Development of Probiotic-Fermented Black Mulberry (Morus nigra L.) Juice and Its Antioxidant Activity in C2C12 Cells

Abstract

Black mulberry (Morus nigra L.) is considered a medicinal and food-homologous plant in China. An obstacle to its widespread use is that its annual season and shelf life are extremely short. In this paper, fermented black mulberry juice (FBMJ) was prepared with various probiotic strains, and response surface methodology was used to determine the optimum production conditions for achieving the maximum active substance content in the resulting product. The fermentation process increased levels of biological enzymes and total phenols in the resulting juice. When the ratios of the Lactobacillus inocula to the total inoculum were 27.96% for L. paracasei, 15.37% for L. casei, 16.64% for L. plantarum, and 5.12% for L. delbrueckii, the B. animalis subsp. lactis content reached 15.83%, the L. fermentum content reached 19.08%, and the superoxide dismutase (SOD) activity reached 310 U/g. To study the antioxidant characteristics of the juice, C2C12 cells were treated with H₂O₂ to induce oxidative stress and the cytoprotective activity of FBMJ was subsequently evaluated. After treatment with FBMJ for 24 h, cell viability was found to be protected under H₂O₂ exposure, while SOD and glutathione peroxidase (GSH-Px) levels were increased. The reactive oxygen species level and malondialdehyde content were also decreased. These results provide molecular evidence for the antioxidant effect of FBMJ and demonstrate that lactic acid bacteria fermentation has a positive effect on black mulberry juice (BMJ).

1. Introduction

As one of the main corridors of the ancient Silk Road, Xinjiang in northwest China has a long history of sericulture and contains a preserved population of a wide variety of mulberry trees. These include black mulberry (Morus nigra L.), which was first developed in Iran and later planted in the southern Xinjiang region. The species only has a two-week harvest period in August each year [1]. In China, black mulberry is a medicinal and food-homologous material used in traditional Chinese medicine. It comprises a rich source of natural antioxidants, such as polyphenols, flavonoids, organic acids, pectin, and other carbohydrates [2]. These components have been found to exert direct and indirect effects on diabetes [3], oxidative stress [4], inflammation, tumor, hepatic diseases, immunomodulation, hyperlipidemia [5], neural damage [6], and chronic diseases [7], either synergistically with other materials or in their pure form. Unfortunately, black mulberry is a seasonal fruit that features a fragile pulp and high sugar content, thereby making it extremely difficult to preserve, store, and transport.

In recent years, researchers have demonstrated that lactic acid bacteria fermentation is a cheap and effective value-adding processing method for edible substances [8]. The fermentation process produces organic acids and other bioactive compounds that help extend the shelf life of fresh juices [9]. Furthermore, the pharmacological activities of many bioactive substances become enhanced under the action of probiotic biotransformation [10]. Tang et al. [11] used Lactobacillus plantarum to ferment mulberry pomace and found that its antioxidant activity was greatly improved. Kim et al. [12] used lactic acid bacteria to biotransform mulberries and found that the fermented mulberries had enhanced antibacterial and anti-inflammatory activities. Meanwhile, compared to single-strain fermentation, the metabolic mechanism of mixed-strain fermentation is more complex and produces a higher abundance of fermentation products [13]. Due to the different characteristics and substrate adaptability of individual strains, it is necessary to select relevant dominant bacteria that are beneficial to the fermentation of black mulberry in order to optimize the process of mixed bacterial fermentation.

In this paper, several fermented black mulberry juice (FBMJ) products containing different lactic acid bacteria were developed. Firstly, 6 bacterial strains with high acid production capacity, SOD production capacity, total phenol production capacity, and the ability to produce a desirable sensory evaluation profile were screened by a single-factor test, and the inoculum concentration was determined using a unified design test. Subsequently, based on the preliminary experiments, the fermentation process was optimized based on SOD activity and total phenol content using response surface methodology [14]. In addition, different concentrations of FBMJ were used to protect cells subjected to hydrogen peroxide-induced oxidative stress, and the effect of different doses of BMJ and FBMJ on levels of cellular reactive oxygen species (ROS) in C2C12 cells was determined by flow cytometry. SOD activity, glutathione peroxidase (GSH-Px) activity, and the malondialdehyde (MDA) content in the cells were measured to study the mechanism by which FBMJ prevents damage caused by oxidative stress.

