extfieldsofstudy: Chemistry Medicine

text:
Relief Role of Lysine Chelated Zinc (Zn) on 6-Week-Old Maize Plants under Tannery Wastewater Irrigation Stress

Tannery wastewater mainly comes from leather industries. It has high organic load, high salinity, and many other pollutants, including chromium (Cr). Tannery wastewater is generally used for crop irrigation in some areas of Pakistan and worldwide, due to the low availability of good quality of irrigation water. As tannery wastewater has many nutrients in it, its lower concentration benefits the plant growth, but at a higher concentration, it damages the plants. Chromium in tannery wastewater accumulates in plants, and causes stress at physiological and biochemical levels. In recent times, the role of micronutrient-amino acid chelated compounds has been found to be helpful in reducing abiotic stress in plants. In our present study, we used lysine chelated zinc (Zn-lys) as foliar application on maize (Zea mays L.), growing in different concentrations of tannery wastewater. Zinc (Zn) is required by plants for growth, and lysine is an essential amino acid. Maize plants were grown in tannery wastewater in four concentrations (0, 25%, 50%, and 100%) and Zn-lys was applied as a foliar spray in three concentrations (0 mM, 12.5 mM, and 25 mM) during plant growth. Plants were cautiously harvested right after 6 weeks of treatment. Foliar spray of Zn-lys on maize increased the biomass and improved the plant growth. Photosynthetic pigments such as total chlorophyll, chlorophyll a, chlorophyll b and contents of carotenoids also increased with Zn-lys application. In contrast to control plants, the hydrogen peroxide (H2O2) contents were increased up to 12%, 50%, and 68% in leaves, as well as 16%, 51% and 89% in roots at 25%, 50%, and 100% tannery water application, respectively, without Zn-lys treatments. Zn-lys significantly reduced the damages caused by oxidative stress in maize plant by decreasing the overproduction of H2O2 and malondialdehyde (MDA) in maize that were produced, due to the application of high amount of tannery wastewater alone. The total free amino acids and soluble protein decreased by 10%, 31% and 64% and 18%, 61% and 122% at 25%, 50% and 100% tannery water treatment. Zn-lys application increased the amino acids production and antioxidant activities in maize plants. Zn contents increased, and Cr contents decreased, in different parts of plants with Zn-lys application. Overall, a high concentration of tannery wastewater adversely affected the plant growth, but the supplementation of Zn-lys assertively affected the plant growth and enhanced the nutritional quality, by enhancing Zn and decreasing Cr levels in plants simultaneously irrigated with tannery wastewater.

crop for humans and animals. Previously, no study has reported the effects of Zn-lys on maize crop irrigated with tannery wastewater. Thus, in this experiment, we used lysine chelated Zn as foliar spray on maize irrigated with tannery wastewater, to see the possible positive effects of this chelated compound on the properties of plant growth and photosynthetic content. The central aims of the study were (1) to assess the toxic effects of tannery wastewater in maize and monitor uptake of Cr in different parts of maize, (2) to determine the effects of Zn-lys foliar spray on maize growth that was irrigated with tannery wastewater, and finally (3) to increase growth, photosynthetic content and antioxidative properties of maize with the application of Zn-lys irrigated with tannery wastewater.

Experimental Site
Experiment was carried out in botanical garden of Government College University, Faisalabad (GCUF). While the analysis were carried out in the Nuclear Institute for Agriculture and Biology (NIAB) Faisalabad, Pakistan.

Pot Experiment
Preparation of soil for growing maize was the first step of the experiment. Soil was air dried for one week without direct sunlight and then it was sieved through a 30 mm sieve. Sieved soil was then filled in pots, and the average weight of soil in each pot was 8 kg. The physio-chemical properties of soil have been shown in Table 1. Maize seeds for sowing were obtained from Ayub Agricultural Research Institute, Faisalabad. These seeds were washed with distilled water before sowing in plastic pots.  4 2− (mmol·L −1 ) 6.67 Ca 2+ + Mg 2+ (mmol·L −1 ) 3.5

Treatments
Plants were irrigated with tannery wastewater, in concentrations of 0% 25% 50%, and 100% and sprayed with three different concentrations of Zn-lys (0 mM, 12.5 mM, 25 mM). The properties of tannery wastewater have been shown in Table 2. Three replicates for each treatment were done. Pots arrangement was according to complete randomized design with factorial arrangement. Zn-lys was synthesized by following the standard procedure [33] by using zinc sulfate heptahydrate and L-lysine monohydrochloride, as Zn and lysine salts, respectively.

Leaf Area
Leaf area was measured by leaf meter after six weeks of treatment.

Chlorophyll Contents Determination
Leaf samples were collected after 6 weeks of treatment. Chlorophyll a, chlorophyll b, total chlorophyll and carotenoid contents were determined by using a spectrophotometer (Halo DB-20/DB-20S, Dynamica Company, London, UK) [34]. The extended top leaves were firstly weighed on an electric balance and then dipped in 85% (v/v) aqueous acetone, under dark conditions. The supernatant was collected and centrifuged at 4000× g rpm for ten minutes. In order to make spectrophotometric measurements feasible, 85% aqueous acetone was added for dilution. The measurements were taken at three different absorbances, of 452.5, 644 and 663 nm, against a blank (85% liquid acetone). The following formulae were used for the calculation of photosynthetic pigments: For All of these pigment fractions were calculated as fresh weight in mg g −1 .

Free Amino Acids Determination
Free amino acid was calculated by following the procedure of Hamilton et al. [35] Firstly, 0.1 g of fresh leaf was grinded in buffer solution, with a pH of 7.0. This mixture was then centrifuged for ten minutes at 8000× g rpm. Then, 1.0 mL of supernatant + 1.0 mL of 10% pyridine + 1.0 mL of 2% ninhydrine were mixed in test tubes. This solution was then heated for thirty minutes in a bath of boiling water, and finally, the violet color was obtained. Then, the solution was diluted with distilled water, and the absorbance at 570 nm was measured on a spectrophotometer.

Analysis of Antioxidant Enzymes
Spectrophotometer was used to determine the antioxidant enzyme's activities, including SOD, peroxidase (POD), CAT, and APX, in roots and leaves of maize plant.
Fresh roots and leaf samples (0.5 g each) were taken after six weeks of treatments. These samples were ground in beads beater, and then the samples were homogenized in phosphate buffer (0.05 M), having pH 7.8 in chilled conditions. Supernatant was centrifuged at 12,000× g for duration of 10 min at 4 • C. SOD, POD activities and malondialdehyde (MDA) contents were measured using the methods of Zhang [36].
The method of Aebi [37] was taken up for catalase (CAT, EC 1.11.1.6) activity evaluation. The 3.0 mL reaction mixture was comprised of H 2 O 2 (300 mM) 100 µL and enzyme extract 100 µL, which was ground in buffer containing a volume of 2.8 mL phosphate buffer (50 mM) with 2.0 mM EDTA (pH 7.0). Catalase activity was calculated by assessing the absorbance reduciton on a spectrophotometer at 240 nm, as a result of the disappearance of hydrogen peroxide (ε = 39.4 mM −1 ·cm −1 ).

Determination of Soluble Protein
Method of Bradford [39] was used to determine the soluble protein contents in leaves. The dye used was albumin and Coomassie Brilliant Blue G-250, was the standard. In liquid nitrogen, frozen samples of leaves (0.5 g) were crushed with a pestle and mortar. Afterwards, these were homogenized in 50 mm sodium phosphate buffer (10 mL) having 7.0 pH, and which also contains l.0 mm EDTA-Na 2 and polyvinyl pyrrolidine-40, also known as PVP-40, 2% (w/v). Additionally, the homogenate was centrifuged for duration of 15 min at 11,000× g at 4 • C. After that, the supernatant was used for analyzing the activity of an antioxidant enzyme. Furthermore, the addition of of Bradford solution (1 mL) was added in 100 µL crude extract. The absorbance was then measured at 595 nm wavelength for the determination of the entire protein content. A standard curve of BSA was used for determining the protein concentration.

