Enhanced antioxidant activity of Chenopodium formosanum Koidz. by lactic acid bacteria: Optimization of fermentation conditions.

In this study, different probiotics commonly used to produce fermented dairy products were inoculated independently for Chenopodium formosanum Koidz. fermentation. The strain with the highest level of antioxidant activity was selected and the fermentation process was further optimized via response surface methodology (RSM). Lactobacillus plantarum BCRC 11697 was chosen because, compared to other lactic acid bacteria, it exhibits increased free radical scavenging ability and can produce more phenolic compounds, DPPH (from 72.6% to 93.2%), and ABTS (from 64.2% to 76.9%). Using RSM, we further optimize the fermentation protocol of BCRC 11697 by adjusting the initial fermentation pH, agitation speed, and temperature to reach the highest level of antioxidant activity (73.5% of DPPH and 93.8% of ABTS). The optimal protocol (pH 5.55, 104 rpm, and 24.4°C) resulted in a significant increase in the amount of phenolic compounds as well as the DPPH and ABTS free radical scavenging ability of BCRC 11697 products. The IC50 of the DPPH and ABTS free radical scavenging ability were 0.33 and 2.35 mg/mL, respectively, and both protease and tannase activity increased after RSM. An increase in lower molecular weight (<24 kDa) protein hydrolysates was also observed. Results indicated that djulis fermented by L. plantarum can be a powerful source of natural antioxidants for preventing free radical-initiated diseases.

Introduction

Djulis (Chenopodium formosanum Koidz.) is a traditional crop from the same genus as quinoa (Chenopodiun quinoa), and it is cultivated and consumed as food or used as a wine starter in Taiwan [1]. Reports have shown that djulis exhibits beneficial effects on anti-inflammation, anti-diabetes, anti-oxidation, and immune regulation [1, 2]. Bioactive components and pigments such as peptides, betacyanin (red), betaxanthins (yellow), and polyphenols contribute to the aforementioned effects. Other ingredients, such as rutin and chlorogenic acid, can also restore the injury from UVB on HaCaT cells by reducing the level of interleukin-6 and reactive oxygen species (ROS) [3].

Lactic acid bacteria (LAB) are widely known strains of probiotics. Several studies have indicated that LAB exhibit multiple functions, such as modulating gut health, improving liver function, and decreasing cholesterol levels and blood pressure [4-6]. LAB can also enahnce the flavor of fermented products and increase the amount of antioxidative compounds in dairy products through bioconversion [7]. For example, Hsieh et al. [8] reported that heat killed cells and cytoplasmic fraction forms of Lactobacillus acidophilus BCRC 14079 grown in taro waste medium showed enhanced anti-tumor and immune-modulatory properties. Bianchi et al. [9] reported that synbiotic fermented beverages combining quinoa and soy had favorable nutritional, rheological, and sensory characteristics.

Traditionally, optimal fermentation condition are determined using a one-factor-at-a-time approach [10]. However, this method is both time-consuming and costly in terms of materials and human resources. In worst-case scenarios, the interactions among parameters are often overlooked, resulting in misleading conclusions. As an alternative, response surface methodology (RSM) is a statistical method for simultaneously validating the effects of and interactions among different parameters [8, 11]. RSM has been used in various fermentation applications such as wine making [12], bioethanol production [13], exopolysaccharides [14], and biomass production [8, 15].

The aim of the present study was to select suitable LAB strains for djulis fermentation to enhance antioxidant activity for the development of health-promoting beverages. Antioxidant activity of LAB-fermented djulis was evaluated according to 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) and 2,2-diphynyl-1-picrylhydrazyl (DPPH) free radical scavenging ability. We employed RSM to determine the optimal fermentation parameters (initial pH, agitation speed and cultivation temperature) for achieving the highest level of antioxidant activity. Possible causes for the increased bioactivity after djulis fermentation and composition analysis of djulis samples before and after fermentation were also investigated.

Materials and methods

Materials

Domestic djulis was purchased from Pingtung, Taiwan. Lactobacilli MRS Broth was provided by Hardy diagnostics (Santa Maria, CA, USA). We purchased 95% Ethanol and methanol from Echo Chemical, Co., Ltd. (Taipei, Taiwan), and 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), gallic acid, Folin–Ciocâlteu phenol reagent, and α, α-diphenyl-β-picrylhydrazyl (DPPH) from Sigma-Aldrich Co. (St. Louis, MO, USA). Agar and peptone were provided by Bioshop Inc. (Burlington, ON, Canada).