2. Materials and Methods

2.1. Materials

Fresh black mulberries were purchased from a shop at a Kuqa market, Xinjiang, China. Ethanol, gallic acid, and Folin-Ciocalteu phenol were obtained from Tianjin Shengao Chemical Reagent Co., Ltd. (Tianjin, China). The MRS (DeMan, Rogosa and Sharpe) culture medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd. (Beijing, China). Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Weissella spp., Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus salivarius, Lactobacillus brevis, Leuconostoc lactis, Pediococcus acidilactici, Pediococcus pentosaceus, Leuconostoc mesenteroides, Wickerhamomyces anomalus, Kluyveromyces lactis, Streptococcus lactis, and Lactobacillus johnsonii, Bifidobacterium animalis subsp. lactis were obtained from the China Microbial Culture Preservation Center (Beijing, China). Pectinase, celluclast, and hemicellulase were purchased from Novozymes^®, Ltd. (Beijing, China). All strains were activated twice and incubated at 37 ℃ for 24 h before use. C2C12 cells were obtained from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China). Pectinase, cellulase, and hemicellulase were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and phosphate- buffered solution (PBS) were purchased from Biological Industries Company (Shanghai, China). Trypsin-EDTA (0.05%) and dimethyl sulfoxide (DMSO) were purchased from HyClone Corporation (Logan, UT, USA). Assay kits, including SOD, glutathione peroxidase (GSH-Px), and malondialdehyde (MDA), were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.2. Preparation of Black Mulberry Juice and Inoculum

Black mulberries were washed with distilled water to remove dust and surface impurities. Then, the black mulberries were ground into a homogenate with a beater. Next, 0.2% (w/v) pectinase, 0.1% (w/v) cellulase, and 0.1% (w/v) hemicellulase were added to induce enzymatic hydrolysis for 3.5 h at 55 °C. The digested juice was pasteurized in a 70 °C water bath for 20 min and then cooled to room temperature. A volume of the strain suspension was added to the homogenate. Finally, the juice was fermented for 18 h at 37 °C.

2.3. Selection of the Fermentation Strains and Determination of their Proportions

Twenty strains were used to ferment the black mulberry homogenate, respectively. The monoculture inoculum was maintained at a bacterial concentration of 1 × 10⁶ CFU/mL, and the total soluble solid (TSS) content was adjusted to 18.5 °Brix. Then, fermentation was carried out in a 37 °C incubator for 20 h. After fermentation, the pH, SOD activity, total phenol concentration (TPC), and sensory score were determined for each resulting product, and the dominant strains were selected based on assessments of SOD, acid production capacity, TPC, and sensory score. The optimization test used a uniform design table U10 × (10⁸), which is described as follows. Ten levels of six dominant strains in the total inoculum volume were set as factors. The uniform design table is shown in

Table 1. Uniform design table for the experiment.

No.	L. paracasei X1 (%)	L. casei X2 (%)	L. plantarum X3 (%)	L. delbrueckii X4 (%)	B. animalis X5 (%)	L. fermentum X6 (%)
1	0.15	0.02	0.08	0.08	0.13	0.34
2	0.16	0.04	0.11	0.13	0.09	0.33
3	0.17	0.06	0.14	0.07	0.16	0.32
4	0.18	0.08	0.06	0.12	0.12	0.31
5	0.19	0.10	0.09	0.06	0.08	0.30
6	0.20	0.01	0.12	0.11	0.15	0.29
7	0.21	0.03	0.15	0.05	0.11	0.28
8	0.22	0.05	0.07	0.10	0.07	0.27
9	0.23	0.07	0.10	0.04	0.14	0.26
10	0.24	0.09	0.13	0.09	0.10	0.25

. SOD activity was used as the response value and SPSS 26.0 software was used to conduct a quadratic polynomial stepwise regression analysis to obtain the fermentation regression equation, with maximum SOD activity as the target. The equation was then solved using programming solver functions available in Excel. Finally, the percentages of the six strains in the inoculum were optimized.

2.4. Optimization of the FBMJ Fermentation Process Using Response Surface Methodology (RSM)

After the fermentation strains and ratios were determined, SOD activity and TPC values obtained after fermentation were used as indicators. The single-factor experimental conditions for FBMJ production were as follows: soluble solid content (18.5, 19, 19.5, 20, and 20.5 °Brix), inoculum volume (1, 3, 5, 7, 9, and 12 × 10⁶ CFU/mL), fermentation temperature (27, 32, 37, 42, and 47 °C), and fermentation time (3, 6, 9, 12, 15, 18, 21, and 24 h).

