Determining Fermentation Conditions to Enhance Antioxidant Properties and Nutritional Value of Basil Seeds Using Lactobacillus plantarum

Abstract

Fermented seeds and their bioactive compounds have captivated global interest due to their functional properties. Basil seeds are used worldwide in the food, cosmetic, and pharmaceutical industries, but their functional and nutritional properties after fermentation are not known. The aim of this study was to optimize the effect of fermentation on the improvement in the functional properties of basil seeds by Lactobacillus plantarum. Basil seed samples were categorized into seven water volumes (named A–G) and fermented for 24, 48, 72, and 96 h with L. Plantarum. The results show that the pH and total microbial content (TMC) significantly changed after 24 h of fermentation (p < 0.05). Fermentation significantly increased the antioxidant properties and niacin content of basil seeds compared with non-fermented control samples (p < 0.05). Fermented samples G-24, G-48, and G-72 (samples with a ratio of 1 g (basil):10 mL (water) fermented for 24,48 and 72 h), exhibited the highest DPPH and ABTS^• scavenging activity. The total polyphenol content (TPC) was most prominent in the samples G-72, G-24, and G-48, respectively. Sample G-48 showed the highest antioxidant activity. Notably, sample G-24 showed a significant increase in niacin content (64 µg/mL). These results underscore that varying moisture levels and fermentation durations have a significant impact on the nutritional/functional value of basil seeds. Overall, fermenting basil seeds with L. plantarum increased their functional properties with greater antioxidant and TPC activities as well as increased nutritional value.

1. Introduction

Over time, the interest in the relationship between food and health has increased, and the purpose of food has evolved to enhance human health through added functionality known as functional food [1]. While there is no universally accepted definition, functional food is generally understood to be food supplemented with additional ingredients such as vitamins, proteins, fibers, probiotic bacteria, or other food additives [2]. A recent strategy to achieve functional food involves using fermentation with probiotic bacteria as “food additives” [1,2].

Fermentation is a traditional method that imparts a unique aroma, flavor, and texture to food, enhances digestibility and water-soluble vitamin contents, degrades anti-nutritional factors, converts phytochemicals such as polyphenols into more bioactive and bioavailable forms, produces antioxidant components that are able to decrease/eliminate reactive oxygen species (ROS) and oxidative stress, and enriches the nutritional quality of food [3,4]. ROS and oxidative stress are key chemical processes that can increase the production of oxygen radicals [4,5]. These radicals can cause damage to proteins and DNA and activate intracellular matrix metalloproteinases (MMPs) [6]. Water-soluble vitamins such as niacin/nicotinamide (B3) enhance moisture content, fortify the skin barrier function, and prevent skin damage and scarring [7,8,9]. Lactic acid bacteria (LAB), particularly probiotics, can contribute to the proliferation of nutritional quality (e.g., increasing vitamin levels) and show a positive effect on addressing oxygen radicals during the fermentation process [10,11,12]. This is why interest in using fermented foods has surged in recent decades [13,14,15,16]. A biologically safe method that is conducted on a solid substrate without free moisture but with sufficient water content is one fermentation method [17,18,19]. The environmental conditions, such as the water content and fermentation time, are crucial in determining the final properties of fermented products [15,20,21].

One of the medicinal plants rich in high dietary fiber and protein is basil seeds (Ocimum basilicum L.), which belong to the Lamiaceae family and are increasingly common in Asian and Central American countries [22]. They are recognized for their antioxidant, antimicrobial, anti-inflammatory, and antidepressant properties and their role in preventing type 2 diabetes [22,23]. They are used in different ways, e.g., in food, pharmaceuticals, and cosmetics [24]. The healthy properties of basil can be enhanced through fermentation [22,25]. The influence of optimizing fermentation variables, including moisture and time, on basil seeds’ antioxidant properties, phenolic compounds, and nutritional value, such as niacin content, has not yet been reported.

Therefore, this research aimed to optimize the environmental conditions of basil seed fermentation, including varying the amounts of inoculum water and fermentation times. The hypothesis was that fermenting and optimizing basil seed fermentation can result in a functional diet enriched with antioxidant components and enhanced nutritional value, thereby promoting health.