Microorganisms and medium

Bifidobacterium infantis BCRC14602, Bifidobacterium adolescentis BCRC14606, Bifidobacterium bifidum BCRC14615, Bifidobacterium longum BCRC14634, Bifidobacterium breve BCRC11846, Lactobacillus rhamnosus GG BCRC16000, Lactobacillus delbrueckii subsp. bulgaricus BCRC10696, Lactobacillus plantarum BCRC11697, Lactobacillus acidophilus BCRC14079, Streptococcus salivarius subsp. thermophiles BCRC14085 were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu city, Taiwan). All LAB strains were grown in MRS medium (Sigma-Aldrich, MI, USA). For storage, stock cultures were kept in 20% glycerol at -80°C. Viable cells were grown in MRS medium at 37°C for 20 hours as inoculum and sub-cultured twice a month [16]. The standard growth curve was measured at 600 nm using a Multiskan GO microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Djulis fermentation

Djulis was crushed into powder, filtered using a 0.6 mm mesh and then stored at -20°C until use. Djulis powder was combined with 10 times the amount of ddH2O (w/v) and sterilized at 90°C for 10 minutes. After cooling to room temperature, samples were inoculated with 1% LAB (~7 log CFU mL -1) as a seed culture and then fermented at 37°C for 48 hours. To determine the optimal fermentation time, LAB samples were taken every 12 hours and monitored for their pH, bacteria number, total phenolic content (TPC), ABTS, and DPPH. The optimal LAB was chosen based on the ABTS and DPPH assay results.

Anti-oxidant activity

DPPH assay

Fermented djulis samples were freeze-dried and diluted to 5 mg/mL (deionized water). They were then mixed with DPPH ethanol solution (100 μM) at 1:1 ratio in a 96-well tissue culture plate to carry out the reaction in the dark for 30 minutes. Finally, the samples were analyzed at the 517 nm wavelength using the microplate spectrophotometer [17]. DPPH scavenging activity of the djulis extracts was calculated as follows: where A0 = DPPH (without samples), A1 = Sample + DPPH, and A2 = Sample (without DPPH). We used this method to screen the optimal LAB for djulis fermentation. Once the optimal fermentation protocol was evaluated, the IC50 was calculated to provide an absolute number. IC50 of the djulis samples was obtained from the regression curve between concentration and DPPH scavenging activity.

ABST assay

Fermented djulis samples were freeze-dried and diluted to 5 mg/mL (80% methanol). A 2.5 mM K2O8S2 solution was prepared with K2O8S2 and 7 mM 2,2’-azinobis (3-ethylbenzothia-zoline-6-sulfonic acid) solution. The solution was placed in the dark for 12−16 hours until it became blue-green due to the formation of ABTS‧+. The ABTS‧+ solution was diluted with 0.2 M phosphate buffer solution (pH 7.4) until its OD734 reached to 0.7±0.02. Each sample (3 μL) was added to ABTS‧+ solution (300 μL) in the dark for 6 minutes [17]. After the reaction was completed, the samples were analyzed at 734 nm using the microplate spectrophotometer. The ABTS scavenging activity of the djulis was calculated as follows: where AC = ABTS (without samples); AS = Sample + ABTS.

Determination of the TPC

The TPCs was determined using Folin–Ciocâlteu’s reagent following the methods described by Wu et al. [13] but with slight modifications. Briefly, each extract (100 mg) was dissolved in a solution of 5 mL of 3% HCl in methanol/deionized water (1:1), and the resulting mixture (100 μL) was added to 100 μL of 10% aqueous sodium carbonate solution. After 2 minutes, 100 μL of 50% Folin–Ciocâlteu’s reagent was added to the mixture. After the solution had stood for 30 minutes, absorbance was measured at 750 nm against a blank. TPC was calculated based on the calibration curve of gallic acid, and this is reported as mg gallic acid equivalent per 1 g of dry djulis powder (mg GAE/1 gdw).

Enzyme activity

Protease activity was determined by taking aliquots of 100 μL of the fermented liquid and adding 100 μL of 0.1 M sodium phosphate buffer (pH 5.7). To this mixture, 100 μL of substrate was added and incubated for 30 minutes at 50°C for the two cultivars. The reaction was stopped by adding 500 μL of trichloroacetic acid at 10% (v/v) and centrifuged at 10,000 x g for 5 minutes. We then added 200μL of 1.8 M NaOH to the supernatant. Readings were taken using a spectrophotometer at 280nm. For quantification, an enzymatic unit was considered the amount of enzyme required to increase the absorbance by 0.01 [18]. To measure the tannase activity, the sample solution (100 μL) was incubated with 300 μL of 1.0% (w/v) tannic acid within a 0.2 M acetate buffer (pH 5.0) at 40°C for 30 minutes. The reaction was then terminated at 0°C by adding 2 mL bovine serum albumin (1 mg/mL), causing the remaining tannic acid to precipitate out of the solution. The samples were then centrifuged (5,000 x g, 10 min), and the precipitate was dissolved in 2 mL of sodium dodecyl sulfate (SDS)–triethanolamine (1% w/v, triethanolamine) solution. Absorbency was measured at 550 nm after addition of 1 mL of FeCl3 (0.13 M). One unit of tannase was defined as the amount of enzymerequired to hydrolyze 1μ mole of ester linkage of tannic acid in 1 minute under specific conditions [19].