Contents of Hydrogen Peroxide
To observe the H 2 O 2 content in plants, 50 mg of fresh leaf and root was used and grinded in 3 mL of phosphate buffer (50 mM), having a pH of 6.5, and further centrifuged at 6000× g for a duration of 25 min.
In order to find out the H 2 O 2 content, the extracted solution in a quantity of 3 mL was mixed, with 1 mL of 0.1% titanium sulphate in 20% (v/v) H 2 SO 4 , and then it was centrifuged at 6000× g for a 15-min duration. At 410 nm, the intensity of yellow color of the supernatant was determined. H 2 O 2 content was measured with the use of the extinction coefficient (0.28 µmol −1 ·cm −1 ).

Chromium and Zn Concentration Evaluation
With all the three replications, the harvesting of maize plants was done after six weeks. They were then washed extensively with water, distilled water, and finally with the deionized water, to clean them. Leaves, roots and stems were separated from the main plant body. Plant biomass was first dried for 48 h in an oven, with a temperature of 80 • C. The dried biomass was then ground into powder. Each sample was taken in 0.5 g quantity in 100 mL flasks, and 15 mL of concentrated HNO 3 were added in each flash using a pipette. The acid and samples were then mixed, and then the flasks were placed on a hot plate. The temperature was slowly increased up to 275 • C, which resulted in the formation of thick yellow fumes in the flask. when these fumes became low in quantity, then hydrogen peroxide was added carefully in flasks, until dense yellow fumes totally vanished. The flasks were then removed from the hot plate, and these were shifted to the lab, where they became colorless. Their volume was made up to 25 mL with distilled water. For the measurement of the concentration of the Cr and Zn contents in different plant parts, flame atomic absorption spectrometry (Nova 400 Analytik Jena, Jena, Germany) was used. For quality control and quality assurance (QA/QC), for Cr and Zn determination in the plant samples, blanks were placed parallel to the samples, containing acids without samples, and were digested in a similar manner to sample digestions, and these were also analyzed for Cr and Zn, and the calculations were adjusted accordingly. The standards of Cr and Zn with different known concentrations were also run on the instrument, and random standards were also analyzed after every ten samples during analyses, to ensure authenticity of the analyses.

Statistical Analysis
The analysis of variance (ANOVA) was carried out by using a statistical package. SPSS version 16.0 (SPSS, Chicago, IL, USA). For the determination of the significant difference between values, Tukey's test was used.

Effects on Plant Growth and Yield
The application of tannery wastewater to maize substantially reduced the morphological growth of maize ( Figure 1). However, Zn-lys application modulated the growth under contaminated soil conditions. For instance, on average, the plant height, root length, number of leaves, and leaf area decreased up to 27% and 49%, 15% and 43%, 28% and 53%, and 35% and 50%, at 50% and 100% tannery wastewater, compared with the control. The average values for all morphological parameters were recorded as higher and/or similar, but not up to a significant level at 25% tannery wastewater, as compared with control. Moreover, with increasing tannery water treatment percentage in the irrigation water, the effects of Zn-lys were more prominent as compared to lower doses of tannery wastewater, as such effects were also more positive, with an increased Zn-lys application dose. Moreover, 100% tannery wastewater treatment with no Zn-lys spray showed the lowest growth and biomass, including root length, plant height, leaf area and number of leaves per plant Figure 1). colorless. Their volume was made up to 25 mL with distilled water. For the measurement of the concentration of the Cr and Zn contents in different plant parts, flame atomic absorption spectrometry (Nova 400 Analytik Jena, Jena, Germany) was used. For quality control and quality assurance (QA/QC), for Cr and Zn determination in the plant samples, blanks were placed parallel to the samples, containing acids without samples, and were digested in a similar manner to sample digestions, and these were also analyzed for Cr and Zn, and the calculations were adjusted accordingly. The standards of Cr and Zn with different known concentrations were also run on the instrument, and random standards were also analyzed after every ten samples during analyses, to ensure authenticity of the analyses.

Statistical Analysis
The analysis of variance (ANOVA) was carried out by using a statistical package. SPSS version 16.0 (SPSS, Chicago, IL, USA). For the determination of the significant difference between values, Tukey's test was used.

Effects on Plant Growth and Yield
The application of tannery wastewater to maize substantially reduced the morphological growth of maize ( Figure 1). However, Zn-lys application modulated the growth under contaminated soil conditions. For instance, on average, the plant height, root length, number of leaves, and leaf area decreased up to 27% and 49%, 15% and 43%, 28% and 53%, and 35% and 50%, at 50% and 100% tannery wastewater, compared with the control. The average values for all morphological parameters were recorded as higher and/or similar, but not up to a significant level at 25% tannery wastewater, as compared with control. Moreover, with increasing tannery water treatment percentage in the irrigation water, the effects of Zn-lys were more prominent as compared to lower doses of tannery wastewater, as such effects were also more positive, with an increased Zn-lys application dose. Moreover, 100% tannery wastewater treatment with no Zn-lys spray showed the lowest growth and biomass, including root length, plant height, leaf area and number of leaves per plant Figure 1). The biomasses of stem, leaves and roots (fresh and dry) were substantially lowered under tannery wastewater application, and such reductions were more severe, with increasing the tannery wastewater concentration, except at 25%, whilst exogenous Zn-lys application improved the fresh and dry biomass, either with or without tannery wastewater treatments. The highest fresh and dry biomass was recorded in Zn-lys application at 25 mM dose under control, and each tannery wastewater treatment ( Figure 2). The biomasses of stem, leaves and roots (fresh and dry) were substantially lowered under tannery wastewater application, and such reductions were more severe, with increasing the tannery wastewater concentration, except at 25%, whilst exogenous Zn-lys application improved the fresh and dry biomass, either with or without tannery wastewater treatments. The highest fresh and dry biomass was recorded in Zn-lys application at 25 mM dose under control, and each tannery wastewater treatment ( Figure 2). The biomasses of stem, leaves and roots (fresh and dry) were substantially lowered under tannery wastewater application, and such reductions were more severe, with increasing the tannery wastewater concentration, except at 25%, whilst exogenous Zn-lys application improved the fresh and dry biomass, either with or without tannery wastewater treatments. The highest fresh and dry biomass was recorded in Zn-lys application at 25 mM dose under control, and each tannery wastewater treatment (Figure 2).

Effects on Photosynthetic Pigments
The photosynthetic pigments, i.e., chlorophyll a, chlorophyll b, total chlorophyll contents and carotenoids, decreased with increasing concentrations of tannery wastewater, especially as the extent of such reductions were obviously lower in Zn-lys applied plants than non-applied plants. The application of tannery wastewater specifically at 50% and 100% led to 18% and 52%, 36% and 63%, 24% and 56% and 36% and 56% reductions in Chl a, Chl b, total Chl contents and carotenoids, respectively. The values were marginally different than for the control at 25% wastewater treatment. The lowest values for Chl a, Chl b, total Chl contents and carotenoids under each wastewater treatment were recorded in plants without Zn-lys application, whilst Zn-lys application at 25 mM led to a prominent increase in photosynthetic pigments, as compared with non-Zn-lys applied plants ( Figure 3).

Effects on Photosynthetic Pigments
The photosynthetic pigments, i.e., chlorophyll a, chlorophyll b, total chlorophyll contents and carotenoids, decreased with increasing concentrations of tannery wastewater, especially as the extent of such reductions were obviously lower in Zn-lys applied plants than non-applied plants. The application of tannery wastewater specifically at 50% and 100% led to 18% and 52%, 36% and 63%, 24% and 56% and 36% and 56% reductions in Chl a, Chl b, total Chl contents and carotenoids, respectively. The values were marginally different than for the control at 25% wastewater treatment. The lowest values for Chl a, Chl b, total Chl contents and carotenoids under each wastewater treatment were recorded in plants without Zn-lys application, whilst Zn-lys application at 25 mM led to a prominent increase in photosynthetic pigments, as compared with non-Zn-lys applied plants (Figure 3).