2. Materials and Methods

2.1. Chemical and Reagents

Basil seeds (O. basilicum) were purchased from Spicy World Company (Stafford, TX, USA). 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS^•) were obtained from Sigma-Aldrich (Sigma–Aldrich, St. Louis, MO, USA) and Fischer Scientific for evaluating antioxidative characteristics. Folin and Ciocalteu’s phenol reagent, sodium carbonate, and gallic acid for measuring TPC were purchased from Sigma-Aldrich Company (Sigma–Aldrich, St. Louis, MO, USA). The lyophilized culture of L. planetarum was activated from the lab’s microbial collection (University of South Florida, USA). De Man–Ragosa–Sharpe agar (MRSA), chloroform (99.0–99.4% CHCl₃), methanol (>99.0%), potassium persulfate (K₂O₈), and niacin standard (>99.0%, C₆H₅NO₂) were purchased from Sigma-Aldrich (Sigma–Aldrich, St. Louis, MO, USA).

2.2. Sample Preparation

To optimize the fermentation of the basil seeds, we considered various fermentation durations and water volumes to determine the best conditions.

Table 1. Description of fermented and unfermented basil samples. The aliphatic characteristics indicate the ratio of basil powder (g) to water (mL), and the numbers determine fermentation time (h).

Name of Samples Based on Ratio of Basil Powder (g)–Water (mL)	Fermentation Time (h)	All Samples’ Abbreviations
A: adding 1 g of basil powder to 4 mL of water	0: without fermentation (control) 24: 24 h fermentation 48: 48 h fermentation 72: 72 h fermentation 96: 96 h fermentation	A-0	A-24	A-48	A-72	A-96
B: adding 1 g of basil powder to 5 mL of water		B-0	B-24	B-48	B-72	B-96
C: adding 1 g of basil powder to 6 mL of water		C-0	C-24	C-48	C-72	C-96
D: adding 1 g of basil powder to 7 mL of water		D-0	D-24	D-48	D-72	D-96
E: adding 1 g of basil powder to 8 mL of water		E-0	E-24	E-48	E-72	E-96
F: adding 1 g of basil powder to 9 mL of water		F-0	F-24	F-48	F-72	F-96
G: adding 1 g of basil powder to 10 mL of water		G-0	G-24	G-48	G-72	G-96

presents the descriptions of the samples of fermented and unfermented basil. The basil samples were prepared at 7 levels of moisture with ratios including 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 based on the weight of the meshed basil powder samples (g) and volume of water (mL) (this amount of water was chosen based on the physical properties and water-holding capacity of the basil seeds). After adding the appropriate volume of water, each mixture was autoclaved at 121 °C for 15 min and then cooled to room temperature (28 °C). After inoculating 5% (mL/mL) of activated L. planetarum into the sterilized basil media for the final bacterial concentration of approximately 1 × 10⁸ cfu/mL, the samples were incubated at 37 °C for 24 h, 48h, 72 h, and 96 h [26].

To activate L. planetarum, 1 mL of bacterial culture were inoculated into 9 mL of MRS medium for 16 h at 37 °C. The activated bacteria were centrifuged at 4300 rpm/4 °C. The supernatant was discarded, and the pellet was washed with saline. This process was repeated. The pellet was diluted by water to reach an OD of 1.6 at a 600 nm wavelength using a Microplate Reader (Perkin Elmer; Waltham, MA, USA) [10,27].

After incubating the basil samples with the L. planetarum for 24 h, 48 h, 72 h, and 96 h, the pH values and microbial counts were measured. Subsequently, the samples were dried at 40 °C for 16 h [24]. The dried fermented basil seeds were blended, meshed, and stored at −80 °C for further analysis. The procedure for the preparation of the fermented basil samples is shown in Figure 1.

2.3. Moisture, pH, and Microbiological Quality Measurement

The moisture contents of the unfermented basil samples with different volumes of water were determined according to procedures of the Association of Official Agricultural Chemists, with some modifications [28]. Approximately 0.1 g of each sample was dried at 105 °C until a consistent weight was achieved. A sensitive balance was used to measure the weights of the samples before and after drying to determine their moisture contents.

