on 20, 60 and 90 das. No significant effect of N fertilizer on the LA was found at any time (day) of observation, and a significant interaction between site elevation and N fertilizer was observed only on 40 das. The response of LA to the site elevation on day 20, different from that of TDW, showed a tendency of increase as the site elevation increased from 320 m up to 850 m, but then decrease at a higher elevation (Fig. 4). On day 60 and 90, LA showed no clear-cut responses of LA that tended to decrease with an increase in the site elevation from 320 m to 850, then relatively constant or to increase slightly at a higher elevation (Fig. 4).
The development of leaf area (LA) per plant with time was similar to that of TDW and independent of site elevation and the rate of nitrogen fertilizer. The LA of plants grown at the optimal elevation (E1, 1700 m asl) increased slowly during the first 20 das, and rapidly thereafter (Fig.  5). At other elevations, the LA increased slowly during about the last 30 days of growth following slow and rapid growth in the previous stages as observed also in the plants at the optimal elevation. The use of a Richards's model was good to capture the development of the LA with time with a high coefficient of determination for each site elevation (P < 0.01). The development of LA with time is closely related to that of leaf number (LN) with time independent of site elevations (Fig. 6). This relationship suggests that the development of LA with time was due mainly to an increase in LN (R 2 = 0.827), and the contribution of individual LA (leaf area per leaf) was < 20%. The relationship between TDW and LA, in general, was significant for each site elevation particularly during the first 60 days of growth (Fig. 7).
Copyright © 2020 Universitas Brawijaya  The present study designed to explore the adaptability of beetroots to suboptimal elevations (< 1700 m asl) for the purpose of large scale cultivation in the tropics. As this crop, known as a cool-weather crop, requires a temperature range of 12-200C for optimum growth (ARC, 2013; Starke Ayres, 2014), heat stress is likely to be the major factor limiting its cultivation in tropical regions. The effect of heat stress could be increasingly significant with time in the tropics due to the phenomenon of global warming (Stocker et al., 2014). The frequency and duration of high temperatures are also considered to increase with dramatic effects on agriculture, economy, and ecology (Easterling et al., 2000;Tripathi, Tripathi, Chauhan, Kumar, & Singh, 2016;Wang, Heckathorn, Mainali, & Tripathee, 2016). On the other hand, the potential impact of climate change (higher temperatures) is not sufficiently understood in most agriculture systems including vegetable crops (Leisner, 2020).
The key to beetroot adaptability is the level of the growth process to tolerate heat stress at the suboptimal elevations where air temperatures are higher than the optimal temperatures. The growth process encompasses all processes leading to the synthesis of plant biomass consisting mainly of photosynthetic products through photosynthesis process. Photosynthesis, the most fundamental physiological process in plants, on the other hand, is highly sensitive to heat stress (Mathur, Agrawal, & Jajoo, 2014;Song, Chen, Ci, Shao, & Zhang, 2014).
It was found in the present study that the growth of beetroots is quite adaptable to suboptimal elevations (< 1700 m asl) down to about 300 m in the tropical areas of study. The growth development of beetroots, the accumulation of TDW with time, showed a normal pattern as observed in the plants at the optimal elevation (1700 m) that can be captured by a Richards's model (Fig. 1). No such information has been found in the literature so far. A similar pattern was also reported in previous studies carried out at the elevation of 1700 m (Sitompul & Zulfati, 2019;Sitompul, Roviq, & Riedo, 2019).
The analysis of TDW observed at harvest (90 das) in relation to the site elevation with a linear regression in the plants with 1.2 g N/ha showed a decrease of only 10 g/plant with a decrease of 1000 m in the site elevation in the range of 1700-320 m Fig. 7. Relationship between total dry weight (TDW) and leaf area (LA) per plant of beetroots grown at 1700 m/E1 and 850 m/E2 (A), and 520 m/E3 and 320 m/E4 (B). Each point in the figures is the average of four replicates and represents each treatment combination. The lines are linear relationships for each site elevation: y = 0.049x -4.432, R 2 = 0.897** (E1); y = 0.028x -1.3122, R 2 = 0.805* (E2); y = 0.028x -1.743, R 2 = 0.787* (E3) and y = 0.028x -1.448, R 2 = 0.915** (E4). Simbol * and ** is significant at P≤0.05 and P≤0.01 respectively.