Based on the single-factor test, optimization (

Table 2. Uncoded and coded levels of independent variables and combined model design expert test matrix and experimental data for responses in FBMJ.

Factors			Code	Levels
				−1	0	1
Fermentation time (h)			A	12	15	18
Fermentation temperature (°C)			B	32	34.5	37
Soluble solid content (°Brix)			C	3	6	9
Inoculum concentration			D	19	19.5	20
Run	Fermentation time (h)	Fermentation temperature (°C)	Inoculum concentration (106 CFU/mL)	Total soluble solid content (oBrix)	SOD (U/g)	TPC (mg/g)
1	12	32	6	19.5	214.88	7.68
2	18	32	6	19.5	288.90	8.31
3	12	37	6	19.5	256.23	7.53
4	18	37	6	19.5	308.28	8.07
5	15	34.5	3	18	275.44	8.23
6	15	34.5	9	18	296.78	8.78
7	15	34.5	3	20	286.20	8.62
8	15	34.5	9	20	322.79	9.09
9	12	34.5	6	18	250.56	7.65
10	18	34.5	6	18	305.23	8.47
11	12	34.5	6	20	275.89	8.12
12	18	34.5	6	20	320.98	8.93
13	15	32	3	19.5	254.11	8.17
14	15	37	3	19.5	275.36	8.16
15	15	32	9	19.5	285.56	8.75
16	15	37	9	19.5	311.11	8.39
17	12	34.5	3	19.5	243.34	7.78
18	18	34.5	3	19.5	285.68	8.43
19	12	34.5	9	19.5	277.60	8.22
20	18	34.5	9	19.5	325.10	8.84
21	15	32	6	18	265.78	8.31
22	15	37	6	18	285.98	8.02
23	15	32	6	20	280.45	8.57
24	15	37	6	20	305.80	8.31
25	15	34.5	6	19.5	317.96	8.7
26	15	34.5	6	19.5	324.80	9.06
27	15	34.5	6	19.5	311.23	9.03
28	15	34.5	6	19.5	315.22	8.83
29	15	34.5	6	19.5	319.56	8.93

) was performed using RSM [15,16]. The Box–Behnken test was designed to obtain higher quality FBMJ. The factors and levels of the Box–Behnken test design are shown in Table 2.

2.5. Measurement of Physicochemical Indicators

The total soluble solid (TSS) content was measured using a handheld Brix meter. SOD activity was determined according to the instructions provided by the Nanjing Jiancheng kit. TPC was measured using the method reported by Rosl et al. [17], with the following modifications: in the pretreatment step, 85% anhydrous ethanol was slowly added to the juice and mixed with a vortex oscillator, which was followed by ultrasonic extraction carried out for 30 min at 25 °C in an ultrasonic extractor. After extraction, the supernatant was collected by centrifugation at 6000 r/min for 15 min. The resulting centrifugal precipitate was ultrasonically extracted with 85% ethanol 2–3 times, centrifuged at 6000 r/min for 15 min, combined with the supernatant, and stored in a 100 mL volumetric bottle. This ensured that the oligosaccharide components in each sample were removed. The TPC was calculated from the standard curve equation. Sensory evaluation was performed using the method reported by Liu et al. [8] combined with an assessment of the liquid-fermented juice sensory characteristics. Ten people who had received sensory evaluation training rated the appearance, flavor, smell, taste, and acceptability of the FBMJ products. During training and evaluation, approximately 20 mL of the respective BMJ samples were randomly served at room temperature. Water and white bread were provided for palate cleansing. Sensory evaluation of the 21 samples was carried out in duplicate. Training and evaluation were conducted in accordance with the guidelines of the International Organization for Standardization (I.S.O., 1993) in a sensory laboratory fulfilling the requirements provided by the American Society for Testing and Materials. The sensory evaluation scores are shown in

Table 3. Sensory attributes for fermented black mulberry juices.