The pH of all the basil samples (fermented and unfermented samples) was measured using a high-precision pH meter (Optional Ub-5 Meter, 115 VAC, Cole-Parmer, Chicago, IL, USA) after 24 h, 48 h, 72 h, and 96 h of fermentation. The pH meter was primarily calibrated using commercial pH 4 and 7 standards.

The microbiological quality of the fermented samples was verified by determining the cell counts of L. palntarum in the MRS agar. For this reason, after 24 h, 48 h, 72 h, and 96 h of fermentation, 0.1 g of each fermented sample was added to 0.9 mL of saline and then plated on MRS agar by preparing a serial dilution. Each sample’s colony-forming units (CFUs) were enumerated after incubation at 37 °C for 48 h. Data were expressed as log CFU/mL [10,11,24].

2.4. Antioxidant Properties

2.4.1. Extracting Basil Samples

To measure the antioxidant properties, three tests were examined. Additionally, selecting the extraction solvents and methods was critical for determining the antioxidant properties/polyphenols. In this study, pure methanol was used to extract the fermented basil seeds.

To prepare the basil extract samples, 2.5 mL of pure methanol was added to 250 mg of dried and meshed basil samples. They were completely mixed for 1 min and then kept in a dark place for 2 h. After 2 h, the samples were remixed and centrifuged at 1500× g for 10 min. The supernatants were filtered at 0.45 µm, and the samples were stored at 4 °C for further chemical analysis.

2.4.2. DPPH and ABTS^• Scavenging Assay

An amount of 60 µL of each extracted sample was mixed with 1.5 mL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and kept at room temperature for 30 min. The absorbance of the samples was read at 513 nm using a Microplate Reader (Perkin Elmer; Waltham, MA, USA). The percentage of DPPH free radical inhibition was calculated by Equation (1):

DPPH scavenging activity (%) = (A^{control 513} − B^extract513) × 100/A^{control 513}

(1)

A^control: Pure methanol + DPPH read at ‘0’ min/513 nm; B^extract: Extract of samples + DPPH.

The ABTS^• scavenging ability of the sample extracts was measured according to the method described by Silveira Coelho et al., with some modifications [29]. ABTS^• was prepared by reacting an ABTS^• aqueous solution (7 mM) with potassium persulfate (2.45 mM, final concentration) in a dark place for 16 h. Then, ethanol was used to adjust its concentration to around 0.700 at Abs 734 nm. Then, 50 µL of each sample extract was added to 150 µL of the ABTS^• solution, and the absorbance was measured at 734 nm after 6 min of incubation at room temperature. Gallic acid was used as a standard, and the gallic acid equivalent antioxidant capacity (GAEAC) was subsequently calculated based on the standard curve [30]. The free radical scavenging ability was expressed as gallic acid equivalents in mg per mg of basil mass.

$${\mathrm{ABTS}}^{•} \mathrm{radical} \mathrm{scavenging} \mathrm{activity} (\%)=\left({\displaystyle \frac{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{A}\mathrm{b}\mathrm{s}-\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{A}\mathrm{b}\mathrm{s}}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{A}\mathrm{b}\mathrm{s}}}\right)\times 100$$

(2)

Control Abs: ABTS^• + solvent (pure methanol), Samples Abs: ABTS^• + Extract of sample.

2.4.3. Total Polyphenol Components (TPCs)

Total Polyphenol concentration (TPC) was determined according to the Folin–Ciocalteu method of Shirvani et al. (2016) with a slight modification [31]. An amount of 25 µL of each extracted sample was mixed with 125 µL of Folin–Ciocalteu’s reagent (1:10, diluted with distilled water) and 100 µL of a 7.5% sodium carbonate solution. The samples were incubated at 45 °C for 15 min. After reaching room temperature, the absorbance was determined at 765 nm using a Microplate Reader (Perkin Elmer, Waltham, MA, USA). A calibration curve was prepared using a standard solution of gallic acid (>0.99). The result was expressed as ug of gallic acid equivalents (GAEs) per 100 g of the sample.

