Enhanced DPPH Radical Scavenging Activity and Enriched γ-Aminobutyric Acid in Mulberry Juice Fermented by the Probiotic Lactobacillus brevis S3

Abstract

Mulberries, known for their high sugar content and rich bioactive compounds, have attracted attention for their potential health benefits. γ-Aminobutyric acid (GABA) is an amino acid with multiple physiological functions. To increase the GABA content and enhance the antioxidant capacity in fermented mulberry beverages, we screened a high-yielding strain, Lactobacillus brevis S3, known for its probiotic properties. L. brevis S3 demonstrated an excellent tolerance to simulated gastric acid, gastric juice, intestinal fluid, bile salts, osmotic pressure, and phenol, making it a safe and valuable probiotic candidate for mulberry fermentation. We attempted the addition of different nutritional components to enhance the GABA content in mulberry juice, including 1% yeast extract; 0.5% peptone; 0.01% metal ion complex (magnesium sulfate, manganese sulfate, and ferrous sulfate); combinations of yeast extract and peptone, and all three components. Mulberry juice supplemented with all three components reached a viable cell count of 1.2 × 10¹⁰ CFU/mL after 72 h. The antioxidant capacity and GABA titer were enhanced. The DPPH free radical scavenging capacity increased by 1.62 times, and the GABA content reached 7.48 g/L. By utilizing L. brevis S3 with excellent probiotic properties and supplementation with nutritional components, it is possible to produce low-sugar mulberry functional beverages with a high DPPH free radical scavenging capacity that are rich in GABA.

1. Introduction

Mulberry, a dried fruit cluster from the Morus species, belongs to the Moraceae family [1]. It is available in different varieties, including black mulberry (Morus nigra L.), white mulberry (Morus alba L.), and red mulberry (Morus rubra L.) [2]. With its delightful sweet and sour taste, mulberry is a popular fruit for consumption due to its high sugar content. It is not only appreciated for its flavor but also valuable for its medicinal properties. Mulberry is known to contain more than 150 compounds, including anthocyanins, polyphenols, polysaccharides, and other compounds [2,3,4]. It prevents cardiovascular diseases, treats chronic obstructive pulmonary diseases, has anti-inflammatory effects, alleviates diarrhea, and improves blood circulation disorders, which can be related to its dominant anthocyanin [5]. Anthocyanins play a crucial role as coloring substances in mulberry, offering characteristics such as a high color intensity, water solubility, and safety. Moreover, mulberry anthocyanins can serve as natural antioxidants and natural food colorants, as they offer good stability and are suitable for coloring mildly acidic foods. Preventing atherosclerosis, lowering cholesterol levels, cardiovascular prevention, and providing radiation protection are related to polyphenol enrichment [6,7,8,9,10,11,12]. Mulberries contain a significant amount of polysaccharides, which serve as important functional factors with high biological activity [3,13]. Various extraction methods and purification techniques have identified multiple types of polysaccharides in mulberry [3]. Mulberry polysaccharides have been studied for their potential applications in the food and pharmaceutical industries due to their physiological and pathological activities, such as immunoregulation and blood sugar management [14,15]. Mulberries are rich in vitamins A, B, and C; carotenoids; and various amino acids, which are beneficial for anticancer and antiaging activities [9,16,17]. These nutritional components make mulberries highly valuable for both nutritional and medicinal benefits, and they hold great potential for further development.

While mulberries are nutritionally rich and exhibit various physiological activities, mulberry plants encounter challenges in circulation due to their susceptibility to damage and decay [18]. To address this issue, the partial decomposition of active proteins through microbial fermentation improves their absorption by the human body [11]. Yeast or lactic acid bacteria (LAB) are excellent strains that can be fermented individually or in combination to enhance the taste and nutrition of fermented beverages [11,19]. LAB are widely present in the human intestinal tract [20]. They play a crucial role in metabolizing carbohydrates to produce lactic acid. Most LAB contribute to a variety of physiological functions. They regulate the intestinal microbiota, promoting the growth of beneficial bacteria while inhibiting putrefactive bacteria. This mechanism improves gastrointestinal function and aids in the clearance of intestinal waste [21]. Moreover, LAB offer numerous health benefits. They help prevent and manage lactose intolerance, inhibit cholesterol absorption, lower blood pressure and lipid levels, and exhibit potential antitumor properties [20]. One application of LAB is found in probiotic-fermented fruit and vegetable beverages [22,23]. These beverages, primarily based on LAB, not only retain the nutritional components of the raw materials but also leverage the dual advantages of probiotics, offering the potential for improved gut health and overall well-being. Therefore, LAB demonstrate significant potential in promoting health and are a valuable component in probiotic-fermented beverages.

γ-Aminobutyric acid (GABA) plays a crucial role as a neurotransmitter in the nervous system and possesses various functions, such as antioxidative, anxiolytic, antiaging, hepatoprotective, and blood pressure-lowering effects [24,25,26,27]. This versatile compound has found wide-ranging applications in various sectors, including the food and pharmaceutical industries. GABA is produced through fermentation by LAB, particularly the predominant strains Lactobacillus (L.) brevis, L. plantarum, L. pentosus, and L. buchneri [28,29,30,31]. Compared to using commercially complex culture media to produce GABA, using pure fruit juices as the culture medium did not yield high levels of GABA in the fruit juice [22,32,33,34]. It is important to note that using pure fruit juice as the sole substrate for LAB cultivation may not promote bacterial growth and the synthesis of GABA, resulting in lower GABA production.