Category	Attributes	Score
Appearance	Red color	0–10
	Violet/black color	0–10
	Clarity	0–10
Flavor	Black mulberry flavor	0–10
	Sour flavor	0–10
	Wine bouquet	−10–0
Taste	Sweetness/Sourness (acid-sugar ratio)	0–10
	Astringency	0–10
	Black mulberry taste	0–10
	Wine taste	−10–0
Overall	Acceptability	0–10
	Purchase intention	0–10

Note: 0–3: the characteristic was difficult to perceive; 4–6: the characteristic was moderately perceived; 7–10: the characteristic was strongly perceived.

. It is noteworthy that the fermented juices produced in this study had points deducted if they had an alcoholic flavor (there is a clear limit on the degree of alcohol contained in products in China’s JIAOSU standards).

2.6. Analysis of Cell Viability with the CCK-8 Assay

CCK-8 is a low-toxicity, high-sensitivity, and rapid detection kit based on WST-8, which is widely used to study cell proliferation and toxicity [18]. The effect of FBMJ on the viability of C2C12 cells was detected using the CCK-8 method. The CCK-8 experimental operation steps were as follows. The cell suspension was inoculated in a 96-well plate with 200 μL per well at a cell density of 6 × 10⁵ cells/well. BMJ and FBMJ were then added at concentrations of 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 mg/mL to different well plates, respectively. Six duplicate wells were set for each concentration of each monomer, and blank control wells were set up in tandem with only medium added. Following administration of the treatments, the well plates were placed in an incubator. After culturing in the incubator for 6 h, the well plates were removed and quickly aspirated from the treated cell culture medium. Then, a volume of 100 μL of high-glucose medium (without serum) and 10 μL of CCK-8 solution were added per well. No air bubbles were generated during the process of changing the medium and adding the CCK-8 solution. The 96-well plate containing CCK-8 solution was then placed in an incubator for 2 h. A Bio-Tek microplate reader was used to measure the optical density (OD) value of each well at a wavelength of 450 nm. Cell viability was calculated using the following formula:

cell viability(%) = (A_Sample − A_Blank)/(A_Control − A_Blank) × 100%.

(1)

The groups were treated as follows:

(i) sample group: the OD value was taken from wells containing FBMJ-treated cells and CCK-8 solution.

(ii) blank group: the OD value was taken only from wells containing medium and CCK-8 solution.

(iii) control group: the OD values were taken from wells containing cells and CCK-8 solution without treatment.

2.7. Cell Culture and Treatment

C2C12 cells were maintained in DMEM medium containing penicillin (100 U/mL), streptomycin (100 g/mL), and 10% FBS under an atmosphere of 95% relative humidity and 5% CO₂ at 37 °C. The medium was changed every other day. The cells were divided into two groups: cells treated with H₂O₂ (oxidative stress model group) or left untreated (control group). The appropriate concentration of H₂O₂ was selected by treating cells with different concentrations of H₂O₂ (0, 125, 250, 500, 1000, and 2000 μmol/L) for 12, 24, and 48 h, and then evaluating cell viability using the CCK-8 assay (Section 2.6). In cells treated with 500 μmol/L H₂O₂ for 24 h, the cytotoxicity was approximately 27%; therefore, this condition was subsequently used for the experimental induction of oxidative stress.

To investigate the cytoprotective effect of FBMJ against oxidative damage, C2C12 cells were cultured in a 96-well microplate at 6 × 10⁵ cells/well for 24 h and then divided into the following eight groups: control (culture medium only), H₂O₂ (culture medium for 24 h + H₂O₂ for 24 h), BMJ (0.5, 1, or 2 mg/mL for 24 h) + H₂O₂ (for 24 h), and FBMJ (0.5, 1, or 2 mg/mL for 24 h) + H₂O₂ (for 24 h).

2.8. Evaluation of Antioxidant Activity

The cell suspensions were centrifuged at 1200 rpm for 4 min at 37 °C. The activities of MDA, SOD, and GSH-Px were evaluated using a commercial assay kit (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s instructions. Intracellular ROS generation was detected by staining with DCFH-DA (BL714A; Biosharp; Anhui, China). Briefly, C2C12 cells were seeded in a 6-well plate (6 × 10⁵ cells/well) and cultured for 24 h. Following the different treatments, the cells were washed with phosphate-buffered saline and incubated with DCFH-DA (at a final concentration of 10 vM) in complete medium at 37 °C for 30 min. The cells were then analyzed by flow cytometry (CytoFLEX; Beckman Coulter, Brea, CA, USA).