Effects on Photosynthetic Pigments
The photosynthetic pigments, i.e., chlorophyll a, chlorophyll b, total chlorophyll contents and carotenoids, decreased with increasing concentrations of tannery wastewater, especially as the extent of such reductions were obviously lower in Zn-lys applied plants than non-applied plants. The application of tannery wastewater specifically at 50% and 100% led to 18% and 52%, 36% and 63%, 24% and 56% and 36% and 56% reductions in Chl a, Chl b, total Chl contents and carotenoids, respectively. The values were marginally different than for the control at 25% wastewater treatment. The lowest values for Chl a, Chl b, total Chl contents and carotenoids under each wastewater treatment were recorded in plants without Zn-lys application, whilst Zn-lys application at 25 mM led to a prominent increase in photosynthetic pigments, as compared with non-Zn-lys applied plants (Figure 3). Statistical analyses were carried out by ANOVA, followed by Tukey's Post-Hoc test.

Effects on H2O2 and MDA Production
Tannery water application caused a remarkable increase in the H2O2 and MDA contents in the leaves and root of maize. As compared to the control, content of H2O2 and MDA were increased by up to 12%, 50%, and 68%, and 11%, 29% and 48%, as well as 16%, 51% and 89% and 14%, 48% and 79% in leaves and root, at 25%, 50%, and 100% tannery water application, respectively. Moreover, Zn-lys treatments led to a substantial reduction in H2O2 and MDA in leaves and root of maize than non-Zn-lys applied plants, whilst the H2O2 and MDA contents gradually lowered, with an increase in the concentration of Zn-lys (Figure 4). Statistical analyses were carried out by ANOVA, followed by Tukey's Post-Hoc test.

Effects on H 2 O 2 and MDA Production
Tannery water application caused a remarkable increase in the H 2 O 2 and MDA contents in the leaves and root of maize. As compared to the control, content of H 2 O 2 and MDA were increased by up to 12%, 50%, and 68%, and 11%, 29% and 48%, as well as 16%, 51% and 89% and 14%, 48% and 79% in leaves and root, at 25%, 50%, and 100% tannery water application, respectively. Moreover, Zn-lys treatments led to a substantial reduction in H 2 O 2 and MDA in leaves and root of maize than non-Zn-lys applied plants, whilst the H 2 O 2 and MDA contents gradually lowered, with an increase in the concentration of Zn-lys ( Figure 4).  Statistical analyses were carried out by ANOVA, followed by Tukey's Post-Hoc test.

Effects on H2O2 and MDA Production
Tannery water application caused a remarkable increase in the H2O2 and MDA contents in the leaves and root of maize. As compared to the control, content of H2O2 and MDA were increased by up to 12%, 50%, and 68%, and 11%, 29% and 48%, as well as 16%, 51% and 89% and 14%, 48% and 79% in leaves and root, at 25%, 50%, and 100% tannery water application, respectively. Moreover, Zn-lys treatments led to a substantial reduction in H2O2 and MDA in leaves and root of maize than non-Zn-lys applied plants, whilst the H2O2 and MDA contents gradually lowered, with an increase in the concentration of Zn-lys (Figure 4).

Effects on Free Amino Acids, Soluble Protein, Soluble Sugar and Free Proline
The up-regulations in concentration of the soluble protein and total free amino acids were noticed with the foliar application of Zn-lys, under tannery wastewater treatment in maize plants. Compared with control (0% wastewater treatment), the total free amino acids and soluble protein contents gradualy increased by 10%, 31% and 64%, and 18%, 61% and 122%, at 25%, 50% and 100% tannery water treatment. In addition, the soluble sugars increased by 8% at 25% tannery wastewater, whilst being reduced by 18% and 45% at 50% and 100% tannery wastewater, respectively. On the other hand, the free proline contents increased with the progression of the application dose of tannery wastewater, i.e., increased by 14%, 39%, and 68% at 25%, 50% and 100% tannery wastewater, as compared with control. Overall, total free amino acids, soluble sugars, soluble protein and free proline were examined to be high in plants treated with the foliar spray of Zn-lys, as compared to plants without any Zn-lys application ( Figure 5).

Effects on Free Amino Acids, Soluble Protein, Soluble Sugar and Free Proline
The up-regulations in concentration of the soluble protein and total free amino acids were noticed with the foliar application of Zn-lys, under tannery wastewater treatment in maize plants. Compared with control (0% wastewater treatment), the total free amino acids and soluble protein contents gradualy increased by 10%, 31% and 64%, and 18%, 61% and 122%, at 25%, 50% and 100% tannery water treatment. In addition, the soluble sugars increased by 8% at 25% tannery wastewater, whilst being reduced by 18% and 45% at 50% and 100% tannery wastewater, respectively. On the other hand, the free proline contents increased with the progression of the application dose of tannery wastewater, i.e., increased by 14%, 39%, and 68% at 25%, 50% and 100% tannery wastewater, as compared with control. Overall, total free amino acids, soluble sugars, soluble protein and free proline were examined to be high in plants treated with the foliar spray of Zn-lys, as compared to plants without any Zn-lys application ( Figure 5).

Effects on Antioxidant Enzymes Activities
The irrigation with tannery water altered the antioxidant enzyme activities, including SOD, POD and CAT, in both the leaves and root of maize. Overall, the antioxidant activities were increased from 0 to 50% application of wastewater, followed by an abrupt decline at 100% wastewater treatment, in both leaves and root of maize plants. On the other hand, the application of Zn-lys at all wastewater treatments improved the antioxidant enzyme activities, whereas the antioxidant activities were remained the maximum at the highest level of Zn-lys application ( Figure 6). whilst being reduced by 18% and 45% at 50% and 100% tannery wastewater, respectively. On the other hand, the free proline contents increased with the progression of the application dose of tannery wastewater, i.e., increased by 14%, 39%, and 68% at 25%, 50% and 100% tannery wastewater, as compared with control. Overall, total free amino acids, soluble sugars, soluble protein and free proline were examined to be high in plants treated with the foliar spray of Zn-lys, as compared to plants without any Zn-lys application ( Figure 5). Statistical analyses were carried out by ANOVA, followed by Tukey's Post-Hoc test.

Effects on Antioxidant Enzymes Activities
The irrigation with tannery water altered the antioxidant enzyme activities, including SOD, POD and CAT, in both the leaves and root of maize. Overall, the antioxidant activities were increased from 0 to 50% application of wastewater, followed by an abrupt decline at 100% wastewater treatment, in both leaves and root of maize plants. On the other hand, the application of Zn-lys at all wastewater treatments improved the antioxidant enzyme activities, whereas the antioxidant activities were remained the maximum at the highest level of Zn-lys application ( Figure 6).
Furthermore, the lowest antioxidant enzyme activities were found in the control (without wastewater treatment and Zn-lys application) for both leaves and root of maize plants ( Figure 6). Statistical analyses were carried out by ANOVA, followed by Tukey's Post-Hoc test.
The irrigation with tannery water altered the antioxidant enzyme activities, including SOD, POD and CAT, in both the leaves and root of maize. Overall, the antioxidant activities were increased from 0 to 50% application of wastewater, followed by an abrupt decline at 100% wastewater treatment, in both leaves and root of maize plants. On the other hand, the application of Zn-lys at all wastewater treatments improved the antioxidant enzyme activities, whereas the antioxidant activities were remained the maximum at the highest level of Zn-lys application ( Figure 6). Furthermore, the lowest antioxidant enzyme activities were found in the control (without wastewater treatment and Zn-lys application) for both leaves and root of maize plants ( Figure 6).

Effects on Zn and Cr Uptake
The application of Zn-lys increased the Zn contents in leaves, stem and roots of maize, whilst it reduced the Cr contents in respective plant parts substantially under tannery wastewater application, as the tannery wastewater had been added, which led to an increase in the concentration of Cr. As the tannery wastewater volume increased, the concentration of Cr also increased, like a dose-additive phenomena which led to contamination of the plant tissues with Cr. Zn-lys (12.5, 25 mM) helped the plants to decrease the contamination of Cr. Thus, the Cr concentrations decreased by using Zn-lys at each tannery wastewater treatment. Due to the addition of Zn-lys, Zn concentration was increased in all parts of maize plant (Figure 7). Furthermore, the lowest antioxidant enzyme activities were found in the control (without wastewater treatment and Zn-lys application) for both leaves and root of maize plants ( Figure 6).