In this study, the goal was to find a safe and suitable Lactobacillus sp. strain for high-sugar mulberry fermentation that is capable of producing high levels of GABA. By adding various nutrient components, including nitrogen sources (yeast extract or peptone) and metal ion mixtures (Mn²⁺, Fe²⁺, and Mg²⁺), to mulberry juice, the cell viability, nutritional composition, antioxidative properties, and GABA production were analyzed. This comprehensive analysis will provide valuable insights into the key components that affect cell viability and GABA enrichment in mulberries. Ultimately, the findings will serve as valuable references for the preparation of fermented beverages using high-sugar mulberries, allowing for the development of functional beverages with enhanced nutritional value and the potential health benefits of GABA.

2. Materials and Methods

2.1. Materials

Mulberries (Morus nigra L., Moraceae) were obtained from the Sericultural Research Institute, Chinese Academy of Agricultural Sciences. Yeast extract and peptone were purchased from OXOID Co., Ltd. (Basingstoke, UK). Standard power (GABA), boric acid, methanol, and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA). O-phthaldialdehyde was purchased from Agilent Technologies Inc. (Santa Clara, CA, USA). The other reagents were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). LAB were preserved in our laboratory and isolated from the fermented system of the Chinese traditional liquor Baijiu.

2.2. Medium and Solution Configuration

GYP medium: 10 g/L glucose, 10 g/L yeast extract, 5 g/L peptone, 2 g/L sodium acetate, 0.1 g/L NaCl, 0.1 g/L FeSO₄, 0.1 g/L MnSO₄, and 0.2 g/L MgSO₄. MRS medium: 2% glucose, 0.5% yeast extract, 1% peptone, 1% beef extract, 0.2% KH₂PO₄, 0.2% ammonium citrate, 0.5% sodium acetate, 0.058% MgSO₄, 0.025% MnSO₄, and 1 mL Tween 80, pH 6.2, all expressed in mass/volume%. Columbia blood agar plates were purchased from Best One Base Experimental Mall (Nanjing, China). DNase agar plates containing toluidine were purchased from Ritsu Biotechnology Co., Ltd. (Yancheng, China).

Base culture medium for biogenic amine production: 0.5% yeast extract; 0.5% beef extract; 0.5% peptone, 0.25% NaCl, 0.05% glucose, 0.1% Tween 80; 0.02% MgSO₄·7H₂O, 0.005% MnSO₄·H₂O, 0.04% ferrous sulfate, 0.2% ammonium citrate tribasic, 0.01% CaCO₃, 0.005% V_B, 0.2% KH₂PO₄, and 2.0% agar, pH 5.5. Simulated gastric fluid (pH 2.0): 16.4 mL of 1 mol/L HCl, 800 mL of distilled water, and 10 g of pepsin brought up to 1000 mL. Simulated intestinal fluid: 6.8 g potassium dihydrogen phosphate, and 10 g pancreatin, pH adjusted to 6.8, brought up to 1000 mL.

2.3. Preparation of Mulberry Juice

Fresh mulberries were collected, and the stems were removed. A grinder was used to crush the mulberries. The crushed mulberries were filtered through two layers of sterilized cheesecloth to obtain mulberry juice. The filtered mulberry juice was divided into portions and sterilized at 100 °C for 10 min to maintain hygiene and freshness.

2.4. Screening and Identification of LAB

LAB (25 strains) stored at −80 °C were streaked onto GYP agar plates and incubated at 37 °C for 48 h. A single colony was inoculated into 5 mL of GYP medium and incubated at 37 °C for 24 h as the primary seed culture. This primary seed culture was then inoculated at 10% (v/v) into GYP medium and incubated at 37 °C and 200 rpm under aerobic conditions for 15 h to obtain the secondary seed culture. The secondary seed culture was centrifuged at 4 °C and 5000 rpm for 5 min to remove the fermentation supernatant. The bacterial pellet was washed twice with physiological saline, resuspended in physiological saline with an equal volume as the fermentation solution, and gently mixed to prepare the fermentation seed culture. The fermentation seed culture was inoculated at 10% (v/v) into sterilized mulberry juice and incubated at 37 °C and 200 rpm under aerobic conditions for 48 h. After fermentation, the cell density (OD₆₀₀) and the GABA content were measured.

To observe the cell morphology, Gram staining and scanning electron microscopy were performed. The cells were then ground using a grinder to disrupt the cell walls. Genomic DNA was extracted from the disrupted cells using a Bacterial Genomic DNA isolation kit (Shanghai, China). Full-length 16S rDNA was amplified using the universal primers 27F-AGTTTGATCMTGGCTCAG and 1492R-GGTTACCTTGTTACGACTT. The PCR product was purified and sequenced. The sequences were compared using the BLAST database on NCBI to analyze sequence similarities, and the homologous sequences for each strain were identified. Multiple sequence alignment was performed using Mega 11 software, and the neighbor-joining algorithm (NJ) was employed to construct the phylogenetic tree.

2.5. Influence of Nutritional Components on Fermentation of L. brevis S3 in Mulberry Juice

The strain that was cultured for 24 h was collected by centrifugation at 4 °C and 5000 rpm for 5 min. The cells were then washed twice with physiological saline solution and resuspended to achieve an optical density (OD₆₀₀) of 100. The cell counts were 2.1 ± 0.2 × 10¹¹ CFU/mL. This resulting cell suspension was used as a fermentation starter for mulberry juice fermentation with a 1% (v/v) volume ratio. In the mulberry juice fermentation process, different supplements were added to the mulberry juice. The fermentation starter was inoculated into each of these mulberry juice samples and incubated at 37 °C with agitation at 200 rpm for 72 h. Each resulting fermented sample was designated as follows: Y group: mulberry juice supplemented with 1% (w/v) yeast extract; P group: mulberry juice supplemented with 0.5% (w/v) peptone; M group: mulberry juice supplemented with 0.01% (w/v) metal ions (MgSO₄, MnSO₄, and FeSO₄); Y + P group: mulberry juice supplemented with 1% yeast extract and 0.5% peptone; and Y + P + M group: mulberry juice supplemented with 1% yeast extract, 0.5% peptone, and 0.01% metal ions. Fermented mulberry juice without any supplement served as the control group.