Optimization for djulis fermentation

RSM using the Box-Behnken design was performed to select the optimal conditions for djulis fermentation. Three variables, namely the initial pH (5, 6, 7), agitation speed (50, 100, 150 rpm), and cultivation temperature (20, 25, 30°C), were optimized based on the results of a set of experiments. A total of 15 runs were performed to establish a model and predict the optimal conditions. Three levels of design were introduced: low, medium, and high (respectively labeled as -1, 0, and 1 in Table 1. Minitab software was used to predict the optimal values of the three variables according to the following second-order polynomial equation: where Y is the dependent variable and represents the predicted response on ABTS ability; B0 represents the fitted response at the design’s center point; B, B, and B are the coefficient for linear, quadratic, and cross-product regression, respectively; and X and X (with j = i + 1) are the coded independent variables (X1 = initial pH, X2 = agitation speed, and X3 = fermentation temperature).

Table 1

Range and corresponding levels of the independent variables.

Variable	Range values of coded levels
	−1	0	1
pH (X1)	5	6	7
Rpm (X2)	50	100	150
Temperature (X3, °C)	20	25	30

SDS gel electrophoresis

Djulis samples weighing approximately 0.1 g were mixed with 15 mL of deionized water and stirred for 30 minutes at room temperature. Next, 0.1 M NaOH was added to adjust the pH to 9.0, and the samples were stirred for another 30 minutes, centrifuged at 4,500 × g for 20 minutes, and then 0.1 N HCL was added to adjust the pH to 5.0. After removal of the supernatant, the samples were mixed with 500 μL of 63 mM Tris-HCl solution (pH 8.0). The obtained djulis protein samples were quantitated with a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) [20]. The total proteins were used for SDS gel electrophoresis analysis, and all samples were subjected to 15% SDS-polyacrylamide gel electrophoresis (SDS−PAGE) for 180 minutes at 60 V (stacking gel) and 120 V (separating gel). The separating gel was shaken and washed with deionized water three times at 70 rpm. After the deionized water was removed, RAPIDStain was added to completely submerge the gel, and it was shaken at 70 rpm for 1 hour. Finally, the gel was washed with two to three times deionized water at 70 rpm (10–15 minutes/time).

Composition analysis of fermented djulis samples

Fermented djulis samples were analyzed for their moisture, carbohydrate, protein, fat, and ash content following methods described in the literature [13] but with slight modifications. Crude protein content (g/100 g dry matter) was evaluated using the Kjeldahl method on the basis of nitrogen level and multiplied by 5.7. Crude fat content (g/100 g dry matter) was measured using Soxhlet extraction with petroleum ether. Moisture was determined by oven-drying at 105°C. Ash content was determined by placing samples overnight in a furnace at 600°C. Total carbohydrate content (g/100 g dry matter) was obtained by taking the difference between 100 and the sum of the ash content, moisture, crude fat, and crude protein.

Statistical analysis

All experiments were conducted with three independent evaluations and with three replications for each sample. Values are expressed as the mean ± SD. Minitab software (Minitab Inc., University City, Pennsylvania, USA) was used to perform one-way ANOVA and Duncan’s new multiple range tests as well as RSM evaluation and analysis. Differences were considered statistically significant differences where p < 0.05.

