Different drying temperatures modulate chemical and antioxidant properties of mandai cempedak ( Artocarpus integer)

Background: Mandai, the fermented inner skin of cempedak (Artocarpus integer), may have further use as an industrial ingredient while maintaining its antioxidative capacity. The starter culture of Lactobacillus casei may induce the Mandai fermentation. This research was carried out (i) to investigate the effect of temperature on yield, chemical properties, and antioxidant activity of starter induced fermented mandai powder, (ii) to find the best drying temperature for the powder, and (iii) to find correlations between phenolic contents and antioxidant activity of the powder. Methods: The drying temperature was used as the variable, and was set at 45, 50, and 55°C at a fixed duration of 18 hours. The control was spontaneously fermented mandai dried at 50°C for 18 hours. Total phenolic content (TPC), hydrolyzed tannic content (HTC), and total flavonoid content (TFC) were spectrophotometrically measured, expressed gallic acid (GAE), tannic acid (TAE), and catechin (CAE) equivalents. The DPPH assay measured antioxidant capacity. Results: The best mandai powder had total phenolic content of 348.8±55.6 mg GAE kg -1, HTC of 143.8±9.3 mg TAE kg -1, TFC of 17.5±1.3 mg CAE kg -1, antioxidant activity (IC 50) of 56.96 ppm, ash content of 4.0±0.7%, pH value of 5.0±0.8, and yield of 9.3±0.8%. There was a strong correlation between TPC, HTC, TFC, and antioxidant activity. Conclusions: Drying temperature affected all observed parameters but not yield, ash and pH. The temperature of 45°C emerged as the best treatment to produce mandai powder from L. casei-inoculated mandai cempedak fermentation. The phenolic components contributed to the antioxidant activity of mandai cempedak.


Amendments from Version 1 Introduction
In South and East Kalimantan, Indonesia, people historically consume the lactic acid bacteria (LAB)-fermented inner skin of cempedak (Artocarpus integer), traditionally termed dami or mandai 1 . The use of the inner skin of cempedak in functional food production may partly reduce agricultural waste 2 . Mandai may contain phenolics, flavonoids, tannins, and antioxidant activity.
The unfermented cempedak inner skin (Artocarpus integer) contains bioactive components such as phenolics, flavonoids, and carotenoids 3 . It has antioxidant activity that is potentially higher than the flesh and seeds of cempedak. Mandai with L. casei as the starter had better antimicrobial activity against S. aureus and E. coli in comparison to spontaneous (L. plantarum) mandai 4 . Besides, mandai may function as a probiotic food 5 . While maintaining its antioxidative capacity, fermented mandai may have further use as an industrial ingredient primarily as an exotic tropical flavor and a flavor enhancer.
Mandai powder is produced through a drying process. The right drying temperature is required to produce good quality mandai powder. Drying at the correct temperature minimizes antioxidant damage, implicating the ability to reduce free radicals will be higher, as seen in sinom beverage powder 6 , and cumari peppers 7 .
This research aims (i) to measure chemical and antioxidant properties of L. casei-fermented mandai at 45, 50, 55°C of drying temperature with constant time of drying at 18 hours, which the results are then compared with spontaneously fermented mandai and dried at 50°C for 18 hours; (ii) to find the best drying temperature on starter induced fermented mandai powder; and (iii) to find correlations between phenolic contents and antioxidant activity on starter induced fermented mandai powder.

Methods
Producing mandai powder from the inner skin of cempedak Cempedak was peeled and separated from its husk and flesh, then washed and cut into pieces. The pieces of cempedak inner skin were boiled at 100°C for 5 minutes to remove the sap. The sample was drained and then boiled once more in a sealed container at 100°C for 5 minutes to soften the texture. The sample was then cooled until the temperature was less than 40°C. L. casei strain Shirota isolated from Yakult® as starter culture was inoculated at the concentration of 2% (v/v). For spontaneous fermentation, the mandai was directly stored without inoculation. The spontaneous and inoculated mandai were stored for two weeks at a temperature of 8±2°C to allow slow fermentation to occur. After incubation, mandai was drained and blended. The puree of mandai then was dried for 18 hours at the 45, 50, and 55°C, then ground and screened with an 80-mesh sieve. The 18 hours drying time opted from the result of the preliminary research. All reagents and corresponding suppliers are listed in Supplementary File.