2.9. Statistical Analysis

To obtain higher quality FBMJ, optimization (Table 2) using RSM was performed employing Design-Expert software (version 13). Box-Behnken design (BBD) was applied to determine the impact of the four independent variables: fermentation time (A), fermentation temperature (B), soluble solid content (C), and inoculum volume (D). SOD activity (Y₁) and TPC (Y₂) were set as responses for the FBMJ. The statistical significance of the terms in the regression equations was examined. The significant terms in the model were determined by analysis of variance (ANOVA) for each response (

Table 4. Analysis of variance of SOD activity and TPC in FBMJ.

	SOD Activity (U/g)		TPC (mg/g)
Source	F-Value	p-Value	F-Value	p-Value
Model	52.81	<0.0001 **	34.45	<0.0001 **
A	286.42	<0.0001 **	126.09	<0.0001 **
B	67.36	<0.0001 **	13.06	0.0028 *
C	113.61	<0.0001 **	54.67	<0.0001 **
D	36.28	<0.0001 **	36.17	<0.0001 **
AB	4.16	0.0607	0.18	0.6737
AC	0.23	0.6392	0.021	0.8881
AD	0.79	0.3887	2.28×10−3	0.9626
BC	0.16	0.6957	2.8	0.1166
BD	0.23	0.6399	0.021	0.8881
CD	2.01	0.1786	0.15	0.708
A2	118.81	<0.0001 **	154.61	<0.0001 **
B2	146.32	<0.0001 **	136.98	<0.0001 **
C2	33.54	<0.0001 **	3.99	0.0655
D2	14.84	0.0018 **	9.57	0.0079 **
Lack of Fit	1.19	0.4688 Ns	0.3	0.9450 Ns
R2	0.9814	-	0.9718	-
Adj. R2	0.9628	-	0.9436	-
Pred. R²	0.9125	-	0.9054	-
Adeq Precision	26.4759	-	21.1733	-
C.V. %	1.86	-	1.24	-

Note: A: fermentation time (h), B: fermentation temperature (°C), C: soluble solid content (°Brix), and D: inoculum concentration. * significant at 95% confidence interval. ** significant at 99% confidence interval. Ns, non-significant at 95% confidence interval.

). The reliability of the model was based on the coefficient of determination (R²) and adjusted coefficient of determination (R²-adj). Analysis of the significance of each factor under single fermentation with different strains and analysis of variance (ANOVA) of the uniform design regression equation were performed using SPSS software (version 26.0) (IBM Corp., Armonk, NY, USA). The significance analysis of the cell assay content was performed using the t-test in Prism software (version 8.0.2.) (GraphPad, San Diego, CA, USA). All figures were drawn using Prism v8.0.2 software. All experiments were conducted using three or more parallel experiments and repeated three times.

3. Results

3.1. Selection of Fermentation Strains and Determination of the Proportions of Strains

In this experiment, 20 strains were used to ferment BMJ and the highest values of SOD activity, TPC, and sensory evaluation scores were determined during the fermentation process. The moderate sweet-acid ratio was determined by pH and TSS values. Six strains were ultimately selected for mixed fermentation (

Table 5. Physicochemical properties of MBJ fermented by twenty strains.