Results and discussions

Strain selection for djulis fermentation

To improve the functional properties, nutritional value, and taste of djulis, we fermented djulis grains using different strains of LAB. Djulis samples were inoculated with 10 strains of LAB, and were measured for their DPPH and ABTS radical scavenging activity after 24 hours of fermentation. The results showed that all 10 strains of LAB promoted the antioxidant activity of djulis. However, amongst all samples, L. plantarum BCRC 16000 and 11697 exhibited relatively higher performance for DPPH activity (93.2%) (Fig 1A). Additionally, the ABTS radical scavenging activity assay demonstrated that L. plantarum BCRC 11697 showed significantly higher ABTS radical scavenging activity (76.9%) compared to the control (64.2%) (p < 0.05). Fig 1C shows the TPC was in line with the results regarding L. plantarum BCRC 11697 fermentation and antioxidant activity. L. plantarum BCRC 11697 produced more phenolic compounds than did the other strains. Past studies have reported that a correlation coefficient of R = 0.966 between ABTS and TPC, and R = 0.939 between DPPH and TPC [21]. Turkan [22] also reported that polyphenols are antioxidants that reduce ROS and reactive nitrogen species. This could explain why L. plantarum BCRC 11697 performed the best in the DPPH and ABTS tests. L. plantarum is a common LAB strain used in fermented plant-based foods, and it is often applied to the metabolic bacteria model of phenolic compounds [23]. Moreover, it can degrade phenolic compounds in food, and produce some compounds that affect food flavor and enhance antioxidant activity [23]. For example, all strains of L. plantarum secretes TanBLp (tannase), and tannase can hydrolyze the ester bond of gallic acids and protocatechuic acids [23]. Furthermore, feruloyl esterases are involved in releasing enzymes from plant cell walls and promoting antioxidant activity [24]. ABTS and DPPH assays are widely used methods for assessing antioxidant activity in natural herbal products. Both assays are spectrophotometric techniques based on the quenching of stable colored radicals (ABTS or DPPH) and show the radical scavenging ability of antioxidants even when present in complex plant extracts [25]. Both methods are rapid, simple, inexpensive and widely used to measure the ability of compounds to act as free radical scavengers or hydrogen donors, and to evaluate the antioxidant activity of complex extracts. Several studies have also adopted ABTS and DPPH assays to evaluate the quinoa antioxidant activity [17, 26, 27].

Fig 1

To determine the optimal djulis fermentation time for L. plantarum BCRC 11697, we carried out 54-hour cultivation with samples collected every 6 hours. As shown in Fig 1(A), all LAB strains exhibited similar DPPH scavenging effect, while BCRC11697 showed significantly higher ABTS amongst all the strains (Fig 1(B)); as such, we chose BCRC 11697 for the remainder of the study and used ABTS as the indicator.

Fig 2 shows the results of the ABTS radical scavenging activity, CFU, and pH values during fermentation. ABTS activity in the fermented djulis samples peaked after 24 hours of fermentation (77%) and then decreased gradually after 48 hours. Therefore, 24 hours was selected as the fermentation time for the remainder of the study. Moreover, lactic acidification improved the extraction of total phenols when the selected strain was used, and this has also been reported in previous research [26]. Esterase activity released the complex glycosylated-phenolic compounds into the corresponding phenolic acids during fermentation.

Fig 2

RSM optimization

A set of individual experiments was conducted for each variable (X1: initial pH, X2: agitation speed, and X3: temperature) in order to elucidate the specific effect of each parameter on ABTS antioxidation ability. We investigated initial pH values ranging from 2 to 10 for solutions of djulis powder mixed with deionized water for cultivation. An initial pH of 6 yielded the highest ABTS antioxidant activity (IC50: 2.59 mg/mL) among the fermented samples (S1A Fig). Initial pH values of 5, 6, and 7 were chosen as experimental values. Different agitation speeds were also evaluated for ABTS antioxidant ability. Results showed that samples obtained at 100 rpm of agitation had the best ABTS (IC50: 2.39 mg/mL). Therefore, agitation speeds of 50, 100 and 150 rpm were chosen as experimental values (S1B Fig). For the temperature parameter, we evaluated temperatures ranging from 15°C to 40°C. Results showed the best fermentation temperature to be 25°C, which gave the lowest ABTS IC50: 2.44 mg/mL). Therefore, fermentation temperatures of 20°C, 25°C, and 30°C were chosen as experimental values (S1C Fig). Based on the results from the one-factor-at-a-time approach, RSM was applied to determine and to optimize the three fermentation parameters in order to achieve the highest ABTS anti-oxidation ability in the fermented djulis product (Table 1). The results are summarized in Table 2. Multiple regression was applied to the experimentally determined data in Eq (1) to estimate the regression coefficients, and the following second-order polynomial equation was obtained using Minitab software: where Y = ABTS free radical scavenging ability-IC50; theory value: X1 = initial pH; X2 = agitation speed (rpm); X3 = fermentative temperature (°C).

Table 2

Experimental range and values in the central composite design for optimizing the fermentation conditions.