2.6. Safety Evaluation of L. brevis S3

2.6.1. Hemolysis

The strain was streaked onto Columbia blood agar plates and incubated at 37 °C for 48 h. The plates were then examined for the presence of hemolysis.

2.6.2. DNase Production

The strain was streaked onto DNase agar plates containing toluidine blue and incubated at 37 °C for 48 h. The presence of a rose-pink transparent zone around the colonies was observed.

2.6.3. Biogenic Amine Production

The strain of the primary seed culture was inoculated into MRS medium containing 0.1% arginine, lysine, and histidine, followed by incubation at 37 °C and 200 rpm for 24 h. After three consecutive transfers, the culture was spread onto a bottom layer medium for biogenic amine production to culture at 37 °C. The top layer medium for biogenic amine production was added to the cells. The presence of a purple or red color around the colonies was observed.

2.6.4. Antibiotic Susceptibility

The second seed culture was streaked onto a MRS plate. A filter paper disk with a diameter of 6 mm was soaked with 1 μL of antibiotic solution, placed onto MRS plates, and then incubated at 37 °C for 24 h. The antibiotic solution included erythromycin (40 μg/μL), penicillin (50 μg/μL), rifampicin (4 μg/μL), cefalexin (30 μg/μL), tetracycline (30 μg/μL), chloromycetin (30 μg/μL), vancomycin (30 μg/μL), and kanamycin (30 μg/μL).

2.6.5. Autoaggregation Ability

The strain cultured for 24 h was vigorously vortexed to ensure thorough mixing and then incubated at 37 °C without disturbance. The bacterial cell density in the supernatant was measured every 2 h for 12 h.

2.6.6. Gastric Acid Tolerance Verification

The strain cultured for 24 h was centrifuged at 4 °C and 5000 rpm for 5 min to collect the cells. The cells were resuspended in physiological saline solution to achieve an optical density (OD₆₀₀) of 10. The cell count was 2.0 ± 0.5 × 10¹⁰ CFU/mL. The bacterial suspension was then inoculated into MRS medium at pH 2 and pH 3 with a 10% inoculum volume. The cultures were incubated at 37 °C and 200 rpm for 0 to 4 h, with samples taken every 2 h. The cell counts were recorded as log₁₀ CFU/mL.

2.6.7. Simulated Gastric Fluid and Intestinal Fluid Tolerance

The cells cultured for 24 h were resuspended in physiological saline to achieve an OD₆₀₀ of 10, resulting in a cell count of approximately 2.0 × 10¹⁰ CFU/mL. A 10% volume ratio of the cell suspension was inoculated into simulated gastric fluid or simulated intestinal fluid. The culture was incubated at 37 °C and 200 rpm, and then, the cell count was recorded as log₁₀ CFU/mL.

2.6.8. Cholate, Hypertonic Stress, and Phenol Tolerance

Using the same cell culture (OD₆₀₀ = 10), a 10% (v/v) inoculation volume was inoculated into MRS medium containing 0%, 0.3%, 0.6%, and 1% (w/v) sodium cholate or 0%, 1%, 2%, and 4% (w/v) salt. The mixture was cultured at 37 °C for 2 h, and then, the cell count was recorded. For phenol tolerance, the same cells were inoculated into MRS medium containing 0%, 0.4%, and 0.6% (w/v) phenol and cultured for 24 h, and then, the cell count was recorded as log₁₀ CFU/mL.

2.7. Analysis of Mulberry Fermentation

The fermentation supernatant and cells were collected by centrifugation at 4 °C and 5000 rpm for 5 min from the samples taken during the fermentation process. The cells were used for cell viability testing, while the fermentation supernatant were used for the component analysis.

2.7.1. Cell Density and Cell Viability

The cells were resuspended in an equal volume of physiological saline and gently mixed, and the cell density was detected at 600 nm using a multimode reader (Spectra Mas i3 R-3, BUCHI, Flawil, Switzerland).

The cells were diluted with physiological saline using a 10-fold dilution method to achieve suitable concentrations. The diluted cell suspension (100 μL) was plated onto a MRS plate and incubated at 37 °C for 48 h. The cell count on the plate was recorded as CFU/mL (cell survival in mulberry juice) or log₁₀ CFU/mL (safety evaluation).

2.7.2. Measurement of pH

The pH of the solution was detected by a pH meter (METTLER TOLEDO).

2.7.3. Determination of Polysaccharides

Two hundred microliters of the fermented supernatant were taken and combined with 100 μL of 5% phenol and 500 μL sulfuric acid. After thorough mixing, the reaction was allowed to proceed for 15 min, followed by measurement of the absorbance at 490 nm. Glucose standards (0~0.1 mg/mL) were prepared for comparison.

2.7.4. Determination of Flavone

The fermentation supernatant (100 μL) was combined with 20 μL of 5% sodium nitrite and 20 μL of 10% aluminum nitrate, with a 5-min interval between the addition of different reagents. Subsequently, 80 μL of 4% sodium hydroxide was added to the mixture, followed by a 15-min reaction at room temperature. The absorbance was measured at a wavelength of 510 nm. Rutin standards (0.0~1.6 mg/mL) were prepared for comparison.