Effects on Zn and Cr Uptake
The application of Zn-lys increased the Zn contents in leaves, stem and roots of maize, whilst it reduced the Cr contents in respective plant parts substantially under tannery wastewater application, as the tannery wastewater had been added, which led to an increase in the concentration of Cr. As the tannery wastewater volume increased, the concentration of Cr also increased, like a dose-additive phenomena which led to contamination of the plant tissues with Cr. Zn-lys (12.5, 25 mM) helped the plants to decrease the contamination of Cr. Thus, the Cr concentrations decreased by using Zn-lys at each tannery wastewater treatment. Due to the addition of Zn-lys, Zn concentration was increased in all parts of maize plant (Figure 7). The application of Zn-lys increased the Zn contents in leaves, stem and roots of maize, whilst it reduced the Cr contents in respective plant parts substantially under tannery wastewater application, as the tannery wastewater had been added, which led to an increase in the concentration of Cr. As the tannery wastewater volume increased, the concentration of Cr also increased, like a dose-additive phenomena which led to contamination of the plant tissues with Cr. Zn-lys (12.5, 25 mM) helped the plants to decrease the contamination of Cr. Thus, the Cr concentrations decreased by using Zn-lys at each tannery wastewater treatment. Due to the addition of Zn-lys, Zn concentration was increased in all parts of maize plant (Figure 7).

Discussion
Tannery wastewater often consists of various toxic metals that could severely affect the morpho-physiological growth of crop plants. The application of tannery wastewater to crops could contaminate agricultural lands, as well as food products. When edible plants are grown in contaminated soil, many heavy metals can accumulate in plant parts, which are toxic to plants and also cause harm to the health of human beings [10,40,41]. The tannery wastewater used in the present study has higher values of Cr, and other different parameters (Table 2). Previous study also reported the higher values of Cr, and other studied attributes of tannery wastewater used for the irrigation of plants [42]. The higher values of COD and TOC (Total organic carbon) of tannery wastewater may also cause negative effect on plant growth, which might be due to the alteration of nutrient dynamics in soil and their uptake by plants [15,42,43]. It has been reported that the removal of COD and TOC from tannery wastewater is a difficult task and requires suitable techniques [44]. Thus, most of the industries are discharging wastewater without any proper treatment, and farmers are using this unsuitable water without any treatment. Present study investigated the effects of tannery wastewater on morpho-physiological features of maize plants, and the part of Zn-lys application in modulating the early growth and related physio-biochemical attributes of maize plants. Tannery wastewater application reduced the growth attributes, as well as the biomass of maize (both fresh and dry) plants considerably, whereas Zn-lys application improved the early growth and biomass accumulation of maize (Figures 1 and 2). Tannery wastewater related decline in growth may possibly be due to the pools of heavy metals, especially Cr in the tannery wastewater, that may not only interfere with the plant nutrient uptake, but also disrupt the normal plant metabolism. The Zn-lys application might increase the uptake of more nutrients from the soil, and provide relief against Cr stress. Moreover, comparatively higher growth and biomass accumulation was observed at a lower concentration of tannery wastewater, i.e., 25%, than other concentrations applied in the study. Lower concentrations of tannery wastewater could promote the plant growth, as it contains differential amounts of micro-and macro-nutrients, and could also be a good fertilizer source. Previously, Calheiros et al. [15] reported that the application of tannery wastewater at lower doses showed the positive effects on growth related attributes of Trifolium pretense. On the other hand, the growth and biomass accumulation of Spirodela polyrrhiza was substantially reduced under higher doses of tannery wastewater application [45]. Moreover, Zn-lys being an amino-chelated fertilizer could hinder the mobility of otherwise readily available Cr, as it forms complexes with various heavy metals, including Cr [46]. Zn-lys application induced the improvement in growth and biomass accumulation under higher doses of tannery wastewater, which was also found in wheat [47] and rice [43] previously.
The chlorophyll contents in plants applied with tannery water were considerably lower, whilst the foliar application of Zn-lys modulated the leaf chlorophyll contents of maize ( Figure 3). Metallic ions (ie. Cr) present in leather tannery wastewater showed enormous interference with different vital micronutrients that eventually resulted in the subsequent degradation of chlorophyll contents [48,49]. Cr-induced reductions in chlorophyll biosynthesis were previously reported in different crops, like maize, barley, and spinach [42,50,51]. An enhancement in the chlorophyll contents in Zn-lys applied plants may be ascribed to the higher Zn contents, with subsequent decreases in Cr uptake and accumulation in Zn-lys applied plants, as Zn is actively involved in the structural and functional integrity of chloroplast ultra-structures and chlorophyll biosynthetic in plants [52,53]. Zn-induced improvements in the chlorophyll contents were also observed in Raphanus sativus [54] and wheat [47].
The application of tannery wastewater resulted in significant increase in the production of H 2 O 2 and MDA levels, whereas exogenous Zn-lys application relieved the oxidative damange by reducing the over-production of H 2 O 2 and MDA contents in growing maize plants (Figure 4). The uptake of various metallic ions present in tannery waste most often leads to the over-production of ROS, and hence is the cause of oxidative stress in plants. Increased MDA contents exhibited the higher lipid per-oxidation rate, whereas Cr, being a potent heavy metal in tannery waste, also generates the free radicals that oxidize biological membranes by reacting with them, and hence caused structural and functional damage to plants [10,42]. On the other hand, Zn-lys proved to be a stress alleviator, as it not only reduced the Cr uptake, but also had a ROS scavenging role. Previously, Mohammadi et al. [23] reported that the application of amino-acids chelated Zn helped in the reduction of oxidative stress in lettuce.
Regulation in osmolyte accumulation and anti-oxidative enzyme activities are an innate resonse of the plan towards stress conditions. This study reported the rise in total free amino acids, soluble proteins and free prolines, with a decrease in soluble sugars in tannery wastewater treated plants, whereas the contents of soluble protein, total free amino acids and soluble sugars were comparatively higher in Zn-lys applied plants than non-Zn-lys applied plants ( Figure 5). Furthermore, the antioxidant activities, i.e., SOD, POD and CAT, were increased from 0 to 50% application of tannery wastewater, followed by a sudden decline at full dose (100%) of wastewater treatment. Unexpected severe oxidative stress resulted in decline of activities related to the antioxidant enzymes as studied by Adrees et al. [55,56]. Comparable results were observed by Dey et al. [57] Overall, Zn-lys application improved the antioxidant activities in plants, both under tannery wastewater and control treatments ( Figure 6). Increased soluble sugars and proline might be an innate response of plants against tannery water-induced stress conditions. On the other hand, heavy metals may disrupt amino acids and proteins, thus causing structural and functional damage to the plants. Plant often accumulates differential amounts of various osmolytes and proteins, in stress conditions that not only protect the biological structures, but also help in the cleansing of ROS [41]. Moreover, Zn-lys application improved the antioxidant enzyme activities, and thus, decreased the subsequent oxidative stress in various plant system, as specified by the decline of H 2 O 2 and MDA contents. Recently, Zaheer et al. [42] reported that the application of Zn-lys chelated fertilizer improved the POD, SOD and CAT activities in roots and leaf of spinach grown in tannery wastewater contaminated soil.  [58]. This antioxidant-related ROS quenching mechanism, that was further enhanced by Zn-lys application, might help the plants to better tolerate the tannery wastewater related stress conditions. Exogenous Zn application at 25 µM enhanced the antioxidant activities significantly in bean plants [59]. In addition, Zn-lys application enhanced the Zn contents in different plant parts of maize, whereas it reduced the Cr concentrations in the subsequent plant parts (Figure 7). Higher concentrations of Cr in plants without Zn-lys may be related to the higher doses of tannery wastewater application, whereas amino-acid chelated Zn has the capability to form complexes with various metallic ions, and thus reduced their uptake and translocation in different plant organs [60]. Furthermore, the reductions in the Cr contents in lysine-chelated Zn applied plants were previously noted in rice, wheat and spinach [42,43]. Overall, the Zn-lys application improved the early growth, biomass accumulation, physio-biochemical attributes and Zn contents in different plant parts, whilst it reduced the Cr contents.