No.	Strains	SOD Activity (U/g)	TPC (mg/g)	pH	Sensory Evaluation (Score)	Time to Reach 108 CFU/mL (h)
1	Black mulberry juice	255.19 ± 0.17n	7.14 ± 0.01g	3.43 ± 0.01defg	80.00 ± 0.00ef	/
2	L. plantarum	290.70 ± 0.31c	7.66 ± 0.35de	3.41 ± 0.01ijk	88.33 ± 0.58b	21.03 ± 0.06c
3	L. casei	278.60 ± 0.52e	8.16 ± 0.02b	3.43 ± 0.01efghi	81.00 ± 1.00e	18.17 ± 0.29i
4	L. paracasei	305.36 ± 0.39b	7.89 ± 0.06c	3.44 ± 0.01cbef	80.33 ± 0.58e	19.33 ± 0.29gh
5	Weissella spp.	306.59 ± 0.52a	7.75 ± 0.05cde	3.38 ± 0.01l	70.00 ± 1.00ij	17.33 ± 0.29j
6	L. fermentum	275.36 ± 0.45g	8.51 ± 0.22a	3.41 ± 0.01ijk	80.33 ± 0.58e	17.17 ± 0.29j
7	L. acidophilus	280.59 ± 0.55d	7.65 ± 0.04de	3.38 ± 0.01l	70.00 ± 0.00j	18.50 ± 0.00i
8	L. reuteri	257.63 ± 0.55l	7.59 ± 0.85e	3.45 ± 0.01abc	81.00 ± 1.00e	23.33 ± 0.29b
9	L. delbrueckii subsp. bulgaricus	266.63 ± 0.55i	8.15 ± 0.49b	3.44 ± 0.01bcd	85.00 ± 1.00c	20.17 ± 0.29e
10	L. helveticus	263.40 ± 0.62j	8.09 ± 0.26b	3.44 ± 0.00cde	78.33 ± 0.58g	21.00 ± 0.00d
11	L. salivarius	267.07 ± 0.67i	7.10 ± 0.17g	3.43 ± 0.01defgh	81.00 ± 1.00e	18.17 ± 0.29i
12	L. brevis	271.35 ± 0.56h	7.69 ± 0.16de	3.42 ± 0.01hijk	72.33 ± 0.58i	19.00 ± 0.00h
13	Leuconostoc lactis	255.40 ± 0.63n	7.77 ± 0.02cd	3.41 ± 0.01ijk	82.67 ± 0.58d	21.83 ± 0.29c
14	P. acidilactici	279.47 ± 0.45e	7.16 ± 0.01fg	3.42 ± 0.01fghij	80.00 ± 0.00ef	23.33 ± 0.29b
15	P. pentosaceus	275.52 ± 0.46g	7.15 ± 0.03fg	3.46 ± 0.01a	82.67 ± 0.58d	19.50 ± 0.00fg
16	L. mesenteroides	256.53 ± 0.46m	8.13 ± 0.32b	3.40 ± 0.01k	77.67 ± 0.58g	26.17 ± 0.29a
17	W. anomalus	277.26 ± 0.23f	7.67 ± 0.19de	3.41 ± 0.01ijk	65.68 ± 0.58l	21.50 ± 0.00c
18	K. lactis	231.45 ± 0.39p	8.13 ± 0.32b	3.41 ± 0.01jk	67.68 ± 0.58k	20.83 ± 0.29d
19	S. lactis	260.20 ± 0.18k	7.70 ± 0.26de	3.42 ± 0.01ghij	78.67 ± 0.58fg	19.33 ± 0.29gh
20	L. johnsonii	252.48 ± 0.45o	7.32 ± 0.46f	3.43 ± 0.01defg	75.00 ± 0.00h	19.83 ± 0.29ef
21	B. animalis subsp. lactis	271.30 ± 0.34h	7.17 ± 0.42fg	3.46 ± 0.01ab	90.33 ± 0.58a	21.50 ± 0.00c

Note: Data are expressed as means ± SDs. Different letters next to data in the same column indicate significant differences between data (p < 0.05).

).

SOD has a strong free radical scavenging ability. BMJ is naturally rich in SOD, which is also produced in the process of lactic acid bacteria metabolism [19]. Table 5 shows that the bacteria that generated the highest SOD values were Weissella spp., L. paracasei, L. plantarum, and L. acidophilus; their SOD values increased by 51.4, 50.17, 35.51, and 25.4 U/g, respectively, compared to the black mulberry stock solution (255.19 U/g). Fermentation by L. reuteri and L. mesenteroides showed an effect on SOD activity, but L. reuteri and L. mesenteroides exhibited the longest times to proliferate to 8 × 10⁶ CFU/mL, thereby demonstrating that their growth ability in BMJ was inferior to that of the other strains.

Mulberry (Morus) fruit contains high levels of anthocyanins, quercetin glycosides, and chlorogenic acid. Mulberry polyphenols have been shown to confer benefits to human health. Different fermentation environments produce different total phenolic contents [20]. The bacteria that generated the greatest increase in total phenolic content were L. fermentum, L. delbrueckii, and L. casei, with increases of 1.37, 1.01, and 1.02 mg/g, respectively.

Lactobacillus strains feature diverse metabolisms. Among them, L. delbrueckii [21], L. acidophilus, and P. acidilactici utilize sugar to produce lactic acid and galactose, while L. mesenteroides [22], L. brevis, L. fermentum, and B. animalis subsp. lactis [23] further use acid as a carbon source to produce metabolites such as carbon dioxide, ethanol, and acetic acid. The strains L. casei, L. paracasei, and L. plantarum [24] are not as capable of producing lactic acid as these previous types, but they also produce lactic acid.