2.7.5. Determination of Polyphenol

The fermentation supernatant (55 μL) was mixed with 100 μL of the Folin–Ciocalteu reagent and thoroughly mixed. After 45 min, 200 μL of 1 M sodium carbonate solution were added to the mixture. The resulting mixture was then shielded from light for 1 h before measuring the absorbance at 760 nm. Gallic acid standards (10~100 μg/mL) were prepared and used to establish a standard curve.

2.7.6. Determination of GABA

GABA detection used high-performance liquid chromatography (HPLC, LC-10A, Shimadzu, Koyot, Japan), followed by the derivative using o-phthaldialdehyde. After derivatization, 10 μL of the sample was injected. Chromatography was conducted using a C18 column (SB-C18, 250 mm × 4.6 mm, 5 μm, Agilent, Santa Clara, CA, USA). Mobile phase A consisted of 4.52 g/L sodium acetate in the aqueous phase, while mobile phase B consisted of 22.6 g/L sodium acetate in the organic phase, with the addition of 40% acetonitrile and 40% methanol. The analysis temperature was set at 40 °C. The detection wavelength was 338 nm. The analysis program was as follows: 0 min, the mobile phase B composition was 8%; from 0 to 20 min, it increased to 46.5% B; from 20 to 22 min, it reached 100% B; and it remained at 100% B from 22 to 25 min. From 26 to 30 min, the mobile phase B composition decreased back to 8%. The total duration of the analysis was 30 min. The standard curve was established by plotting the concentration of GABA against the corresponding peak area. By measuring the peak area of the sample, the GABA concentration in the sample could be calculated using the standard curve.

2.8. Detection of Antioxidant Properties

2.8.1. Reducing Power

The fermented supernatant (100 μL) was added to 150 μL of phosphate buffer (0.2 mol/L, pH 6.6) and 0.5 mL of 1% potassium ferricyanide. The mixture was reacted at 50 °C for 20 min. The reaction mixture was added to 0.5 mL of 10% trichloroacetic acid and then left to stand at room temperature for 5 min. Then, 0.25 mL of the supernatant was taken, and 0.25 mL of distilled water and 0.05 mL of 1% FeCl₃ were added to measure the absorbance at 700 nm.

2.8.2. DPPH Radical Scavenging Ability

The fermented supernatant (25 μL) was added to 250 μL of DPPH solution (0.025 mg/mL) and then reacted in the dark for 20 min to measure the absorbance at 517 nm.

2.8.3. Superoxide Anion Radical Scavenging Ability

The fermented supernatant (100 μL) was added to 0.6 mL of Tris-HCl buffer solution (50 mmol/L, pH 8.2) to adjust the volume to 900 μL with water. The mixture was reacted at 37 °C for 10 min. The mixture was added to 100 μL 3.5 mmol/L pyrogallol solution at 37 °C for 6 min and immediately terminated using 50 μL (8.0 mmol/L hydrochloric acid). The mixture was measured for absorbance at a wavelength of 320 nm.

2.9. Statistical Analysis

Three biological replicates were performed for each experiment. At the same time or under the same conditions, analysis of variance (ANOVA) was performed with multiple culture conditions as the main influencing factors. SPSS software version 26 was used for data analysis. The confidence level of the ANOVA was 95%. p < 0.05 was considered statistically significant. The figures were derived from GraphPad Prism 9 software. The data were expressed as the mean and standard deviation of three biological replicates. The error bar represented the standard deviation of the mean.

3. Results and Discussion

3.1. Screening GABA-Producing LAB Suitable for Mulberry Juice

Microorganisms capable of producing GABA are known as GABA-producing strains. GABA-producing strains include LAB, yeast, Escherichia coli, and mold [35,36,37,38]. Among them, LAB are commonly used in the food industry for the fermentation and production of GABA [35,38]. To screen for LAB suitable for fermenting mulberry juice to produce GABA, 25 GABA-producing isolates were isolated from Baijiu, Daqu and inoculated into mulberry juice containing monosodium glutamate. As shown in Figure 1a, all 25 strains grew well in mulberry juice with a cell density of OD₆₀₀ ranging from 1.5 to 2, demonstrating that mulberry juice provides the necessary nutrients for the growth of LAB. However, significant differences in the GABA production capacity were observed among the strains (Figure 1b). GABA production ranged from 2.3 to 4.8 g/L, with strain S25 exhibiting the lowest production (2.31 ± 0.09 g/L) and strain S3 displaying the highest production (4.84 ± 0.49 g/L GABA). Notably, despite a similar cell density (OD₆₀₀), strain S3 outperformed strain S25 in GABA production by 2.1 times. These results signified that mulberry juice could support the growth of LAB, but the efficiency of GABA production was strain-specific. Further investigation is warranted to unravel the mechanisms underlying efficient GABA production in mulberry juice. Consequently, strain S3 was selected as the focal point for subsequent research endeavors.

3.2. Identification and Probiotic Characteristics of GABA-Producing Strain S3

3.2.1. Morphological and Molecular Identification

We performed Gram staining on GABA-producing strain S3. As shown in Figure 2, strain S3 is Gram-positive, with cells appearing as short rod-shaped and cylindrical, measuring 10–20 µm in length and 5–6 µm in width. Strain S3 was further subjected to 16S rDNA gene sequence identification. After PCR amplification and gel recovery, the full-length 16S rDNA sequence of strain S3 was obtained and sequenced. The 16S rDNA gene was 1463 bp. A molecular phylogenetic tree constructed using sequences from the NCBI nucleotide database revealed that strain S3 was identical to Lactobacillus brevis ATCC 14869 (Accession No. EU194349), indicating that strain S3 belongs to Lactobacillus brevis (Figure 3). Studies have shown that GABA-producing strains are mainly found in L. brevis, L. plantarum, and L. buchneri [31,39,40], among others, with L. brevis being widely recognized as a high GABA producer. This is consistent with our study, where L. brevis S3 was identified as a GABA-producing strain suitable for fermenting mulberry juice.