2.5. The Extraction Procedure for Nicotinamide (Niacin)

We followed Hämmerle et al.’s method to measure niacin with some modifications [32]. An amount of 100 mg of each of the top three samples in terms of antioxidant properties was extracted with 10 mL of methanol, using a H₂O–solvent volume ratio of 80:20 (%) with the same procedure mentioned in Section 2.3. To optimize the GC measurements, the sample extracts were dissolved in chloroform with a ratio of 1:1 (500 µL:500 µL) by vortexing for 3 min and transferred into GC vials for analysis (1.00 μL injection). Standards and each replicate sample were run twice in the GC vials for the best extractants. The calibration curve for the niacin standard was in the range of 0.5–10 μg/mL, and 1.00 μL of each sample was injected directly into the GC vials [32].

Detection, Quantification, and Confirmation of Nicotinamide Content by Gas Chromatography (GC-FID)

The samples in chloroform were analyzed using a GC-2023 gas chromatograph (Shimadzu) controlled via GC Real-Time Analysis Software (Shimadzu, Kyoto, Japan), equipped with an AOC-30 i auto-injector (Shimadzu, Kyoto, Japan) and a flame ionization detector (FID). A BPX-5 capillary column (30 m × 0.25 mm I.D. 0.25 μm) was used with the following temperature parameters: the injector was at 330 °C; the column at 120–280 °C at 15 °C/min with a 2 min hold at the final temperature; and the detector at 280 °C. Helium served as the carrier gas at a flow rate of 15 mL/min. The extracted samples (1 μL) were automatically injected in spitless mode (2:1). For all samples, the full-scan mass was obtained by scanning from m/z 78 to 122. The niacin content in each sample was identified by comparing the retention time with the standard and quantified by the peak area integration using the GC PostRun Software (Shimadzu, Kyoto, Japan) [32].

2.6. Statistical Analysis

The analytical procedures were performed using R software 4.4.1. The results are presented as means ± the standard error of the means (SEMs; n = 3). The Shapiro–Wilk test and outlier test were used to assess the normality of the data distribution and the homogeneity of variance, respectively. The samples were analyzed using a one-way analysis of variance (ANOVA) at a significance level of p < 0.05. All experiments were conducted in triplicate. The figures were created using GraphPad Prism 8.0.

3. Results

3.1. Moisture Content, Changes in Colony Count (log CFU/mL), and pH

The moisture contents of the samples ranged from 79% to 91% (

Table 2. Percentages of unfermented basil seed samples’ moisture contents.

Samples	Moisture%
A	79 ± 0.3
B	84 ± 0.3
C	86 ± 0.7
D	88 ± 0.3
E	89 ± 0.60
F	90 ± 0.6
G	91 ± 0.4

). Increasing moisture can affect other health properties and optimizing it can improve the fermentation quality. The initial pH of the basil seeds was near neutral and then decreased over time as the number of bacteria grew. At the beginning of fermentation (T0), all the samples had the same microbial concentration (8.9 log CFU g⁻¹) since they were obtained from the same preparation bulk. Most samples showed a rapid increase in microbial concentration after 24 h, reaching the highest total microbial count (TMC) after 96 h of fermentation (Figure 2a). However, the highest TMC of sample G was observed after 24 h (approximately 12 log CFU/g), which then slightly decreased after 96 h. The highest TMC was observed in sample F (13.1 log CFU g⁻¹) after 96 h. The highest increase after 24 h was seen in samples E, F, and G (12.26 log CFU g⁻¹, 12.38 log CFU g⁻¹, and 12.73 log CFU g⁻¹, respectively). However, there were no significant differences between the samples at 24, 48, 72, and 96 h of fermentation.

To understand how the pH of basil changes during fermentation, measurements were taken for all treatments at 0, 24 h, 48 h, 72 h, and 96 h after adding L. plantarum. Figure 2b shows that the pH dropped significantly after 24 h of incubation, reaching a range of 5.2–5.11 for all samples. After that, the change in pH was moderate and did not significantly differ at 48 h, 72 h, and 96 h. Sample E exhibited the lowest pH (5.11), while sample G had the highest pH after 96 h (5.26). The changes in pH and TMC are shown in

Table 3. Changes in pH and TMC during different fermentation times (hours).