StdOrder	RunOrder	PtType	Blocks	Initial pH	RPM	Tm. (°C)	ABTS-IC50(mg/mL)
12	1	2	1	6	150	30	3.50
8	2	2	1	7	100	30	3.65
5	3	2	1	5	100	20	3.26
9	4	2	1	6	50	20	3.50
7	5	2	1	5	100	30	3.57
4	6	2	1	7	150	25	3.20
2	7	2	1	7	50	25	3.41
11	8	2	1	6	50	30	4.04
14	9	0	1	6	100	25	2.97
3	10	2	1	5	150	25	3.24
1	11	2	1	5	50	25	3.28
10	12	2	1	6	150	20	3.66
6	13	2	1	7	100	20	3.65
15	14	0	1	6	100	25	3.14
13	15	0	1	6	100	25	2.83

The predicted optimal parameters of X, X, and X were obtained by applying the regression analysis of Eq (2); these were pH 5.55, 104 rpm, and 24.4°C. The predicted value of ABTS-IC50 was 2.42 mg/mL which approximates our experimental result (2.35 mg/mL).

The coefficient of determination of the regression for the response related to the significant effects in the model was R2 = 0.946 (Table 3). Hence, the sample variation of 94.6% for ABTS-IC50 was associated with the three independent variables. The interaction between temperature and agitation speed can be observed from the results (p < 0.05). We hypothesize that is because the heating process can be accelerated by the increased agitation speed, resulting in a favored environment for LAB growth and TPC production. Similar results were reported by Dinarvand et al. [28]. They reported that the interaction between temperature and agitation speed affected the production of invertase from Aspergillus niger. The surface plots for ABTS-IC50 are shown in Fig 3. The initial ABTS-IC50 increased with the initial pH, reaching an optimal ABTS-IC50 value approximately 5.55, which declined gradually above the optimal pH due to inactivation of the tannase, which accords with previous reports [23, 25]. The adequacy of the full quadratic model of liquefaction was also evaluated via ANOVA. The model summary statistics in Table 3 indicate the adequacy of the models including linear, 2-factor interactions and quadratic terms (P < 0.05). The lack-of-fit error was nonsignificant (p = 0.842), verifying the accuracy fit of the second-order model (Eq 2) to the true response of ABTS-IC50.

Table 3

Estimated regression coefficients for ABTS free radical scavenging ability-IC50.

Sources	DF	Sum of squares	Mean squares	F-value	P-value
Model	9	1.24941	0.138824	9.74	0.011	Significant
pH	1	0.0386	0.038597	2.71	0.161
RPM	1	0.04905	0.049049	3.44	0.123
Tm.	1	0.06107	0.061074	4.29	0.093
pH2	1	0.00356	0.023883	1.68	0.252
RPM2	1	0.13241	0.186733	13.1	0.015	Significant
Tm.2	1	0.812	0.812001	56.98	0.001	Significant
pH*RPM	1	0.00728	0.007285	0.51	0.507
pH*Tm.	1	0.02307	0.023075	1.62	0.259
RPM*Tm.	1	0.12236	0.12236	8.59	0.033	Significant
Residual error	5	0.07126	0.014251
Lack of fit	3	0.02082	0.006939	0.28	0.842
Pure error	2	0.05044	0.025219
					R2	94.60%
					Adjusted R2	84.89%

DF refers to degrees of freedom, which differs significantly (p < 0.05). The optimal starting reaction conditions for anti-oxidation were pH 5.55, 104 rpm and 24.4°C.

Fig 3

For the following assay, we chose pH 5.55, 104 rpm and 24.4°C as our fermentation conditions. Between the unfermented and fermented djulis samples, the fermented sample exhibited markedly higher antioxidant ability. For example, the IC50 of ABTS was 3.4 mg/mL before fermentation, and this decreased to 2.35 mg/ml after optimization. In addition, the IC50 of DPPH was 1.11 mg/mL before fermentation, decreasing to 0.33 mg/mL (p < 0.05) after RSM (Table 4). Compared to the TPC in the unfermented djulis samples, that in the fermented samples exhibited a significant increased from 9.33 to 28.97 (mg of gallic acid/gdw) (p < 0.05). (Table 4).

Table 4

Changes in TPC, cell number, pH, and DPPH and ABTS radical scavenging activity in djulis fermented with L. plantarum BCRC 11697.