3.2.2. Safety Evaluation and Potential Probiotic Characteristics of L. brevis S3

L. brevis S3 underwent a series of safety evaluation tests, including hemolysis, DNase enzyme production, and biogenic amine production, which are important to assess the safety of the bacteria [34,41]. L. brevis S3 exhibited no hemolytic activity and was determined to be γ-hemolytic. Additionally, it did not produce DNase enzymes or biogenic amines (

Table 1. In vitro safety evaluations of L. brevis S3.

Characteristics	Production
Hemolysis	-
DNase production	-
Biogenic amine production	-

-, negative.

and Figure S1). It is important to note that hemolysis, DNase enzyme production, and the presence of biogenic amines can have implications for human health. Therefore, the absence of these attributes in L. brevis S3 is indicative of its safety.

L. brevis S3 demonstrated varying degrees of sensitivity to antibiotics (

Table 2. Antibiotic susceptibility of L. brevis S3.

Antibiotics	Concentration (μg)	Inhibitory Clear Zone (mm)	Sensitive
Erythromycin	40	34 ± 0.8 a	H
Chloromycetin	30	0 d	R
Cefalexin	30	20 ± 2.1 c	I
Vancomycin	30	0 d	R
Penicillin	50	24 ± 1.1 b	H
Tetracycline	30	20 ± 2.0 c	I
Rifampicin	4	26 ± 0.3 b	H
Kanamycin	30	0 d	R

The diameter of the inhibitory clear zone was 0 mm, resistance (R); <10 mm, low sensitivity (L); 10~20 mm, mid-sensitivity (M); and > 20 mm, high sensitivity (H). The small case letters were used for signification analysis.

). The strain exhibited high sensitivity to erythromycin, penicillin, and rifampicin. In contrast, it showed moderate sensitivity to cefalexin and tetracycline. However, L. brevis S3 did not display sensitivity to chloromycetin, vancomycin, or kanamycin. Furthermore, a comparative analysis showed that the potential probiotic strain Weissella confusa G2 exhibited sensitivity to vancomycin [41]. Additionally, L. brevis F064A demonstrated varying sensitivity to different antibiotics [34]. These results suggest the presence of inter- and intraspecies variations in antibiotic sensitivity among different strains, which could be attributed to their respective ecological niches.

The capacity for potential probiotics to colonize the gastrointestinal tract and inhibit the colonization of pathogenic bacteria is crucial. One important factor in achieving this is the ability of cells to self-aggregate, allowing adherence to intestinal cells and preventing colonization by pathogens [34,42]. To evaluate the adhesion efficacy to intestinal cells, autoaggregation tests were conducted. Autoaggregation above 50% was considered high. As shown in

Table 3. Autoaggregation activity of L. brevis S3.

Time (h)	2	4	6	8	10	12
Autoaggregation (%)	12.03 ± 0.29 f	25.92 ± 0.49 e	37.93 ± 0.15 d	52.96 ± 0.25 c	65.75 ± 0.29 b	75.89 ± 0.48 a

The small case letters were used for signification analysis.

, L. brevis S3 exhibited a significant increase in autoaggregation over time, starting from an initial self-aggregation of 12.03 ± 0.29% and reaching 75.89 ± 0.48%. These results indicate that L. brevis S3 possesses a notable autoaggregation ability.

Probiotics are living microorganisms that colonize the host’s body and provide beneficial effects. Their survival in the intestine depends on their tolerance to low pH and high concentrations of bile salts [43]. The human digestive system presents several challenges to the survival of lactobacilli, including the acidic environment of the stomach, the alkaline nature of intestinal fluid, and the presence of gastrointestinal enzymes [44]. L. brevis S3 displayed a consistent tolerance to gastric acid in both the simulated fasting and satiety states (Figure 4a). The strain exhibited a strong tolerance under pH 2 and pH 3 conditions, with a remaining survival rate of 99% after 4 h of treatment at pH 2 and above 99% at pH 3 (Figure 4a). L. brevis S3 showed a 100% remaining survival rate after 3 h of treatment under simulated gastric fluid (Figure 4b). This indicates that the strain can survive effectively even in the presence of gastric proteases. Furthermore, as shown in Figure 4c, L. brevis S3 was not impacted by simulated intestinal fluid and maintained high survival rates. These experiments demonstrate that L. brevis S3 exhibits a strong tolerance to gastric acid, simulated gastric fluid, and intestinal fluids. The strain’s ability to survive under these conditions highlights its potential as a viable probiotic.

In the human small intestine, the concentration of bile salts ranges from 0.03 to 0.3 g/100 mL [45]. This concentration is crucial for the probiotic effects of LAB, as they must tolerate these bile salt levels to exert their beneficial effects on the body. L. brevis S3 illustrated a decreasing trend in cell viability as the concentration of bile salts increased (Figure 4d). This indicates that a high bile salt stress environment can cause damage to the cells. Despite the damaging effects, the results revealed an astonishing finding. L. brevis S3 displayed an impressive survival capability of 94%, even when exposed to a bile salt concentration of 1% for 4 h. This remarkable tolerance to bile salts demonstrates the resilience of L. brevis S3 and highlights its potential as an effective probiotic strain.