The sensory score was affected by the ratio of sourness to sweetness and various flavor substances. The bacteria that yielded the highest sensory scores were B. animalis subsp. lactis, L. plantarum, L. delbrueckii subsp. bulgaricus, P. pentosaceus, and L. reuteri, with scores of 90.33, 88.33, 85.00, 82.67, and 81.00, respectively. Among them, the pH value of the fermentation broth produced by Weissella spp. and L. acidophilus were both 3.38. These two strains exhibited a strong acid-producing ability, which underlied their lower sensory scores, as high acidity destroys the sweet-acidity of the resulting fermentation broth.

Therefore, based on high TPC, SOD, and sensory scores, two optimal bacteria were selected for each factor. The selected bacteria were chosen based on not conferring negative effects on the other factors. Thus, L. paracasei, L. casei, L. fermentum, L. delbrueckii, L. plantarum, and B. animalis subsp. lactis were determined to be the optimal fermentation strains to ferment BMJ.

Studies have shown that the SOD content in mixed-bacteria fermentation broth is higher than that produced by single-bacteria fermentation [19]. This experiment was conducted using a uniform design table (

Table 6. Results of uniform design experiments.

No.	L. paracasei X1 (%)	L. casei X2 (%)	L. plantarum X3 (%)	L. delbrueckii subsp. bulgaricus X4 (%)	B. animalis subsp. lactis X5 (%)	L. fermentum X6 (%)	SOD Activity (U/g)
1	0.15	0.02	0.08	0.08	0.13	0.34	241.13
2	0.16	0.04	0.11	0.13	0.09	0.33	229.08
3	0.17	0.06	0.14	0.07	0.16	0.32	268.01
4	0.18	0.08	0.06	0.12	0.12	0.31	243.12
5	0.19	0.10	0.09	0.06	0.08	0.30	261.06
6	0.20	0.01	0.12	0.11	0.15	0.29	250.73
7	0.21	0.03	0.15	0.05	0.11	0.28	273.12
8	0.22	0.05	0.07	0.10	0.07	0.27	245.21
9	0.23	0.07	0.10	0.04	0.14	0.26	289.14
10	0.24	0.09	0.13	0.09	0.10	0.25	279.15

). The response values were obtained by mixed bacterial fermentation according to the fermentation process conditions described in Section 2.3.

According to the uniform design of the experimental plan, the quadratic polynomial stepwise regression equation was established using SOD activity as the response value, as follows:

Y = −222.233 − 417.105X₄ + 482.766X₁² + 238.677X₁X₃ + 974.6674X₁X₅ + 81.662X₁X₆ + 1740.59X₂X₃ + 450.63X₄X₆

(2)

p = 0.0098 < 0.01 in the regression equation, thereby confirming that the equation had a good degree of fit. In summary, the optimal inoculation ratios of FBMJ were predicted to be L. paracasei (27.96%), L. casei (15.37%), L. plantarum (16.64%), L. delbrueckii (5.12%), B. animalis subsp. lactis (15.83%), and L. fermentum (19.08%). SOD activity reached 346.18 U/g. In the verification test, SOD activity reached 310 U/g, which was higher than that produced by single-strain bacterial fermentation.

3.2. Response Surface Experimental Results and Analysis of Variance

The lactic acid bacteria in the FBMJ required 6 h to reach the logarithmic growth phase and 12 h to reach the stable phase. The fermentation products in juice increased over time. As shown in Figure 1A, SOD activity rapidly increased from 3–15 h and reached its maximum value (314 U/g) at 15 h. Thereafter, SOD activity showed a slowly decreasing trend. The concentration of total phenols reached 8.98 mg/g at 18 h, and then decreased to 8.53 mg/g with increasing time. Therefore, we chose a fermentation time of 15 h for subsequent study.

The fermentation temperature influences the metabolic activity of lactic acid bacteria, which in turn affects the fermentation quality [25]. As can be seen in Figure 1B, SOD activity reached its highest value (309 U/g) at 37 °C and then showed a decreasing trend with increasing temperature. The concentration of total phenols gradually stabilized at 8.15 mg/g at 37 °C and then showed a rapid downward trend above 37 °C. At high temperatures, bacterial intolerance to heat leads to a decrease in enzyme metabolism and activity [26], which consequently affects the biotransformation of phenolic substances in the system. Moreover, phenolic substances are readily degraded at high temperatures. Therefore, 37 °C was chosen as the optimal fermentation temperature.