Sample/Time	0		24 h		48 h		72 h		96 h
	pH	TMC 1	pH	TMC 1	pH	TMC 1	pH	TMC 1	pH	TMC 1
A	6.05 a ± 0.07	8.92 ± 0.85	5.19 b ± 0.03	11.98 ± 0.81	5.22 b ± 0.13	11.99 ± 0.11	5.24 b ± 0.01	12.20 ± 0.058	5.24 b ± 0.08	12.89 ± 0.55
B	6.23 a ± 0.16	8.92 ± 0.85	5.22 b ± 0.07	11.79 ± 0.91	5.22 b ± 0.02	11.82 ± 0.03	5.21 b ± 0.25	12.00 ± 0.12	5.22 b ± 0. 42	12.48 ± 0.70
C	6.21 a ± 0.04	8.92 ± 0.85	5.12 b ± 0.04	11.61 ± 1.04	5.14 b ± 0.02	11.80 ± 0.32	5.19 b ± 0.01	12.44 ± 0.14	5.20 b ± 0.218	12.62± 0.61
D	6.27 a ± 0.05	8.92 ± 0.85	5.16 b ± 0.02	11.75 ± 2.04	5.17 b ± 0.01	11.90 ± 0.21	5.18 b ± 0.06	12.12 ± 0.25	5.19 b ± 0.11	12.24± 0.16
E	6.24 a ± 0.01	8.92± 0.85	5.11 b ± 0.04	12.26 ± 1.05	5.12 b ± 0.01	12.25 ± 0.33	5.13 b ± 0.02	12.40 ± 0.33	5.22 b ± 0.27	12.72 ± 0.761
F	6.31 a ± 0.02	8.92 ± 0.85	5.17 b ± 0.05	12.38 ± 0.79	5.18 b ± 0.06	12.46 ± 0.33	5.19 b ± 0.01	12.70 ± 0.44	5.22 b ± 0.11	13.15 ± 0.84
G	6.29 a ± 0.03	8.92 ± 0.85	5.17 b ± 0.01	12.73 ± 0.95	5.22 b ± 0.04	12.48 ± 0.12	5.16 b ± 0.07	12.50 ± 0.83	5.26 b ± 0.12	12.48 ± 0.82

¹ TMC unit is cfu/mL. The differences between the two groups are illustrated as letters (^a) and (^b). Samples with no letters are not significantly different.

.

3.2. Impacts of Different Fermentation Conditions on Antioxidant Properties of Basil Seeds

Antioxidant and polyphenolic compounds can enhance the health benefits of seeds through several mechanisms, such as scavenging free radicals, complexing with pro-oxidants, and quenching singlet oxygen [7,25,33].

3.2.1. DPPH and ABTS^• Scavenging Ability

The DPPH scavenging assay revealed that basil fermentation significantly affected antioxidant properties (p < 0.05) (Figure 3). The DPPH scavenging activity percentage ranged from 11 to 55%. All control samples (unfermented) showed the lowest antioxidant properties. Samples G-48 and G-24 exhibited the highest antioxidant activity, with 55.38% and 49.32%, respectively. These values were significantly different from the control sample (G-0) (p < 0.05) but not significantly different from the other G samples, including G-24, G-72, and G-96 (49.32%, 53.49%, and 46.52%, respectively). Considering the average of samples with the same water content (A, B, C, D, E, F, and G), sample G (1 g of basil powder in 10 mL of water) had the highest average (51%) compared with the other samples (A (43%), B (46%), C (46%), D (44%), E (45%), and F (46%)) (Figure 4a). Regarding the fermentation time, the 48 h fermentation showed the highest antioxidant properties (49%) compared with 24, 72, and 96 h (45%, 46%, and 44%, respectively) (Figure 4b).

The second measure of antioxidant potential, the ABTS^• scavenging test was assessed using the standard curve of gallic acid, ranging from 0.01 to 0.5 mg GA/mL (Figure 5a). The ABTS^• antioxidant capacity of the fermented samples was significantly higher than that of the unfermented samples (p < 0.05) (Figure 5b). Among the fermented samples, sample G-72 had the highest ABTS^• radical disappearance (64.68 mg GA/g), followed by G-24 (63.65 mg GA/g) and G-48 (63.14 mg GA/g). On average, sample G had significantly higher values than samples A, B, C, D, E, and F, with averages of 51.04, 53.25, 54.89, 51.57, 52.66, and 53.25 mg GA/g, respectively (Figure 4c). However, the average antioxidant capacity of all fermented samples during 24 h, 48 h, and 72 h (55.84, 55.81, and 55.36 mg GA/g) was not significantly different, but all values were higher than those at 96 h (48.62 mg GA/g) (Figure 4d).