The normal growth and metabolism of microorganisms are influenced by osmotic pressure. The adaptability of microorganisms to osmotic pressure changes varies among species, making high osmotic stress tolerance an important indicator for evaluating excellent probiotics. Within the human gastrointestinal tract, the concentration ranges from 1.0 to 4.0 g/100 mL [46]. As the salt concentration increased, the osmotic pressure intensified, creating a more challenging environment and resulting in a decrease in the viable cell count. Remarkably, L. brevis S3 exhibited a strong tolerance to high osmotic stress (Figure 4e). After 24 h of cultivation in a highly osmotic environment with a salt concentration of 4 g/100 mL, the strain maintained a remarkable survival rate of 94%. These findings demonstrate the resilience of L. brevis S3 to high osmotic stress conditions.

Phenol, a carcinogen, is produced in the intestine during the bacterial deamination process of aromatic amino acids that occurs during the digestion of dietary proteins [47]. Considering the potential harmful effects of phenol, it is crucial for probiotic bacteria to exhibit a tolerance to phenol. As the phenol concentration increased, the survival ability of L. brevis S3 gradually decreased (Figure 4f). However, even under a phenol concentration of 0.6% after 24 h of treatment, the strain demonstrated a notable residual survival capability of 92%. This finding indicates that L. brevis S3 exhibits a robust ability to tolerate phenol, which is an encouraging attribute for its potential use as a probiotic strain. Overall, the above results indicate that L. brevis S3 possesses the ability to withstand several challenging conditions in the human gastrointestinal tract, including gastric acid, gastrointestinal fluids, bile salts, high osmotic pressure, and phenol. L. brevis is a common plant-based lactic acid bacterium found in fermented foods such as kimchi and pickles. It has a resistance to acid and salt, allowing it to survive in harsh environments. Although it is not naturally present in the human body, L. brevis can reach the intestinal tract when consumed live and produce significant amounts of lactic acid to inhibit the growth of harmful bacteria and regulate the gut environment. L. brevis has been associated with several beneficial effects, including enhancing immune function, promoting healthy skin, reducing cholesterol levels, and suppressing allergic reactions. Therefore, based on its safety and potential value, L. brevis S3 is an effective probiotic strain.

3.3. Influence of Nutritional Components on the Survival of L. brevis S3 in Mulberry Juice

LAB, as demanding microorganisms, rely on several essential nutrients, including carbon sources, organic nitrogen sources, and mineral salts, for their growth and metabolic activities. While mulberry juice naturally contains sugars, amino acids, and minerals that can support the growth of LAB, it lacks a sufficient organic nitrogen source [48]. Additionally, we determined whether the lack of mineral salts affects GABA synthesis in the strain. Therefore, based on the composition of commercial LAB culture medium, including organic nitrogen sources and a mixture of metal ions, we explored the factors that influence the high production of GABA by LAB in high-sugar fruit juice. Yeast extract and peptone are among the most commonly used organic nitrogen sources for cultivating LAB. These metal ions (Mn²⁺, Mg²⁺, and Fe²⁺) are commonly found in LAB culture media. This targeted approach of supplementation is expected to significantly improve the overall performance of LAB in mulberry juice fermentation, leading to an enhanced survival ability and ultimately higher GABA production levels.

As shown in Figure 5, which illustrates the changes in cell concentration over time, the viable cell count increased in all groups as the fermentation time was prolonged. Notably, when compared to the control group, all the additive groups demonstrated higher viable cell counts, except for the M group. At 72 h of fermentation, the Y + P + M group exhibited the highest viable cell count, reaching 1.2 ± 0.86 × 10¹⁰ CFU/mL. These findings emphasize the role of appropriate nutritional components in enhancing strain viability in mulberry juice. Specifically, organic nitrogen sources such as yeast extract, peptone, and their combination play a crucial role. Interestingly, the addition of metal ions alone did not improve strain viability. However, when combined with the composite nitrogen source, the viable cell count reached the highest level, indicating a counteracting effect of the composite nitrogen source against the impact of metal ions on the strains. Considering the importance of organic nitrogen sources in LAB growth, especially during the late stage of fermentation, they serve as essential elements to sustain strain viability.

3.4. The Effect of Nutritional Components on the pH of Mulberry Juice Fermented by L. brevis S3

LAB are known for their ability to ferment carbohydrates into lactic acid, and their acid production capacity serves as a reflection of this fermentation ability. In this study, we aimed to assess the acid production capacity of L. brevis S3 by monitoring the pH changes of mulberry juice during fermentation. Initially, the mulberry juice had a pH of approximately 4.9 (Figure 6). The pH of the fermentation broth decreased over time. After 72 h of fermentation, both the control group and the additive groups exhibited a decrease in fermentation pH, ranging from 3.7 to 4.0. Specifically, the control group reached a pH of 4.08 ± 0.01, while the Y, P, M, Y + P, and Y + P + M groups reached pH values of 3.75 ± 0.02, 3.77 ± 0.01, 3.90 ± 0.01, 3.86 ± 0.01, and 3.71 ± 0.01, respectively. These findings indicate that L. brevis S3 effectively utilizes carbohydrates in mulberry juice to produce acid through fermentation. Notably, the fermentation pH environment promotes high GABA production by L. brevis S3, as the synthesis of GABA requires the catalysis of GAD and the maintenance of GAD enzyme activity in a low pH environment [49].