Copyright © 2020 Universitas Brawijaya asl (Fig. 3). This is equivalent to 2.5 t/ha or 27.6% of TDW at the optimal elevation (1700 m asl), and the TDW of beetroots at 320 m asl may reach 22.4 g/plant (~ 5.6 t/ha) or 61.9% of TDW at the optimal elevation. A relatively similar trend was shown by the TDW of plants with 0.4 g N/plant. The TDW of plants without N fertilization was low and showed no response to the site elevation as expected suggesting that nitrogen nutrient has an important role in the adaptability of beetroots to high temperatures. The TDW response of plants with 0.8 g N/plant to a decrease in the elevation was not significant and could be an artifact that needs further investigation. The decrease in the biomass production of plants at suboptimal elevations could be related to a decrease in LA as a consequence of LN decrease (Fig. 6). This is based on a close relationship between TDW and LA with R 2 ≥ 0.787 (Fig. 7) suggesting that the contribution of factors other than LA including the efficiency of photosynthesis (TDW/LA) to TDW was quite small (21.3%). This is in agreement with the previous studies showing photosynthesis as the main process determining the adaptability of plants to heat stress.
In contrast, for instance, apple trees are very sensitive to high temperature (heat stress). Based on the results of a previous study (Sitompul, Sitawati, & Sugito, 2013). The analysis of the linear relationship between fruit yield (fruit fresh weight) and elevation in the study showed a yield decrease of > 10 kg/ tree with a decrease of 100 m in elevation, and the apple trees produced no fruit at the elevation of < 800 m asl. This is consistent with the reality of the cultivated apple trees in the region of Malang even with the varieties have adapted to the local condition that fruits are produced only at elevations above a certain limits.
The adaptability of beetroots to lower elevations found in the present study is in agreement with the results of a study in Brazil (Serra Talhada-PE, 7º57'15" S) that showed a high fresh root yield (46.48 t/ha~6 t/ha of root dry weight) of beetroots at an elevation of 429 m asl (dos Santos et al., 2017). The beetroot crop was grown during the autumn season (March-June) with the supply of 15.6 t/ha green manure of Calotropis procera. With the same application of green manure at sowing, a lower root yield (41.48 t/ha) was obtained from a crop grown during the spring-summer season (October-January). These yields were much higher than that of an experiment (< 17.0 t/ha) conducted in the field of Urutai-GO, Brazil (17029'10"S) at 697 m elevation ((da Silva Curvêlo, Diniz, de Azevedo Pereira, & Ferreira, 2018). This experiment was carried out during the period of October-December 2016 with an average temperature of 230C.
The present study also showed that a sufficient N fertilizer is required to support the adaptability of beetroots to the suboptimal elevations. The results of several studies previously suggest an important role of plant nutrients in the adaptability of plants to heat stress (Liu et al., 2019). It was found, for instance, that the application of N fertilizer (e.g. 300 kg N/ha) may sustain a sufficiently high yield of rice under high temperature (Liu et al., 2019). The role of nitrogen on plant adaptation to high temperatures (heat stress) could be associated with photosynthesis, the most sensitive function of plants (Wang, Heckathorn, Mainali, & Tripathee, 2016). Leaves situated aboveground are exposed directly to air temperature and the main site of the photosynthetic process that determines the production of carbohydrates. The primary target of heat stress on photosynthesis is considered to be photosystem II and Ribulose bisphosphate (RUBP) carboxylase/oxygenase (Rubisco) (Mathur, Agrawal, & Jajoo, 2014). It is therefore that photosynthesis is likely the main process responsible for the negative effect of high temperature on TDW or plant growth (Wang, Heckathorn, Mainali, & Tripathee, 2016). In natural conditions (380-400 ppm CO2), the main factor liming photosynthesis of C3 plants is the formation of RuBP (ribulose 1,5-bisphosphate) and the carboxylation of Rubisco (RuBP carboxylase/oxygenase) (Farquhar, von Caemmerer, & Berry, 1980;von Caemmerer & Evans, 2015). Yamori, Nagai, & Makino (2011) found that CO2 assimilation at 380 µmol/mol CO2 was limited by RUBP carboxylation at low leaf N and by RUBP regeneration at high leaf N. In the present study, the positive effect of N could be associated with an increase in the RUBP carboxylation that needs further study.