Conclusions
In conclusion, tannery wastewater proved beneficial to maize plants if applied at a low concentration, but at higher doses, it reduced the morphological growth and photosynthetic pigments of plants, whilst it enhanced the oxidative stress in terms of H 2 O 2 and MDA contents. Moreover, important activities of vital antioxidant enzyme including SOD, CAT and POD, in the roots and leaves of maize plant were enhanced with the application of tannery water as compared with the control, which has been further modulated by Zn-lys application. In addition, foliar Zn-lys application reduced the Cr contents in different parts of maize plants, while Zn contents were improved under these treatments. Hence, tannery wastewater can be applied as a fertilizer at lower doses (as it contains some micro/macro elements), however, the higher levels may be toxic to maize plants for normal growth. The dose of Zn-lys should be optimized if it needs to be used as a fertilizer source. Lysine chelated Zn can be efficiently used to improve the growth and stress reduction in maize plants, if applied in a suitable quantity to plants growing in tannery wastewater.


extfieldsofstudy: Medicine Chemistry

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

text:
Reduction of the acidity and peroxide numbers of tengkawang butter (Shorea stenoptera) using thermal and acid activated bentonites

Tengkawang fat (Shorea stenoptera), from an indigenous plant of the Kalimantan forest, has excellent potential as an alternative source of vegetable fat because it has a high level of fatty acids composition. Activated natural bentonite can be used as a bleaching agent to improve the quality of tengkawang fat. This research aims to reduce the acidity, peroxide number values and identify the physicochemical properties (fatty acid composition, nutrients, and thermal) of tengkawang butter. Initially, tengkawang samples from Nanga Yen and Sintang were pre-treated using the degumming process with 1% phosphoric acid and the neutralization process with a 1 M NaOH 10% w/w solution. The results show that the acidity (mg NaOH/g) of the tengkawang fat samples was reduced from 11.00 to 3.36 when using bentonite activated at 200 °C. The bentonite activated with 0.5 M HCl reduced the acidity to 3.61. The peroxide number (meq O2/kg) of the tengkawang fat samples was reduced from 9.45 to 4.84 and 3.47 by bleaching with thermal-activated and acid-activated bentonites, respectively. Peroxide value correlates with β-carotene content. The smaller of the β-carotene content, the smaller the peroxide value. The acidity, peroxide number, and iodine number values from tengkawang fat after treatment adhere to the SNI 2903: 2016 standard. The main content of fatty acids in tengkawang fat is palmitic acid, stearic acid, and oleic acid. These results show that both products are suitable for the food industry in terms of the acid and peroxide numbers. The application of this research results will assist local people in increasing the economic value of the product from tengkawang plant, which is an indigenous plant from Kalimantan.

Introduction
Indonesia's fat consumption needs are relatively high, so it must import 20,880,600 kg of solid vegetable fat to fulfil fat needs [1]. One of Indonesia's local plants used as alternative vegetable fat is tengkawang (Shorea stenoptera). Several plants in the Dipterocarpaceae family, such as Shorea stenoptera, S. mecisopteryx, S. pinanga, produce tengkawang fruit [2,3]. Tengkawang fat, known as illipe butter, has excellent potential as an alternative source of vegetable fat because it has a high level of fatty acid composition and is widely used in the community to meet vegetable fat needs. Various ways of tengkawang fat being used as a material for household consumption. For example, tengkawang fat has been used by communities as a material for lipstick [4], wet noodles [5], and for making black butter rice [6]. Tengkawang butter can be used as a mixing agent for chocolate fat, margarine, and lip paint [7]. The properties of tengkawang fats are similar to cocoa fat; it is classified as a cocoa butter substitute and can be used in the cosmetics and food industries [8].
Tengkawang fat produced by individual community members has a quality below the Indonesian national standard (SNI), meaning that further processing is needed. Traditional tengkawang fat processing uses a device known as an "apit" with a production capacity of around 4-5 kg of fat in a single process [9]. Today's industrialized processing of fats and oils frequently includes degumming, neutralization, and bleaching. The degumming process removes impurities, such as phosphatides, from fats [10] and neutralization to reduce excess free fatty acids in oils and fats [11]. The bleaching process removes excessive dyes and impurities, using adsorbents such as bentonite [12].
Bentonite is primarily montmorillonite, and it is a 2:1 form of mineral; the unit layer structure consists of one Al 3þ octahedral [13]. Modified reactions change bentonite's surface and structural characteristics by replacing interlayer cations (e.g., Na þ , K þ , Ca 2þ ) with particular species or sites [14]. Bentonite has reasonably good properties as an adsorbent because it can exchange ions on the surface. The silica layer's charge causes this phenomenon, while the interaction with its filling ions on interlayers such as Al, Ca, and Mg is not so strong that it tends to exchange ions with other cations (protonation) [15]. The cation exchange capacities (CEC) of calcium bentonite range from 40 to 70 mEq/100 g, while those of sodium bentonite is between 80 and 130 mEq/100 g [16]. The bentonite activation process is needed to obtain more optimal results. The activation of bentonite with acids can increase the surface area [17].
Modification of bleaching is needed to achieve the desired butter quality of tengkawang butter [18]. Various ways of modifying the bleaching process activate adsorbents, added chemicals, and heaters [18,19,20,21]. Using adsorbent is the most commonly used because it is relatively useful and low-cost to bleach vegetable oil. One of the adsorbents often used in bleaching vegetable oils is bentonite/bleaching earth [12]. Bentonites/Bleaching Earth are proven to be an adsorbent for bleaching [12,20,22].
Research conducted by Hidayat et al. on tengkawang fat processing with activated carbon obtained acidity values, peroxide numbers, and iodine numbers of 6.12 mg NaOH/g fat, 5.55 mEq O 2 /kg fat, and 31.39 g I 2 /100 g fat, respectively [23]. This research aims to reduce the acidity, peroxide number values and identify the physicochemical properties (fatty acid composition, nutrients, and thermal) of tengkawang butter. In such a way, this research will contribute to the increase of added value to the tengkawang fat products in Kalimantan by meeting the food industry standards.

Preparation of materials
Tengkawang fat for this study was obtained from the Nanga Yen and Sintang Villages, Kapuas Hulu Regency, West Kalimantan, Indonesia. Analytical-grade chemicals, such as NaOH, KI, H 3 PO 4 , ICl, and KIO 3 , were obtained from Merck Millipore (Germany). Bentonite was obtained from Lampung Regency, Lampung Province. The tengkawang fat treatment process in this study involved degumming, neutralization, and bleaching. The bleaching process was carried out using bentonite, which was activated by thermal and acid methods. The pre-treatment process was carried out based on previous research using 20% phosphoric acid for degumming and 1 M NaOH in the neutralization process [23]. However, the bleaching process used thermal and acid activation methods.

Pre-treatment of tengkawang fat
The degumming process was carried out to remove the gum and impurities found in tengkawang fat [23]. Tengkawang fat that had been melted was added to a 20% phosphoric acid solution for 1% (w/w). The mixture was then stirred with a magnetic stirrer for 30 min in a beaker glass. After 30 min, the tengkawang fat was separated from the impurities using warm water at 60-70 C.
The neutralization process is carried out to remove the remaining free fatty acids in tengkawang fat using alkaline reagents [23]. 1 M NaOH of 10% (w/w) was added to degummed tengkawang fat. The mixture of tengkawang fat and base was stirred for 30 min at temperatures around 60-70 C. The soap layer was separated from the oil layer using centrifugation. Warm water was used to dissolve the remaining soap contained in the component. Centrifugation was used to separate the oil (above) and the layer of water (below).

Bentonite activation
Two variations of the bentonite activation process were carried out: acid activation and thermal activation. The method used is based on the method used by Christidis et al. [24]. HCl was used as the chemical reactant in the acid activation method. HCl solution with two concentrations (0.5 N and 1 N) was added to the bentonite with bentonite to the HCl ratio of 1:5 (w/w). The mixture was then stirred with a magnetic stirrer for time variation (1 and 3 h) at 70 C in a beaker glass. After stirring, the bentonite was washed with distilled water to a pH of about 6. The bentonite slurry was then filtered and separated using a vacuum filtration method and a Sartorius Stedim cellulose nitrate membrane with a porosity of 0.8 μm. The bentonite was dried in the oven at 100 C for 3 h to evaporate the bentonite's water content.
The bentonite thermal activation was carried out at a variation temperature of 200 C and 300 C for 60 and 120 min.
The characterization of the bentonite before and after activation was conducted using a Scanning electron microscope (SEM), brunauer-Emmett-teller (BET) surface area analysis, X-ray fluorescence (XRF), and X-ray diffraction (XRD).