3.5. Effect of Nutritional Components on Bioactive Substances in Fermented Mulberry Juice

The abundance of bioactive substances in mulberries, such as polysaccharides, flavonoids, and polyphenols, contributes to their dietary and medicinal properties. To investigate the impact of nutritional components on the active constituents of L. brevis S3 fermented mulberry juice, we examined the levels of polysaccharides, flavonoids, and polyphenols in the fermentation broth. As shown in Figure 7a, the fermentation liquid exhibited a substantial decrease in the polysaccharide content, dropping from 63 to a minimum of 10 after inoculation with L. brevis S3 (Figure 7a). This finding demonstrates the significant reduction in the sugar content achieved by the addition of LAB. Furthermore, the groups supplemented with nutritional components exhibited notable reductions in the polysaccharide content compared to the control group, suggesting the possibility of preparing low-sugar beverages by incorporating suitable nutrient components to support bacterial growth.

As demonstrated in Figure 7b, the presence of the L. brevis S3 strain led to a decrease in the flavonoid content in mulberry juice, particularly in the Y + P and Y + P + M groups. In the control group, the initial decrease in the flavonoid content was followed by a significant increase after 72 h of fermentation, suggesting that L. brevis S3 can utilize the nutritional constituents in mulberry juice to produce more flavonoids. Similarly, in the Y, P, and M groups, the decrease in flavonoid levels was comparable to that observed in the control group. This indicates that the addition of L. brevis S3 and extending the fermentation period through the addition of Y, P, or M nutrient components can be effective in preparing mulberry functional beverages rich in flavonoids. In contrast, although the Y + P group and Y + P + M group exhibited relatively low levels of flavonoids, these levels remained relatively stable after 24 h of fermentation. This suggests that the addition of nutritional components has a comprehensive impact on LAB fermentation.

The findings depicted in Figure 7c reveal that the changes in the polyphenol content following the inoculation of L. brevis S3 align with the patterns observed for polysaccharides and flavonoids. Initially, there was a decrease in the polyphenol content within the first 24 h, followed by a relatively stable or slight increase thereafter. These trends suggest that the rapid growth of L. brevis S3 actively utilizes the bioactive components present in mulberries. Compared to the control group, the Y group resulted in a significant lack of decline in the polyphenol content after 24 h. This suggests that it is possible to craft mulberry fermented beverages with heightened levels of polyphenols by supplementing a yeast extract. In summary, the incorporation of different nutritional components induces a substantial impact on the functional components found in mulberry juice. Through the addition of a single nutrient, fermented mulberry beverages rich in polyphenols and flavonoids can be prepared. Conversely, complex nutrient additions containing nitrogen sources and metal ions can be utilized to produce probiotic fermented beverages with reduced sugar contents.

3.6. Influence of Nitrational Components on the Antioxidant Properties of Fermented Mulberry Juice

To evaluate the antioxidant capacity of mulberry juice fermented by L. brevis S3, we analyzed the total reducing power, hydroxyl radical scavenging ability, and DPPH free radical scavenging capacity of the fermented mulberry juice. As shown in Figure 8a, the total reducing power declined as the fermentation time increased after the L. brevis S3 inoculation. The control group exhibited an increase in the total reducing power from 1.39 ± 0.03 mg Vc/mL to 1.57 ± 0.02 mg Vc/mL after 72 h. The Y group showed an increase to 1.50 ± 0.08 mg Vc/mL, the P group increased to 1.45 ± 0.09 mg Vc/mL, the M group increased to 1.48 ± 0.01 mg Vc/mL, and the Y + P group increased to 1.39 ± 0.02 mg Vc/mL, corresponding to increments of 13%, 8%, 4%, 6%, and 1%, respectively. These findings indicate that the addition of nutritional components can enhance the total reducing power of mulberry juice following fermentation. However, in the Y + P + M group, the total reducing power decreased by 11%. This observation might be attributed to the rapid growth of L. brevis S3, which necessitates the utilization of a greater quantity of bioactive substances present in mulberries.

Likewise, as the fermentation time increased, the hydroxyl radical scavenging ability of both the control group and the groups with additives increased (Figure 8b). The hydroxyl radical scavenging abilities of the control group, Y group, P group, M group, Y + P group, and Y + P + M group reached 9.74 ± 0.15 mg Vc/mL (65%), 9.60 ± 0.54 mg Vc/mL (63%), 8.17 ± 1.43 mg Vc/mL (39%), 8.45 ± 0.17 mg Vc/mL (43%), 8.20 ± 1.65 mg Vc/mL (38%), and 7.04 ± 0.32 mg Vc/mL (19%), respectively (Figure 8b). These results indicate that the fermentation process involving L. brevis S3 significantly enhances the hydroxyl radical scavenging ability of mulberry juice. However, it is worth noting that the rate of increase for each additive group was lower than that of the control group. This discrepancy may be attributed to the utilization of active components in mulberry juice by each specific additive group.

Similarly, as shown in Figure 8c, with a prolonged fermentation time, both the control group and the additive groups displayed a substantial increase in DPPH radical scavenging ability. Particularly, after 72 h, the Y + P group and the Y + P + M group demonstrated a noteworthy enhancement in their DPPH radical scavenging ability, from 1.065 ±0.01 mg Vc/mL to 2.15 ± 0.05 mg Vc/mL (102%) and from 1.055 ± 0.04 mg to 2.76 ± 0.15 mg Vc/mL (162%), respectively. Supplementing nutrient components significantly augments the DPPH radical scavenging ability of mulberry juice. The inclusion of probiotic L. brevis S3 in mulberry juice leads to improvements in the total reducing power, hydroxyl radical scavenging ability, and DPPH scavenging ability of the fermented liquid, particularly with the extension of the fermentation time. Furthermore, the addition of composite nitrogen sources and composite nitrogen sources with metal ions markedly enhanced the DPPH radical scavenging ability of the mulberry fermented liquid. It is notable that the increase in DPPH radical scavenging ability achieved by L. brevis S3 in mulberry juice surpasses that of L. brevis F064A by 26.53% [34]. This suggests that L. brevis S3 serves as the dominant strain for fermenting mulberries.