Extraction of mandai powder
For the analysis of TPC, HTC, TFC, and antioxidant activity, 20 g mandai powder was dissolved in 60 ml 95% ethanol (SmartLab cat no. A1035, Indonesia) and macerated for 24 hours. Mandai was filtered through filter paper (Whatman no. 4), and the liquid extract was dried at 50°C for 16 hours. The duration of liquid drying opted from the preliminary optimation research.

Yield, ash, and pH
The yield was measured as the ratio of mandai powder to initial mandai cempedak (w/w), while ash contents were measured as described 8 . About 2 g of the sample was diluted with distilled water to a volume of 20 ml. This mixture was homogenized and allowed to soak for 15 minutes before the pH was measured.
Phenolic content and antioxidant activity measurement TPC was estimated by the Folin-Ciocalteu assay and expressed as gallic acid equivalents (GAE), as described previously 9,10 . HTC was estimated by the Folin-Ciocalteu assay and expressed in mg kg -1 tannic acid equivalent (TAE), as described 11 . TFC was estimated using the aluminum chloride (AlCl 3 ) method and expressed in mg kg -1 catechin equivalent (CAE), as described 12 . The antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, as described 13 . All standards were purchased from Sigma-Aldrich. Table 1 except IC 50 of antioxidant activity were subjected to analysis of variance (ANOVA), and Fisher's least significant difference test determined the significance of the difference between the averages (at α = 5%, analyzed with GraphPad Prism version 6.0. Values were expressed in average ± standard deviation (SD). IC 50 of antioxidant activity was produced by employing the non-linear fit of the one-phase association method with GraphPad Prism version 6.0. Pearson correlation analysis was performed with Microsoft Excel 2016.

Yield, ash, and pH
The yields of mandai powder at drying temperatures of 45, 50, and 55°C did not differ significantly ( Table 1). The yield of each Yield, ash, pH, total phenolic content (TPC), hydrolyzed tannin content (HTC), total flavonoid content (TFC) are presented in average ± standard deviation.
Letters after the numbers indicate the least significant difference at α=5% treatment did not differ significantly with the yield of mandai powder with spontaneous fermented that dried at 50°C (control). A comparison between the ash content of each treatment with control showed no significant difference ( Table 1).
The pH values of dissolved mandai powder ranged from 4.5±0.5 to 5.3±0.2. The drying temperature did not affect the pH value of the dissolved mandai powder.

TPC, HTC, and TFC
The drying temperature significantly affected the TPC of mandai powder. The highest average value of TPC in mandai powder dried at 45°C. In comparison to control, the TPC of each treatment was significantly different. The drying temperatures in each treatment resulted in significantly different HTC in comparison to that of control mandai. The drying temperature of 45°C produced higher HTC than that of mandai powder dried at 50°C and 55°C. The drying temperature significantly affected the TFC of mandai powder. The TFC of mandai powder, which dried at 45 and 55°C were significantly different from the control, but that of dried at 50°C was not significantly different from control.

Antioxidant activity
Half-maximal inhibitory concentration (IC 50 ) value of DPPH for each treatment was obtained through one-phase association equation. In the concept of IC 50 value, the lower the IC 50 value means the substance is more potent or showing a better antioxidant capacity. The drying temperature significantly affected the antioxidant activity of mandai powder. Control mandai powder extract had the highest IC 50 value, while mandai powder extract dried at 45°C had the lowest IC 50 value of ( Table 2). The TPC, HTC, and TFC have a strong correlation with antioxidant activity. The higher TPC, HTC, and TFC value, the higher the antioxidant activity in mandai powder (Table 3). Lowering the drying temperature may additionally preserve the content of polyphenols, therefore, may further increase the antioxidant capacity.
Correlation of TPC, HTC, TFC, and antioxidant activity Based on Pearson correlation analysis, there was a strong correlation between total phenolic content (r = 0.796) and total flavonoid content (r = 0.783) with antioxidant activity. The  strongest correlation occurred between total tannin content (r = 0.910) and antioxidant activity in mandai powder.