Bleaching process
The bleaching process was conducted by adding 5% thermalactivated and acid-activated bentonite to the tengkawang butter. The mixture of pre-treatment tengkawang and bentonite was stirred for 30 min at a temperature of 60-70 C in a beaker glass. After the stirring process was complete, the bentonite was separated from the tengkawang fat using a vacuum filtration method and a Sartorius Stedim cellulose nitrate membrane with a porosity of 0.8 μm.

Chemical analysis of tengkawang butter
The composition of tengkawang fats was characterized using a Gas Chromatography (GC) instrument to determine the fatty acid profile. Samples of up to 1 g of tengkawang fat from Nanga Yen and Sintang were prepared and dissolved in a 100 mL heptane solvent. For each fat sample, 1 ml was injected into the GC column. Before the fat sample was injected, it had to be confirmed that the mobile phase was running well. The mobile phase used in this study was helium gas. After being heated to a temperature of 300 C, the evaporating component was detected by an FID detector. The detector produced a separation chromatogram of the sample component.
Thermal testing of fat samples was conducted using DSC instruments to determine the thermal properties of the fat. A total of 1 g of sample was put into the DSC container. In this study, samples were heated to a temperature of 70-80 C with a 1 C/min heating rate and allowed to stand for 30 min. Data generated from the thermogram were compared to determine changes in the thermal properties.

Analysis of heavy metal contaminant
Analysis of heavy metals contaminants in tengkawang fat was carried out according to the AOCS 999.11 method [25]. Flame Absorbance Spectroscopy (FAAS) instrument was used to test the heavy metal content with a mixture of acetylene gas -air as a burner. Hollow cathode for heavy metals element (Pb, Hg, As, Cd, Sn, Cu). Concentrated HCl and HNO 3 for diluting heavy metals in the sample. Heavy Metals (Pb, Hg, As, Cu, Sn, and Cd) Standard solution. The sample was drying for 10-20 min in the oven at 100 C. After the drying process, the sample was placed in the furnace. The temperature was increased at a maximum of 50 C/h to 450 C for 8 h or overnight. After the sample turning into ash, dilute sample residue with 10-30 mL 0.1 M HNO 3 and homogenize. The calibration curve was made with a heavy metal standard. The sample was tested with a specific hollow cathode for each heavy metal elements in the AAS instrument.

Physical characterization of bentonite
The type of XRD (X-Ray Diffraction) instrument used in this study was the PANalytical: X'Pert PRO with Cu Kalpha 1.54 Å as the radiation source. XRD characterization was carried out at angles of 2θ.5 o -90 o . Characterization was carried out in both the initial bentonite and after activation (acid and thermal).
The type of XRF (X-ray fluorescence) instrument used in this study was the PANalytical: Epsilon1 with Ag. XRF is the source of radiation. XRF was performed to determine the metal and non-metal content in the bentonite samples. The XRD and XRF instruments belong to the Laboratory of Research, Department of Physics, Faculty of Mathematics and Natural Sciences, Universitas Indonesia.
An SEM test was performed to determine the bentonite microstructure's characteristics at each stage of the process; the test produced each sample's surface image in three dimensions. The instrument used was an SEM (Scanning Electron Microscopy) JEOL JSM 6510 LA belonging to the Scanning Electron Microscope Laboratory of the Department of Metallurgy and Materials, Faculty of Engineering, Universitas Indonesia.
A Brunauer-Emmett-Teller (BET) test was performed to determine the pores' surface area in the bentonite. This study's type of BET instrument was a Quantachrome Quadrasorb-Evo Surface Area & Pore Size Analyzer. The bentonite samples were characterized using a BET analyzer and N 2 gas sorption to determine their surface areas. The BET instrument belongs to the Integrated Laboratory and Research Centre, Universitas Indonesia.

Analysis of acidity, peroxide, and iodine value of tengkawang butter
The analysis of acidity, peroxide, and iodine value was using the SNI method: 01-3555-1998 [26]. Analysis of acidity was using the method of base titration with 0.1 M NaOH as the titrant. The calculation of acidity uses the following equation: where: V ¼ volume of NaOH as the titrant (mL) T ¼ NaOH Normality 40 ¼ Mass molecule of NaOH (gram/mol) m ¼ mass of sample (gram) Iodine number analysis was performed using the titrimetric method with Wijs solution as the reagent, sodium thiosulfate solution as the titrant, and starch as an indicator. The calculation of iodine numbers uses the following equation: where: Peroxide number analysis was performed using the titrimetric sodium thiosulfate solution as titrant and starch as an indicator. The equation for calculating peroxide numbers as follows: where: Vo ¼ Volume of sodium thiosulfate solution 0.l N for blank titration (mL). V l ¼ Volume of sodium thiosulfate solution 0.l N for sample titration (mL) T ¼ Normality of sodium thiosulfate solution (0.1 N) m: the mass of the sample (gram)

Statistical data analysis
Analysis of variance testing (ANOVA) was carried out in this study. If there is a change in the treatment process, further testing is carried out using Duncan's New Multiple Range Test (DMRT) at the 5% level [27].

Physicochemical properties of bentonite
XRD (X-Ray Diffraction) characterization to determine the bentonite's crystal composition and structure. Comparing the XRD diffractogram peaks with the database can determine the type of crystal in the material.
In Figure  was also seen in the study conducted by Temuujin et al. (2004), which used HCl as an acid for activation with reduced montmorillonite peak results [17]. There are still quartz peaks, which indicate that quartz tends to be resistant to acid changes compared to montmorillonite. In a study conducted by Amari et al. (2018), quartz peaks also remained after a leaching process with sulfuric acid [28]. Thermal activation reduces the intensity of montmorillonite crystals in bentonite. The intensity of montmorillonite in natural bentonite reduced from 2096 to 1510 for thermal-activated bentonite.  have reported that thermal activation showed a decrease in montmorillonite crystals' intensity at 2θ 5 o in their study [21]. This phenomenon indicates a dehydration process or a loss of water molecules between the crystal sheet and distortion of the crystalline structure due to the high temperatures used in thermal activation [29]. From the graph, the activation causes a decrease in quartz crystal peaks and kaolinite. This phenomenon occurs because kaolinite and quartz begin to experience anhydrous decomposition at 200 C; the heating temperature causes damage in the crystal structure [30]. Figure 2 shows the difference in morphological structure between natural, acid-activated, and thermal-activated bentonite. Acid-activated bentonite has a smooth surface due to acid leaching. Hydrogen ions from acids dissolve impurities on bentonite's surface, increasing the surface area [31]. Morphologically, thermal-activated bentonite has more pores when compared to natural bentonite. This phenomenon is due to the effect of the activation temperature, which evaporates the bentonite's water content so that the pores that were previously still bound to water become open [32]. Rough surfaces show swelling as a result of the interactions between the montmorillonite crystals in bentonite and water. Rough surfaces occur because thermal activation makes bentonite more porous [33,34]. Previous studies have shown that heating at 250 C for 90 min tends to make bentonite surfaces rougher [35]. Table 1 shows BET testing on thermal-and acid-activated bentonite to determine changes in the bentonite samples' surface area.  (2012), which found that the activation of bentonite with acid (hydrochloric acid) resulted in a change in surface area from 25.0 m 2 /g to 75.5 m 2 /g [36]. Viera et al. (2010) reported a calcination process of bentonite at a temperature of 500 C, increasing the surface area from 78.9 to 89.0 m 2 /g [37]. The acid-activated bentonite's surface area in this study is much higher compared to those of natural and thermal-activated bentonite. This phenomenon was because of the reduced impurity of minerals found on the bentonite's surface that increased the surface area and later served as an adsorbent [38]. Table 2 shows the composition of natural bentonite, acid-activated bentonite, and thermal-activated bentonite. In thermal activation, there was a decrease in the silicate levels and the Si/Al ratio. This phenomenon   was likely due to damage to the bentonite structure, especially the silicate bonds. The decreasing Si/Al ratio data reinforces data from the XRD, where the montmorillonite crystal peaks were smaller than in the natural bentonite. The activation of bentonite with acids caused an increase in the silicate levels (SiO 2 ) due to the octahedral cations' remobilization presented in the aluminosilicate group [39,40]. The displaced octahedral cation was composed of silicate structures due to the bentonite composition's high silicon content [41]. Some studies have also proved that acid activation increases silica levels in bentonite and reduce the impurities. Salem [43]. Based on the data in Figure 1(A and B) and Table 2, there is a correlation between changes in crystal intensity with the composition of acid-activated and thermal-activated bentonite. In figure 1(A and B), there is a decrease in the intensity of montmorillonite, quartz and kaolinite crystals. Crystal intensity in thermal activation bentonite reinforcing the data in Table 2 that reduces the Si/Al ratio composition in bentonite. In acid-activated bentonite, the intensity of montmorillonite crystals decreased slightly, but quartz and kaolinite tended to remain. However, because in the acid activation process, there is a leaching process which reduces the impurity cations. There is cation remobilization which increases the Si/Al content ratio in bentonite after the leaching process. Due to the addition of acid in the activation process, there will be H þ cations in the bentonite composition. H þ cations correlate with an increase in acidity in the system, which results in increase acidity of fatty products in the bleaching process.
Based on the data in Table 1 and Figure 2, the thermal and acid activation processes increase the surface area of bentonite. The larger surface area can absorb more impurities in the oil bleaching process. The impurities such as coloured compounds, organic hydroperoxides and FFA can be absorbed into the pores on the surface of the bentonite. The reduced impurity content will improve the quality of the oil. Therefore, the activation process that increases the surface of bentonite can have a good effect on oil processing.