3.7. Influence of Nutritional Components on GABA Production by L. brevis S3 in Mulberry Juice

The main objective of this study was to enhance the GABA content in fermented mulberry juice using the probiotic strain L. brevis S3. To investigate the impact of nutritional components on GABA production during mulberry juice fermentation, it is important to understand their effect on cell viability. While mulberry juice is already rich in essential amino acids, enriching its GABA content requires the addition of 10 g/L monosodium glutamate (MSG) as a precursor for GABA synthesis. We measured the GABA content of fermented mulberry juice with different additives (control group, Y group, P group, M group, Y + P group, and Y + P + M group) over a 72-h fermentation period. The results, shown in Figure 9, indicated a significant increase in the GABA content in mulberry juice with a prolonged fermentation time. GABA production reached 4.49 ± 0.09 g/L, 6.72 ± 0.44 g/L, 4.91 ± 0.04 g/L, 3.44 ± 0.61 g/L, 7.29 ± 0.01 g/L, and 7.48 ± 0.46 g/L in the respective groups. Compared to other fermented fruit juices, such as litchi juice with only 1.5 g/L GABA produced by L. plantarum HU-C2W and 3.3 g/L GABA produced by L. brevis F064A during the fermentation of Taiwanese mulberry juice for 48 h [34,50], L. brevis S3 is an excellent strain for GABA production during mulberry fermentation.

Figure 8. Effects of various nutrient components on the (a) total reducing power, (b) hydroxyl radical scavenging capacity, and (c) DPPH radical scavenging capacity of S3-fermented mulberry juice. Each experiment was biologically repeated three times. Each point is represented by three repeated mean values and standard deviations (±SDs). The small case letters were used for signification analysis.

Figure 8. Effects of various nutrient components on the (a) total reducing power, (b) hydroxyl radical scavenging capacity, and (c) DPPH radical scavenging capacity of S3-fermented mulberry juice. Each experiment was biologically repeated three times. Each point is represented by three repeated mean values and standard deviations (±SDs). The small case letters were used for signification analysis.

In previous studies, mulberry juice fermentation was conducted using a single culture of either Saccharomyces cerevisiae SC125, resulting in a production of 1.45 g/L GABA, or L. plantarum BC114, with a yield of 1.03 g/L GABA. However, when a coculture of starters was employed, the GABA content increased to 2.42 g/L [51]. This indicates the potential benefits of using multiple cultures together. Similarly, when compared to the control group, the yeast extract group, protein group, composite protein group, and composite protein with metal ion group exhibited increases in GABA production in mulberry fermented juice by 50%, 9%, 62%, and 67%, respectively. However, the M group did not increase the GABA content or cell survival in mulberry juice. It was likely that the mineral salts present in mulberry juice already fulfill the growth requirements of the bacterial strains, and the extra addition of metal ions might hinder their growth. The glutamate decarboxylase system, responsible for converting glutamate to GABA while consuming hydrogen ions, plays a crucial role in this process [52]. Since LAB generate a significant amount of acid from sugars in fruit juice, strains that synthesize GABA using the glutamate decarboxylase system consume a substantial amount of hydrogen ions. As a result, they are protected from acid stress. This explains why the strains with the addition of composite protein and metal ions exhibited the highest viability and maintained high activity.

It is worth mentioning that the use of almost residue-free glutamate in combination with the Y group, Y + P group, and Y + P + M group provides a reference for more consumers to accept mulberry fermented beverages. These GABA-enriched functional beverages serve as high-value-added products and contribute to the processing of high-sugar berries. However, it is important to note that the requirement of adding 2% sodium glutamate for L. brevis F064A to achieve a GABA yield of 3.3 g/L poses a challenge in the acceptance of mulberry fermentation liquids by consumers due to the presence of a significant amount of residual glutamate [34]. Furthermore, the probiotic strain L. brevis S3 used in this study demonstrates good probiotic effects and exhibits a tolerance to various conditions, such as gastric acid, gastrointestinal fluids, bile salts, osmotic stress, and phenol. The functional beverage prepared from fermented mulberries by L. brevis S3, together with compound nitrogen sources, metal ions, and other nutrient components, not only enriches the GABA content but also displays high antioxidant activity.

4. Conclusions

Lactobacillus brevis S3, a strain known for its high GABA production, is an ideal choice for fermenting mulberries due to its capabilities. It displays a remarkable tolerance to various gastric and intestinal components, as well as osmotic stress and phenol. These properties make it a valuable and promising probiotic strain with potential applications in the food industry. When nutritional components are added, L. brevis S3 can maintain high cell viability in mulberry juice while effectively reducing the polysaccharide content in mulberries. Furthermore, the addition of these components enhances the overall quality of fermented mulberry juice, increasing its total reducing power, hydroxyl radical scavenging ability, and DPPH radical scavenging ability. In particular, the combination of yeast extract, peptone, and metal ions led to a significant increase (162%) in DPPH radical scavenging activity. Additionally, this fermentation process achieved the highest GABA production of 7.48 g/L, which represents the maximum value for preparing GABA-rich berry beverages. These findings provide evidence that the probiotic strain L. brevis S3, when combined with the addition of food-safe nutritional components, enables the production of low-sugar functional beverages with a high DPPH radical scavenging capacity and enriched with GABA.