CONCLUSION
The plant growth of beetroots is quite adaptable to suboptimal elevations (< 1700 m asl) in the tropics. A decrease of 1000 m in the site elevation below the optimal elevation (1700 m asl) caused a reduction of only about 30% in TDW. The normality of growth patterns of plants at the suboptimal elevations similar to that at the optimal elevation was observed. The adaptability of Copyright © 2020 Universitas Brawijaya beetroots to heat stress could be enhanced by the application of nitrogen fertilizer in a sufficient amount (e.g. 1.2 g/plant). The leaf area of plants associated mainly with the leaf number is the major factor of plants supporting the adaptability of beetroot growth at the suboptimal elevations.


extfieldsofstudy: Physics Chemistry

text:
Nutrient content and bioactive compounds characterization of Goroho banana (Musa acumunafe sp) stem meal fermented with Trichoderma viride as an alternative feed for broiler chickens

The aim of this study was to determine the nutrient content and to characterize the bioactive compounds of Goroho banana (Musa acumunafe sp) stem meal fermented with Trichoderma viride. Parameter measured were proximate analysis, amino acid, phytochemical, antioxidant potency of goroho banana (Musa acumunafe sp) stem meal fermented. Data were analysed using descriptive method. Results showed that crude protein, crude fibre, crude fat, Ca, P, and gross energy were 4.86%, 22.03, 0.94, 0.42, 0.18 and 3156.67 Kkal/kg, respectively. Amino acid were analyse using HPLC and demonstrated that it was composed by 16 amino acids. Seven of them were essential amino acids; lysine (0.41 %) histidine (0.23 %), leucine (0.56 %), threonine (1.13 %), valine (0.11%), methionine (0.11 %), isoleucine (0.41 %), phenylalanine (0.39 %), and nine of them were non-essential amino acids; serine (0.31 %), aspartic acid (0.56 %), arginine (1.13 %), proline (0.38 %), glutamic acid (0.70 %), glycine (0.33 %), alanine (0.34 %), and tyrosine (0.30 %). Phytochemical screening by colour visualization showed the presence of flavonoid 0.06 % (w/w) and analysis by titrimetric tannin 0.01 %. Vitamins C and E compound were analysed by HPLC, whereas Vitamin C (Ascorbat acid) was 437 mg/kg and Vitamin E (Tocopherol) was < 0.01 mg/100 g. It can be concluded that Goroho banana (Musa acumunafe sp) stem meal fermented with Trichoderma viride can be used as an alternative feed.

Introduction
Goroho banana (Musa acuminafe, sp) is a typical type of banana that is very popular with consumers, especially in the area of the city of Manado, Indonesia, marked by so many places selling fried foods that use it because it has a distinctive taste and it is consumed by diabetics. As a result, many banana stems are just thrown away as trash that disturbs the aesthetics of the environment. Utilization of banana stems in general have not been popular, there is no information. The use of goroho banana stems is possible as a feed because in terms of composition of goroho banana stems contain enough nutrients needed by livestock. Chemical analysis shows that the banana goroho (Musa acuminafe, sp) stem contains protein (2.53%), fat (1.49%), ash (12.93%) and crude fiber (23.48%) and gross energy 3723 kcal [1]. Animal feed sourced from agricultural and plantation wastes has low nutritional value so it needs to be optimized for quality through fermentation technology [2].
The fermentation process can degrade protein molecules into amino acids and peptides. Tripsyn inhibitors can also be graded or modified during the fermentation process and lose activity to increase  [3]. Palm kernel core fermentation with Trichoderma riseiii can increase nutrient content [4]. Acid free amino acids and bioactive peptides can be released due to microbial activity during the fermentation process or are processed by enzymes produced by microbes [5]. Fermented pineapple peel in the ration improved fat and cholesterol on broiler chicken [6].
Generally, natural antioxidants can be found in stems, bark, roots, root bark, fruit, fruit skin, leaves, seeds, and flowers [7]. These compounds are mostly phenol or polyphenol compounds derivatives of cinnamic acid and other organic acids. Studies of Gill et al [8] have shown the antioxidant and antiulcer effects of C. sativus in rats. The vegetables contain several phytochemicals possessing antioxidant activity, and the major groups of phytochemicals include vitamins A, C, E and K, carotenoids, terpenoids, flavonoids, polyphenols, saponins, enzymes and minerals. In the kepok banana (M. paradisiaca normalis) stem and milk banana (M. paradisiaca sinensis) have flavonoid compounds [9]. Antioxidants can fight LDL cholesterol and prevent damage to cells or tissue of blood vessels. Aside from being an antioxidant, flavonoid compounds, alkaloids and tannins have other mechanisms that help reduce LDL cholesterol levels in the blood. In vitro study shows that flavonoids work as HMG-CoA reductase enzymes inhibitors so that cholesterol synthesis decreases [10]. Trichoderma viride as feed alternative for broiler chickens.