Group	TCP(mg of gallic acid/gdw)	ABTS-IC50(mg/ml)	DPPH-IC50(mg/ml)	log CFU/ml	pH	Protease activity(U/mg-protein)	Tannase activity(U/mg-protein)
Unfermented	9.33 ± 0.25 a	3.40 ± 0.32a	1.11 ± 0.20a	7.31 ± 0.04 a	5.55 ± 0.02 a	ND	ND
Fermented	27.68 ± 0.21 b	2.44 ± 0.26b	1.01 ± 0.17a	7.42 ± 0.08 a	4.21 ± 0.03 b	1.69 ± 0.17 a	0.88 ±0.11 a
Fermented-RSM	28.97 ± 0.19 b	2.35 ± 0.46b	0.33 ± 0.02b	8.61 ± 0.32 b	4.09 ± 0.01 b	2.53 ± 0.21 b	1.12 ± 0.09 b

Statistical differences were calculated using Duncan’s new multiple range test. Values are presented as the mean ± SD of three independent experiments with the different superscripts (a, b) indicating significantly differences (p < 0.05). ND: not detected.

The increase of TPC is due to the presence of protease (2.53 U/mg-protein), tannase (1.12 U/mg-protein), and other enzymes used in enzymatic hydrolysis. Solid-state fermentation has also been adopted for cereal grains fermentation using fungi in previous research [29] that reported significantly greater antioxidant properties in the fermented products than unfermented grains. In the case of quinoa fermentation, that study reported an increase of 2.46 mg/g in TPC content after 35 days of fermentation. For our case, the TPC content of fermented djulis increased by 19.64 mg/g after 24 hours, which provided a fast and economically feasible method for the up-scaled production of antioxidant-rich ingredients.

Changes of djulis components through fermentation

Principal components analysis showed that the freeze-dried powder of the fermented djulis contained 68.80% carbohydrates, 17.01% crude protein, 4.12% crude fat, 5.71% ash, and 4.37% moisture (Table 5). A slight decrease in carbohydrates, protein, and fat was observed due to the presence of enzymes partaking in hydrolysis and oxidation, which was discussed in a previous study [29]. LAB utilized the carbohydrates, protein and fat of the djulis since it was the only nutrient within the medium. Some nutrients could be hydrolyzed into small molecules such as peptides, oligosaccharides, and short-chain fatty acids due to the presence of related enzymes.

Table 5

Carbohydrates, protein, fat, ash and moisture of Chenopodium formosanum Koidz. fermented product.

	Carbohydrates(g/100 g)	Protein(g/100 g)	Fat(g/100 g)	Ash(g/100 g)	Moisture(g/100 g)
Quinoa	74	16.3	7	2.7	0
Djulis	70.62 ± 0.15	19.15 ± 0.32	4.34 ± 0.17	2.62 ± 0.08	3.27 ± 0.21
Dry powder of fermented djulis	68.80 ± 0.16	17.01 ± 0.29	4.12 ± 0.54	5.71 ± 0.08	4.37 ± 0.21

Values are presented as mean ± SD of three independent experiments.

Protein hydrolyzation

Fermented grain products are consumed in many countries and are one of the most crucial sources of bioactive peptides [29]. Grains fermented using bacteria (LAB and Bacillus spp.) yield many different types of fermented products that possess a multitudinous array therapeutic properties, such as antioxidant, antihypertensive, antimicrobial, antidiabetic, and anticancer activity [29].

A previous study identified five peptides with antioxidant activity (ABTS and DPPH) after LAB fermentation through the hydrolysis of quinoa protein [26]. The size of each peptide was approximately 5–9 amino acid residues. Another study demonstrated that the bands between 53 and 41 kDa, two bands around 32 to 24 kDa, and bands under 24 kDa potentially have antioxidant activity as well [30]. The present study employed SDS-PAGE to determine the protein distribution of unfermented and fermented djulis. Our results showed that proteins of djulis had been hydrolyzed after fermentation. For example, two bands were between 24 and 32 kDa, one major band under 24 kDa disappeared after fermentation, and more bands with lower molecular weight were observed after fermentation (Fig 4). These results aligned with the behavior of wheat after L. plantarum M616 fermentation [30]. Virtanen et al. [30] also reported that milk whey protein hydrolysate weighing 4–20 kDa showed remarkably high antioxidant activity compared to that of the original milk whey protein.

Fig 4

Conclusions

We applied RSM to determine the optimal fermentation conditions and to evaluate the interaction among the initial pH, agitation speed, and temperature. Our results show that L. plantarum BCRC 11697 is the optimal LAB strain for djulis submerged fermentation among the 10 candidates of LAB strains were investigated. After RSM-assisted optimization, we observed significant improvement in the free radical scavenging activity of DPPH and ABTS and in TPC. The presence of protease and tannase activity also supports that L. plantarum BCRC 11697 enhances free radical scavenging bioactivity through protein hydrolysis and the release of bound-phenolic compounds. In conclusion, fermented djulis using LAB shows potential for commercialization as a beverage. Future studies will need to investigate the up-scaled production of djulis content, determination of the specific mechanisms of antioxidation and identification of bioactive peptides for the findings of this study to be employed in commercial applications.