3.2.2. Total Polyphenol Concentration (TPC)

The total polyphenol concentration was the third antioxidant property tested, measured using the standard curve of gallic acid in the range of 0.01 to 0.5 mg GA/mL (Figure 6a). The fermented samples showed a significant difference in the TPC compared with the control samples (p < 0.005) (Figure 6b). The range of TPCs among the fermented samples was from 1423.09 mg/100 g to 2994.58 mg/100 g. Samples G-48, G-72, and G-24 showed the highest TPCs (1919.74, 1858.0, and 1781.97 mg/100 g, respectively) which were significantly different from that of G-0 (785.68 mg/100 g). Among the other fermentation conditions, the average of the G-samples (2865.367 mg/100 g) showed the highest TPC compared with samples A, B, C, D, E, and F (2564.3, 2658.1, 2663.4, 2578.4, and 2702.9 mg/100 g, respectively), regardless of the time (Figure 4e). In terms of the fermentation time, the average TPC after 48 h (2766.3 mg/100 g) was higher than that after 24, 72, and 96 h (2576.4, 2682.7, and 2697.3 mg/100 g, respectively) (Figure 4f).

3.3. Impacts of Different Fermentation Conditions on Nicotinamide (Niacin) Content of Basil Seeds

To investigate the effects of various basil fermentation conditions on the niacin content, the top three fermented samples were selected based on their antioxidant properties and TPCs (G-24, G-48, and G-72). Their niacin contents were then measured using the GC-MS multiple reaction monitoring (MRM) scan mode. MRM is a powerful method for eliminating matrix interference during GC assays, thereby increasing sensitivity and selectivity by the mass filtering of the precursor and product ions by altering the collision energy. In this study, the MRM parameters were optimized to set the precursor ion and collision energy with a scanning frequency ranging from 78 and 106 m/z. For the niacin content assay of the basil samples, the standard curve was created with pure niacin concentrations from 0.5 to 10 µg/mL (Figure 7a,b). The retention time was 5.480 min. Our results show that the niacin content increased by fermentation (p < 0.05). Samples G-24 (63.97 μg/mL), G-48 h (9.85 μg/mL), and G-72 (5.4 μg/mL) showed increased niacin contents by 63, 9, and 5 times, respectively, compared with unfermented samples (1.14 μg/mL).

4. Discussion

Generally, the efficiency and effectiveness of fermentation are governed by multiple variables, such as microbial culture, time, and percentage of moisture [27,34,35,36]. Additionally, the ability of microbial cultures (LAB) to produce antioxidant properties, TPC, and healthy components such as niacin varies according to the species, strains of bacteria, and fermentation conditions, including temperature, pH, moisture, and time [10,37,38,39]. pH plays an important role in fermentation as it can affect the production and solubility of certain substances [10,19,40]. In our study, the most significant changes in pH and TMC occurred during the first 24 h of fermentation. The significant increase in the number of bacteria within 24 h caused the drop in the pH. Our results are consistent with those of Tangüler et al., who showed that the most significant change in L. plantarum during fermentation occurred within 24 h [41]. Vatansever et al. also reported that the pH of vegetables significantly decreased during fermentation, reaching 5 after 24 h [42]. In some samples, the pH increase did not persist throughout the fermentation processes, slightly rising after 96 h. Based on scientific research, pH increases during fermentation because microorganisms initially consume nutrients and produce organic acids. As fermentation progresses and nutrients become scarce, the microorganisms start consuming the organic acids as a nutrient source, leading to an increase in pH [10,33,43,44]. Our results are consistent with those of Yuan et al., who showed that the pH initially decreased under neutral conditions and then increased throughout the fermentation process [43]. Laaksonen et al., also demonstrated that the pH decreased from 5.9 at the start of fermentation with LAB to values below pH 5 within 24 h and then slightly increased after 48 h [10]. The changes in the pH and TPC during fermentation are shown in Table 3.