Materials and methods
Goroho banana (Musa acuminafe sp) steam: goroho banana stem waste was obtained from in Kawangkoan Village, North Sulawesi Province, Indonesia and was brought to our laboratory for the experiment.

Micro-organism
The filamentous fungi was obtained from Laboratory of Gajah Mada University.

Maintenance medium
Trichoderma. viride was maintained on malt extract agar and stored at 4ºC. The organism was subcultured once for every 3 months.

Inoculum preparation
The fungus was sub cultured on malt extract agar in petri dishes for 5 days. Spore suspension containing approximately 3x10 6 spores per mL.

Substrate
Preparation for product fermented goroho banana stem dried immediately in the oven at 60ºC to constant moisture content. The sample of goroho banana stem fermented, dried and stored in cold dark conditions. Determination of proximate chemical composition: determination of moisture, total ash, crude protein, crude fibre, crude fat were determined by standard method [11], amino acid analyses by Waters AccQ'Tag [12]. Phytochemical screening of the extracts was carried out to know the different constituents present as per the standard procedures. The extracts were tested for alkaloids, sterols, triterpenes, saponins, flavonoids, quinon and tannins [13,14].

Nutritional evaluation
Chemical composition of goroho banana stem fermented are shown in table 1. Results showed that investigations on nutritional evaluation of goroho banana stem fermented are rich in crude protein, crude fat, gross energy and Ca, followed by crude fibre. Our previous study confirmed that proximate analysis on goroho banana stem fermented as shown in table 1 [15]. The fermentation process has been known as one of the cheapest ways to improve the quality of cassava protein [16]. Ogbonnaya et al [17]

Amino acid composition of fermented Goroho banana stem
The amino acid composition are shown in table 2. Fermented banana stem containing essential amino acids include valine, histidine, tryptophan, isoleucine, lysine, leucine, methionine, threonine, and phenylalanine. Essential amino acids are amino acids that cannot be made by the body and must be obtained from food sources of protein. According to Lehninger [18], moulds can secrete protease enzymes into the environment to break down proteins into amino acids. The results of this study has identified the composition of the fermented goroho banana stem amino acid that consisting of 9 essential amino acids: lysine, leosine, isoleusine, phenylalanine valine, methionine, histidine, arginine and threonine and 6 types of non-essential amino acids tyrosine, alanine, glycine, serine glutamic acid and aspartic acid. In regard to the composition of amino acids in fermented goroho banana stem obtained in the present study, it is presumed that this can be used as an alternative feeds for broiler chickens.

Phytochemical screening and flavonoid estimation
In this study, the qualitative phytochemical analysis revealed the presence of bioactive compounds of goroho banana stem fermented (table 3). Results showed that phytochemical screening by colour visualization showed the presence of flavonoid, tannin, saponin and steroid. The quantitative analysis of spectrophotometry found flavonoid total was 0.06% (w/w), and by titrimetry found tannin was the value of 0.01%. The values of Vitamin C (Ascorbate) and Vitamin E (Tocopherol) were analysed using HPLC.
The contained flavonoid that can be considered to a cheap source of flavonoids. According to Saxena [19], flavonoids have been reported to exert multiple biological property including antimicrobial, antioxidant, cytotoxicity, anti-inflammatory, as well as antitumor activity. Flavonoids, mainly present as colouring pigments in plants also function as potent antioxidants at various levels [20,21].
The goroho banana stem fermented in this study also have tannin. This study was in line with Pane [7], who reported that tannins have been found in the banana stem anni (M. paradisiaca normalis). Tannins have astringent properties, hasten the healing of wounds and inflamed mucous membrane. Tannins are potential metal ion chelator, proton precipitating agents and biological antioxidant [22]. Goroho banana stem fermented has been evaluated as anti-oxidant compounds as vitamin C (Ascorbic) and vitamin E (Tocopherol). Antioxidants are inhibitors that work to inhibit oxidation by reacting with reactive free radicals to form non-reactive free radicals that are relatively stable or compounds that protect cells from the harmful effects of reactive oxygen free radicals [22]. Kadam et al [23] reported that natural antioxidants contained in food can be categorized into groups of nutrients consisting of vitamin C, vitamin E and B Carotene.

Conclusion
The present study reveals that Goroho banana (Musa acumunafe sp) stem meal fermented with Trichoderma viride can be used as feed alternative.