(A) Initial pH-The ABTS radical scavenging activity of Chenopodium formosanum Koidz. fermented with Lactobacillus plantarum BCRC 11697. (B) RPM-The ABTS radical scavenging activity of Chenopodium formosanum Koidz. fermented with Lactobacillus plantarum BCRC 11697. (C) Fermentation temperature-The ABTS radical scavenging activity of Chenopodium formosanum Koidz. fermented with Lactobacillus plantarum BCRC 11697. Statistical differences were calculated by Duncan’s new multiple range test. Values are presented as mean ± SD of three independent experiments with different superscripts (a, b, c, d) are significantly different (p < 0.05).

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8 Dec 2020

PONE-D-20-34408

Enhanced Antioxidant Activity of Chenopodium formosanum K oidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions

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"This project was funded by the Ministry of Science and Technology, Taiwan (MOST

109-2628-E-002-007-MY3 and MOST 107-2320-B-255-001-MY3). The funding

grant of BMRPD42 for this study was also provided in part by research grants from

the Chang Gung Memorial Hospital, Chiayi, Taiwan."

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"This project was funded by the Ministry of Science and Technology, Taiwan (MOST 109-2628-E-002-007-MY3 and MOST 107-2320-B-255-001-MY3). HC Kuo received grant of MOST 107-2320-B-255-001-MY3. KC Cheng received grant of MOST 109-2628-E-002-007-MY3. URL: https://www.most.gov.tw/?l=en . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Overall, the manuscript provides a good information of the subject matter. The experimental designs and analyses are standard and appropriate for the study. However, there are some concerns that need to be addressed by the authors.

1. The authors may wish to send the manuscript for a professional proof-read. There are some grammatical errors and inconsistency of sentences. Many of the scientific names of organisms throughout the manuscript are not in italic.

2. Introduction:

i) The sentence 'Lactic acid bacteria (LAB) are responsible for a group of famous strains of probiotics' (page 4, line 62-63) is somewhat misleading.

ii) The motivation/cause for this study is not clearly explained. The authors may wish to further explain the reason why they wanted to enhance the antioxidant activity and also why they selected LAB in the fermentation process. And perhaps explain why they selected ABTS and DPPH assays as the anti-oxidant assays for this study.

3. Materials and methods:

i) Page 7, line 143 - ABTS full name is repetitive.

ii) Page 9, line 193 - stated here that the authors used SAS software. But on page 13, line 300 - the authors stated that they used Minitab. Which statistical software did they actually use? Why did the authors used two different statistical software?

4. Results and discussion: In my opinion, this section is not sufficiently elaborated.

i) Page 12, line 262 - secrets? or secretes?

ii) Page 12, line 267-268 - at this stage, why did the authors left out the DPPH-radical scavenging evaluation?

iii) Page 12, line 271-274 - this statement is redundant. Similar meaning has been stated in line 259-263. The authors should provide another explanation along with appropriate references.

iv) Page 13, line 284 - I wonder why at this stage the authors expressed the results of ABTS scavenging activity in Ic50 but in % at the earlier stage of the study?

v) Page 15, line 336-337 - the sentence 'in the case of quinoa fermentation.....' - please provide reference for this statement.

vi) Page 16, line 367-374 - I think that comparison to yogurt fermentation is inappropriate. In my opinion, it is better if the authors compare their results with other studies on cereal or grains fermentation.

5. Conclusions : It is stated in line 384 that the RSM was applied to evaluate the interaction among the three factors. But I could not find the discussion on the interaction of factors anywhere in the manuscript. I can see in Table 3 that the interaction between temperature and agitation speed is significant. The authors should elaborate in the Results and discussion section.

6. Table 4: Are the superscript letters indicating significance of the data are stated correctly? Please check.

Reviewer #2: Reviewer

The research article entitled “Enhanced Antioxidant Activity of Chenopodium formosanum Koidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions” submitted in your esteemed journal is a good work where the authors have tried to improve the antioxidant activity by lactic acid bacterial and also optimized the fermentation conditions.

Minor Comments: -

What is the method of preparation of bacterial cell suspensions used for fermentation? Also describe about the starter culture for fermentation in material and method section.

In DPPH section what is 1 in the calculation of scavenging activity (%)?

Author cite only one reference for both DPPH and ABTS assay but author used two different calculation method for both DPPH as well as ABTS. Be specific about the citations as well as the calculation.

In section 2.5 author mentioned Wu et al (2020). What is the number of this citation in final list of references???