Fatty acid composition of tengkawang butter
Fatty acid profiles of the tengkawang butter samples from Nanga Yen and Sintang were analyzed to determine the fatty acid composition. The fatty acids contained in the tengkawang fat were predominantly palmitic acid, stearic acid, and oleic acid. Figure 3 is a GC chromatogram for analysis of the fatty acid content of tengkawang fat. Based on the chromatogram, the clear peaks are fatty acids C16 (palmitic acid), C18 (stearic acid), C18: 1 9c (oleic acid), C18: 2 6c (linoleic acid), C20 (arachidonic acid) with RT values of 9.635, 11.469, 11.799, 12.412 and 13.924, respectively. Comparing the retention time (RT) of the sample with the standard retention time (RT) can determine the type of fatty acids [44]. The standard RT can be compared with a specific RT value to determine components in a separated column. Retention time for standard C16, C18, C18:1, C18:2 and C20 are 9.684, 11.676, 11.984, 12.521 and 14.040, respectively. Table 3 shows the main fatty acid composition (%) of each Nanga Yen and Sintang tengkawang fat sample consisted of stearic acid (44.267 and 42.600); oleic acid (31.894 and 31.290), and palmitic acid (19.710 and 20.220). The differences in the tengkawang growth environment and the seed extraction method influence the various tengkawang fat [45]. For the tengkawang fat from Nanga Yen, the method is the traditional steaming/heating process at high temperatures (100-150 C). In Sintang, in contrast, the fogging process was used to process the tengkawang fat. Both processes yield different results regarding the quality of the tengkawang fat produced, seen in the differing acidity and peroxide number values. Apart from the growth sites, the different types of tengkawang also have different fatty acid contents. Based on research conducted by Gusti and Zulnely (2015) using Shorea pinanga and Shorea mecisopteryx, different types of tengkawang have different contents [46]; Shorea pinanga contains stearic acid, oleic acid, palmitic acid, and linoleic acid in quantities of 1.56, 42.79, 11.78, and 22.04, respectively, while Shorea mecisopteryx contains these acids in quantities of 0.80, 31.28, 14.51, and 27.05, respectively.
In the tengkawang butter samples from Nanga Yen and Sintang, the type of tengkawang used was Shorea stenoptera, which has the dominant fatty acid components of stearic acid, oleic acid, and palmitic acid. Significant fatty acid components in S. pinanga and S. mecisopteryx are oleic  acid and linoleate acid. High oleic acid and linoleate acid contents in fat indicate that the fat is susceptible to oxidative degradation reactions triggered by the influence of high temperatures and light [46]. Tengkawang butter has potential as a cocoa butter equivalent (CBE) because it has a fatty acid composition that treats chocolate fat. CBEs are vegetable fats with similar physical and chemical characteristics to cocoa butter (CB) [47]. The primary fatty acids in CBEs are palmitic acid, stearic acid, and oleic acid, similar to those found in CB [48]. Gunstone (2011) have reported that the percentage of palmitic, stearic, and oleic acid in illipe butter makes it a useful component of CBE blends and allows it to be used directly without further processing [49].

Health issue of tengkawang butter
Research on health issues from tengkawang fat is still limited. That is because there is not much research data on nutrient content contained in tengkawang. In this study, we analyzed the nutrient content and metal contamination of tengkawang butter. Table 4 is the nutrient content found in tengkawang fat. The total fat content of tengkawang is very high, which is above 99%. Tengkawang fat has more calories than chocolate fat and shea fat. Consumption of foods with high calories can increase body weight and body fat mass [50].
Therefore, the intake of tengkawang, chocolate, and shea fats should be controlled not to cause obesity.
The vitamin composition of tengkawang fat is also deficient and insignificant. This condition is probably due to the processing at high temperatures and oxidation, which destroys the tengkawang fat vitamins. Vitamins are easily damaged when exposed to hot temperatures and interact with oxidizing agents such as oxygen in the atmosphere [51]. When using tengkawang fat as a food ingredient, it is necessary to add vitamins to increase vitamin levels.
The dominant fatty acid content in tengkawang fat is saturated fatty acids (SFA) such as palmitic acid and stearic acid and mono-unsaturated fatty acids (MUFA) such as oleic acid. The results of the analysis did not show any trans fatty acids in tengkawang fat. Trans fat increases lowdensity lipoproteins (LDL), triglycerides, and insulin levels and reduces beneficial high-density lipoproteins (HDL) [52]. High serum LDL levels, such as cholesterol, will increase the risk of atherosclerosis or coronary heart disease. Although it does not contain trans fat, tengkawang fat is high in SFA. Consumption of saturated fatty acids too high will also increase the LDL content in the body. Joint FAO/WHO recommended total intake of SFA is less than 10% of total energy intake [53]. Therefore, consumption of tengkawang fat must be controlled not to increase LDL levels in the body.   Table 5 are metal contaminants found in tengkawang fat. Heavy metals can harm human health. Cd is a dangerous element that can damage membranes and DNA and accumulate in the body's metabolism for 16-33 years [54]. Cd's daily intake is 0.008 mg, which is about 10.7% of the Reference Dose (RfD), equivalent to 0.001 mg.kg-1 of body weight per day [55]. Pb's toxic effects on several organs, such as the liver, kidney, spleen, and lungs, leading to reduced organ function [54]. Pb's daily intake is estimated to be up to 0.012 mg, equivalent to 0.245 mg per day for a 70 kg weight [55]. Arsenic is an element with a high risk of various complications, including dermatological effects, cardiovascular effects, pulmonary disorders, reproductive effects, and neurological effects [25,56,57,58,59]. Mercury is an environmental pollutant with a high risk to health due to the high toxicity and mobility of mercury in the ecosystem [60,61]. The Joint FAO (Food and Agriculture Organization of the United National)/WHO Expert Committee on Food Additives (JECFA) in 2004 set a limit for the concentration of mercury at 1.6 mg/kg of body weight [62].
In the contaminant analysis of tengkawang butter, the content of heavy metal contaminants (Cd, Hg, As, Pb, Sn) was below the threshold for heavy metal content in food raw materials. Therefore, tengkawang fat in this study is free from the risk of heavy metal contaminants. Tengkawang Nanga Yen and Sintang were obtained from rural areas far from industry and the risk of pollution.