Increasing the number of LAB during fermentation can also increase the efficiency (%) of antioxidant properties and the TPC of extraction [38,45,46,47,48]. In our experiments, the TPC of fermented samples increased by two- to four-fold, depending on the fermentation conditions. Torres-León et al. reported that fermentation could increase antioxidant properties and the TPC by three-fold [49]. Our results show that the TPC increased moderately with an increase in moisture from a ratio of 4:1 to 10:1 (mL/g of seeds to water) (p < 0.05), with group G having the highest TPC with the highest volume of water. Regarding the fermentation time, 24 h fermentation showed lower TPCs in groups A, B, and C with the same water volume. However, as the water volume increased, so did the TPC. This result was consistent with the ABTS^• scavenging test. The samples with the highest water content fermented for 24, 48, and 72 h (G-24, G-48, and G-72) showed the highest TPCs. Among them, sample G-48 had the best average in terms of both the water volume and fermentation time (p < 0.05). According to scientific research, the release of bound phenolic compounds (or polyphenols) during fermentation can enhance the product’s antioxidant ability and health benefits [22,50,51], showcasing their critical role in scavenging free radicals [52]. These results are consistent with the DPPH and ABTS^• scavenging activities, showing a more than three-fold increase in antioxidant properties in the fermented samples compared with the control samples (p < 0.05), with group G showing the highest average radical scavenging potential after 48 h of fermentation. Rocchetti et al. and Hubert et al. also reported that the antioxidant properties and TPCs of fermented samples increased and peaked after 48 h of fermentation [53,54]. In our samples, the lowest ABTS^• scavenging ability among the four groups (A, B, C, and D) was observed after 24 h of fermentation, consistent with the TPC results. However, increasing the water volume up to 10 mL improved the activity of bacteria to produce TPC and showed ABTS^• scavenging ability. The overall DPPH scavenging ability of the samples was similar to the ABTS^• test (Figure 8). However, changes in the fermentation time among groups A–F (with the same water volume) did not show a significant difference in the DPPH scavenging percentage. In the G group, the 24, 48, and 72 h fermentations showed the highest DPPH scavenging ability, with the 48 h fermentation significantly differing from the 96 h fermentation, which showed the lowest ABTS^• and DPPH scavenging ability across all groups. Several factors could explain this phenomenon, including the binding of antioxidant compounds (e.g., TPC) with other constituents and their degradation by microbial enzymes [55]. Therefore, the moisture content and fermentation time are crucial factors influencing the quality, quantity, and antioxidant properties of products [53,56]. By optimizing the moisture and duration of basil seed fermentation, we could enhance their antioxidant properties and TPC. Antioxidant and polyphenolic compounds can improve the health benefits of seeds through various mechanisms, such as scavenging free radicals, complexing with pro-oxidants, and quenching singlet oxygen, thereby reducing their activity [7,33,57,58]. Vitamin B content is another secondary health metabolite that can also increase during fermentation [7,10,14]. Our results show that the niacin content increased with fermentation with the best fermentation times for the highest niacin content being 24 and then 48 h. Optimizing the fermentation conditions was crucial in increasing the niacin content (Figure 8c). Our results are consistent with those of Ngene et al. who reported that L. plantarum could increase the niacin content during fermentation [59].

Based on all results, basil seeds show the potential to improve health due to secondary metabolites like niacin and phenolic chemicals, which possess antioxidant qualities.

5. Conclusions

Our study found that fermenting basil seeds with L. plantarum significantly increased their total polyphenol content and enhanced their radical scavenging ability. Both moderate moisture and fermentation time were crucial factors in achieving a high antioxidant activity and bioactive compound effectiveness. Specifically, a higher moisture content during fermentation promoted TPC and antioxidant properties. A 48 h fermentation period resulted in the highest average TPC and scavenging radical ability among all the samples, while the highest niacin content was observed at 24 h of fermentation. These findings strongly suggest the potential for developing novel plant-based foods using fermented basil seeds. Fermentation could lead to the production of healthier products rich in phenolic compounds with a greater antioxidant capacity and niacin content, making them valuable as functional food additives.