In section 2.7 line space is different as compared to the other text. Kindly follow the uniformity in whole MS.

The figures are not clearly visible.

At line no 310 author highlights the bracket with red colour. Correct it accordingly.

From section 3.3 it showed that fermentation decrease the carbohydrate, protein and fat content. Kindly explain? What is the reason behind this?

In table 4 correct the word TPC instead of TCP.

Correct the MS with a uniform pattern according to the journal guidelines and also formatting it accordingly.

“After reviewing the manuscript, in my opinion that once the corrections will amended by the author than it will be fit and accepted for publication in your esteemed journal”.

Reviewer #3: The paper describes the utilization of RSM in the optimization of fermentation parameters in order to maximize antioxidant activity. The work is scientifically interesting and sound.

It is good that the authors point out that the increase in antioxidant capacity may be partly related to the enhanced extractability of phenolic compounds. It needs to be remembered that human gut is able to effiicently "extract" and utilize also phenolic compounds that - due to non-extractability in sample preparation - may not be observed when total phenolics/antixoidant capacity is measured.

English needs to be slightly improved.

Some clarification is needed in the following:

1) What is the difference between "Fermented" and "Fermented-RSM" in Table 4?

2) What is "quinoa" mentioned in Table 5? Reference to literature source may be needed. Does "Djulis" denote the nonfermented samples?

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: DR. Pardeep Kumar

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Jan 2021

Please see the attached file as our response.

Submitted filename: 20210116 Response R1.docx

Click here for additional data file.

27 Jan 2021

PONE-D-20-34408R1

Enhanced Antioxidant Activity of Chenopodium formosanum K oidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions

PLOS ONE

Dear Dr. Cheng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The general impression is that the authors have put insufficient effort in revising the manuscript. The provided answers to the reviewers' comments are not properly addresses:

R: "And perhaps explain why they selected ABTS and DPPH assays as the anti-oxidant assays for this study."

A: "The reason why we choose ABTS and DPPH assays is because djulis is rich of phenolic compounds."

There are numerous antioxidant assays, which differ in their ability to scavenge particular radicals. The authors have to better explain why they have chosen the stated two. It should be discussed in the manuscript too.

R: "Results and discussion: In my opinion, this section is not sufficiently elaborated."

The authors provided no answer. It is a general remark; however, the Discussion section should be considerably supplemented in several parts where the reviewers' have made their notes.

R: "Conclusions : It is stated in line 384 that the RSM was applied to evaluate the interaction among the three factors. But I could not find the discussion on the interaction of factors anywhere in the manuscript. I can see in Table 3 that the interaction between temperature and agitation speed is significant. The authors should elaborate in the Results and discussion section."

A: "Page 14, lines 323-324: The interaction between temperature and agitation speed can be observed from the results (p < 0.05)."

I cannot accept the provided statement as the elaboration on the concern raised by the reviewer.

A: "We thank the reviewer for the comment, the data presented in % was in comparison to the control based on the equation: DPPH scavenging activity (%) = [1—(A1—A2) / A0] × 100, A0 = DPPH (without samples), A1 = Sample + DPPH, and A2 = Sample (without DPPH). We used this method to screen the optimal LAB for djulis fermentation. When the optimal fermentation protocol was evaluated, IC50 was expressed to provide absolute number to the audience."

This reply should stand in the main text. If two out of three reviewers raised the concern on different presentation of scavenging activities, it is expected readers will also do.

The language must be further polished. Some examples:

L46-47: "...can significantly increase the phenolic compounds," - phenolic compounds cannot be increased. Its amount can instead.

L137 and elsewhere:  "scavenging activity of the djulis" - No article is used when a non-count noun is generic or nonspecific.

L244: "T test" it is rather "t- test"

L363: no contractions such as "it's" are allowed.

Table titles and figure captions: please do not write djulis capitalized.

Figures 3 and 4 should be submitted in the .tiff format. Figure S1 is not a raw image since has been edited.

Significance letters in three histograms representing Figure 1 might be questionable. Please provide statistical working sheet with the raw data as Supporting information.

==============================

Please submit your revised manuscript by Mar 13 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

8 Mar 2021

Please see the attached file as our response

Submitted filename: 20210215 Response R2.docx

Click here for additional data file.

15 Mar 2021

Enhanced Antioxidant Activity of Chenopodium formosanum K oidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions

PONE-D-20-34408R2

Dear Dr. Cheng,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

28 Apr 2021

PONE-D-20-34408R2

Enhanced Antioxidant Activity of Chenopodium formosanum Koidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions

Dear Dr. Cheng:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Branislav T. Šiler

Academic Editor

PLOS ONE