Discussion
Yield, ash, and pH More water in mandai evaporated at higher temperatures. The evaporated water caused the yield of mandai powder to reduce with increasing drying temperature; however, the water content of the final product is also lesser. A previous report documents that the yield may decrease with increased drying temperatures 14 .
The drying temperature did not affect the ash content of mandai powder. Microbes equally used the mineral resources in the spontaneous and L. casei-fermented mandai, so it did not have a different effect on ash content.
During mandai fermentation, the population of LAB increased until day 14, thus lowering pH 1,5 . Inferring from previous research, organic acids such as lactic acid and acetic acid were produced to lower the pH and caused a more acidic environment on day 12 of fermentation 5 . The fermentation medium, duration of fermentation, and the use of starter cultures may play a role in the final pH and organic acid contents of LAB-fermented products. The previous research stated that the pH of spontaneously fermented rye dough was higher than the pH of starter-fermented rye dough 15 . However, cucumber pickle fermented with LAB produced organic acids that were higher than that of spontaneous fermentation 16 .

TPC, HTC, and TFC
TPC of the inner skin of cempedak was at 21.29 mg GAE kg -13 . TPC of mandai powder ranged from 199.2±13.4 to 348.8±55.6 mg GAE kg -1 , higher than that of unfermented cempedak. Phenolic compounds were sensitive to heat treatment so that the drying process reduced the TPC 17 . The TPC of mandai powder dried at 55°C was the lowest value observed when compared to other treatments. The drying process, especially at higher temperatures (i.e., 55°C), combined with the long drying time duration (i.e., 18 hours), resulted in loss of antioxidant activity 18 .
In dry conditions, components in the cell, such as membranes and organelles, clump together, resulting in fewer extracted phenolic compounds 14 . Drying at 50°C quickly disabled the oxidation of polyphenols. However, the initial oxidation of polyphenols might have occurred before drying and led to polyphenol degradation. Phenolic compounds are sensitive, unstable, and susceptible to degradation by oxygen and light 19 . The enzymatic oxidation of polyphenols components is mostly caused by polyphenol oxidase 7 . Injury to the cell membrane liberates and therefore activates these enzymes, which convert phenolic compounds to quinones.
Phenolic contents may vary on the types of heat processing, such as drying, boiling, and steaming 19 . Two consecutive boilings at 100°C for 5 minutes assisted removal of sap in the inner skin of cempedak and texture softening. This two-step boilings can be replaced by one-step boiling at a longer time. However, the use of two consecutive boilings is necessary to reduce the length of heat exposure; therefore, this was included as an essential preparation step in our patent application on mandai fermentation.
Environmental factors affecting phenolic concentrations include weather conditions, seasons, and post-harvest conditions 20 . Phenolic contents are also related to varieties of different fruits, diversity of extraction methods 21 , and the type of phenolic components in the plant and its location in the cell, as well as the type of solvent and method of extraction 22-24 .
L. casei may modify the phenolic component, causing a significant difference between TPC of each treatment and control 25,26 . The fermentation and metabolic activity of LAB may play a role on levels of total phenolic in rye dough and bread 15 and Moringa oleifera leaf powder 27 . HTC was inversely correlated with drying temperature. Also, the duration of drying contributes to the loss of tannins 28,29 , consistent with the results in the drying of yacon (Polymnia sonchifolia) and coffee leaf tea 30,31 . Degradation of flavonoid structures is linked to the degree of heat exposure 30,32 .

Correlation of TPC, HTC, TFC, and antioxidant activity
The antioxidant activity of mandai powder was low when compared to the fresh form. Temperature plays a role in retaining the antioxidant activity of the powder ( Table 2). The temperature had a significant effect on the inhibition of free radicals of DPPH in grass jelly (Premna serratifolia) 33 . A strong correlation was observed between antioxidant activity and the polyphenol contents of mandai powder. Previous studies have reported strong correlations between TPC, HTC, TFC, and antioxidant activity 34,35 . The phenolic chemical structure has a role in the inhibition of free radicals, largely depending on the number and position of the hydrogen donation from the hydroxyl group to the aromatic ring of the phenol molecules 36 .

Conclusions
Drying temperature affected total phenolic, tannin and flavonoid contents, and antioxidant activity, but did not affect ash content, yield, and pH of starter induced fermented mandai powder.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results?

Yes 2
Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com