Thermal analysis of tengkawang butter
Thermal characterization uses DSC to determine the thermal properties of a material. The tengkawang fats from Nanga Yen and Sintang have different characteristics due to their different fatty acid contents.
The DSC of the tengkawang fat samples in Figure 4 shows different peaks for the tengkawang fats from Sintang and Nanga Yen. In the raw tengkawang fat from Nanga Yen, the onset point occurred at 37.7 C and the offset point at 48.4 C, whereas in the raw tengkawang fat from Sintang, the onset point was at 39.1 C, and the offset point was at 48.1 C. There was also a big difference in heat flow between the tengkawang fat samples from Nanga Yen and Sintang. For the Nanga Yen tengkawang fat sample, the peak of heat flow occurred at -28,921 mW, while for the Sintang tengkawang fat sample, the peak of heat flow occurred at -3,156 mW. The enthalpy value of the raw tengkawang from Nanga Yen was 119.34 J/g, while that of the tengkawang from Sintang was only 98.99 J/ g. This phenomenon was probably due to differences in the fat compositions of the tengkawang samples from Nanga Yen and Sintang. The tengkawang sample from Nanga Yen has a larger component of saturated fatty acids, requiring more energy than the samples from Sintang. There are thermogram changes in raw and refined tengkawang fat. Refined tengkawang has a shorter peak than raw tengkawang and confirms the fatty acid profile data for both fats. In refined tengkawang fat, there is a slight increase in unsaturated fatty oleic acid and a decrease in saturated palmitic acid; due to the change in composition, the peak shifts to a lower temperature with a smaller peak height. The energy value needed to melt tengkawang fat before and after purification also changed, from 119 J/g to 91 J/g for the tengkawang from Nanga Yen and 98 J/g to 86 J/g for the tengkawang from Sintang. The enthalpy and temperature values of tengkawang fat are comparable to those of other vegetable fats.
From the results shown in Table 6, the enthalpy value of tengkawang fat (from both Nanga Yen and Sintang) is almost similar to dark chocolate and chocolate fats. This phenomenon is probably due to fatty acids' compositions, which are almost the same for tengkawang butter as for chocolate fat. Table 7 uses a complete randomized design of the first-factor factorial group Nanga Yen and Sintang consisting of thermally activated treatments (300 C -120 min; 300 C -60 min; 200 C -120 min; and 200 C -60 min). The second factor Nanga Yen and Sintang consisted of acidactivated treatment (HCl 0.5 N (1 h); HCl 0.5 N (3 h); HCl 1 N (1 h); and HCl 1 N (3 h). Table 7 shows the results from a statistical analysis of the acidity, peroxide, and iodine number of tengkawang fat. Numbers in the same acidity columns in thermal treatment of Nanga Yen dan Sintang tengkawang butter indicate that interaction was not significantly different according to Duncan's Multiple Range Test (DMRT) 5%. Meanwhile, the acid treatment shows different letters, which indicate the interaction significantly different. Numbers in columns of peroxide number in thermal treatment for Nanga Yen tengkawang butter there are significantly different for 300 C-120 min and 200 C-60 min, and not significantly different for Sintang tengkawang butter. For iodine columns, there are no significantly different for some data and significantly different for the others.

Acidity and peroxide number of tengkawang butter
The quality of tengkawang fat's analysis parameters is acidity, peroxide number, and iodine number. The acidity describes how much free fatty acid is in the fat sample. The acidity significantly influences the acidity of the solution needed to determine the effect of bentonite on the fat sample. The peroxide number is an essential indicator of the rancidity of a sample. The higher the peroxide number's value, the easier it is for fat  to oxidize or turn rancid. The purpose of the bleaching process is to reduce the peroxide value to below ten meq O 2 /kg. The iodine number is a value that shows how many double bonds are in fat. Table 8 shows the comparison of acidity, peroxide, and iodine number with SNI 2903: 2016 of tengkawang fat as raw material. The acidity value of tengkawang fat after acid-activated bleaching treatment has a higher value than after thermal-activated bleaching. In the activation of bentonite, a hydronium ion (H þ ) fills the cation vacancy in the activation process and increases the acidity value. The higher the HCl concentration in bentonite activation, the more acidity in the tengkawang sample increased. This phenomenon is because bentonite acidity is higher from thermal bentonite, and it releases more Hydronium ions (H þ ) and increases in acidity.
The iodine value in the tengkawang sample varied based on the type of bleaching performed. However, the iodine value change still enters the  According to Duncan's Multiple Range Test (DMRT) 5%, the same column's number followed by the same letter is not significantly different. threshold of the tengkawang fat standard, so that it is not a problem in this case. The peroxide value after acid-activated bleaching treatment is smaller than thermal-activated bleaching. Acid-activated bentonite has a higher surface area than thermal-activated bentonite, causing it to absorb impurities. The peroxide value in oil must be below 10, but it will increase due to the thermal decomposition process [63]. The content of compounds with many double bonds, such as β-carotene, influences the number of peroxides. The double bond in β-carotene can undergo an oxidation reaction, which increases radical species in the system [64]. Radical species will add organic peroxide compounds, which increase the value of the peroxide and make fats quickly turn rancid. In previous studies, we have evaluated the use of bentonite to reduce β-carotene levels in tengkawang fat [35]. The β-carotene content was reduced from 125.42 μg/g to 25.51 using a 10% thermally activated bentonite.
Tengkawang butter with bleaching process using 10, 20, and 30% thermal-activated bentonite had levels of β-carotene (μg/g) respectively 25.51, 19.24 and 17.79. The β-carotene content is less with a higher content of bentonite in the bleaching process. Also, the peroxide level decreases with the reduction of β-carotene content. The peroxide value (meq O 2 /kg) in the bleaching process using 10, 20 and 30% bentonite had values of 4.95, 4.87 and 4.78, respectively. The decrease in the β-carotene content influence the reduction of peroxide value. Condition factors such as temperature, pressure, oxygen concentration, and the reaction medium influence the adsorption of β-carotene in the bleaching process [35]. Figure 5 shows the reaction of peroxide radicals with unsaturated components such as β-carotene and fatty acids. The medium's polarity influences the pathways of oxidation of β-carotene. Only radical addition reaction is formed in non-polar solvent pathways, whereas carotenoid radical cations are formed in polar ones [65,66]. Under non-polar conditions, the radical decomposition of β-carotene into non-radical products such as epoxides and cyclic ethers [65]. The products of radical decomposition can also produce aldehyde group products such as apocarotenals [67]. Under polar conditions, β-carotene changes to a peroxyl anion ion (ROO À ) and a carotenoid cation (Car þ ) [65]. Although vegetable fats such as tengkawang fat are non-polar, the negative charge on bentonite, which tends to be polar, will support the reaction process for non-polar components (fatty acids and β-carotene) to interact with bentonite [66]. Based on Silva et al. (2013) research, the β-carotene adsorption process using bentonite is a chemisorption process [68].

Conclusions
The processes of thermal-activated bleaching and acid-activated bleaching can significantly reduce the acidity and peroxide number values for tengkawang fat. The acidity (mg NaOH/g fat) can be reduced from 11.00 to 3.36 by thermal-activated bleaching and 3.61 by acidactivated bleaching. Peroxide numbers (meq O 2 /kg) can be reduced from 9.45 to 4.84 by thermal-activated bleaching and 3.47 by acidactivated bleaching. Peroxide value correlates with β-carotene content. The smaller the β-carotene content, the smaller the peroxide value. The acidity, peroxide number, and iodine number values from tengkawang fat after treatment adhere to the SNI 2903: 2016 standard. The main content of fatty acids in tengkawang fat is palmitic acid, stearic acid, and oleic acid. The fatty acid content of tengkawang is similar to that of chocolate, meaning that tengkawang fat can become a cocoa butter substitute. Specific surface area values (m 2 /g) are 82.27 for natural bentonite, 131.08 for thermal-activated bentonite, and 230.82 for acidactivated bentonite. The results obtained from this study indicate that the quality of the tengkawang process results is in accordance with Indonesia national industry standards (SNI) for food. The processing method in this research can be a reference for the tengkawang fat process for producers in Kalimantan from traditional to an industrial method.

Data availability statement
Data included in article/supp. material/referenced in article.