As shown in Figure 1C, when the inoculum concentration reached 6 × 10⁶ CFU/mL, the growth and metabolism rate of the lactic acid bacteria was moderate, SOD activity reached its maximum value (308 U/g), and TPC also reached its maximum value (8.57 mg/g). There was no significant difference between the inoculum containing 6 × 10⁶ CFU/mL and that containing 9 × 10⁶ CFU/mL. After having reached 1.2 × 10⁷ CFU/mL, the lactic acid bacteria rapidly proliferated and produced acid, thus affecting SOD activity. Appropriately increasing the content of lactic acid bacteria is conducive to the conversion of macromolecular phenols into small molecular phenols by microorganisms and can also increase the concentration of total phenols [27]. However, when the inoculum content is too high, this can cause instability in the phenolic substances and loss of biological enzyme activity. Therefore, the optimum inoculum concentration was set as 6 × 10⁶ CFU/mL.

As shown in Figure 1D, SOD activity and total phenol content showed an upward trend with increasing added sugar. When the TSS content was adjusted to 19.5 °Brix, SOD activity reached its maximum value (306 U/g) and TPC reached 8.58 mg/g. From these results, we selected the TSS value of 19.5 °Brix.

On the basis of the single-factor experiment and according to the Box-Behnken central combinatorial design principle, the fermentation time (A), fermentation temperature (B), inoculum amount (C), and total soluble solid content (D) were selected as response surface optimization factors, while SOD activity (Y₁) and total phenol content (Y₂) were selected as optimization factors. The experimental results are shown in Table 2.

The values of the regression coefficients were calculated and the test variables were related by the following second-order polynomial equations:

Y₁ = 317.75 + 26.31A + 12.76B + 16.57C + 9.36D−5.49AB + 1.29AC − 2.39AD + 1.08BC + 1.29BD + 3.81CD − 23.04A² − 25.57B² − 12.24C² − 8.140D²

(3)

Y₂=8.91 + 0.34A − 0.11B + 0.27C + 0.22D − 0.022AB − 7.500E − 003AC − 2.500E − 003AD + 0.087BC + 7.500E − 003BD − 0.02CD − 0.51A² − 0.48B² − 0.082C² − 0.13D²

(4)

The analysis of variance and significance test results for SOD activity and TPC are shown in Table 4. The final results in terms of R², adjusted-R², C.V.%, and adequate precision are shown in Table 4.

The model regression equation coefficient significance test of Y₁ (Figure 2) showed that the primary terms A, B, C, and D were highly significant; the interaction terms AB, AC, AD, BC, BD, and CD were not significant; and the quadratic terms A², B², C², and D² were highly significant. The Y₂ model (Figure 2) regression equation coefficient significance test showed that the primary terms A, B, C, and D were highly significant; the interaction terms AB, AC, AD, BC, BD, and CD were not significant; the quadratic terms A², B², and D² were highly significant; and the C² term was not significant.

It can be seen from Table 2 that the two regression equation models were highly significant (p < 0.0001), while the result of the lack of fit test was not significant. The correlation coefficient R² value for SOD activity was 0.9814 and the R² value for TPC was 0.9718, indicating a good degree of fit between the measured value and the predicted value and that the process parameters of FBMJ could be analyzed and predicted.

Through the prediction of the regression model, the optimal processing of FBMJ was obtained as follows: a fermentation time of 15.84 h, fermentation temperature of 34.41 °C, lactic acid bacteria inoculum of 8.16 × 10⁶ CFU/mL, and sugar addition of 1.85 °Brix. At this time, the predicted value of the SOD activity of the fermented mulberry enzymes was 332.67 U/g and the predicted value of TPC was 9.14 mg/g.

After comprehensive consideration, the optimal conditions were revised to a fermentation time of 16 h, fermentation temperature of 35 °C, lactic acid bacteria inoculum of 8.2 × 10⁶ CFU/mL, and sugar addition of 1.85 °Brix. Under these conditions, three parallel experiments were performed to verify that the SOD activity of FBMJ was 328.15 U/g and the total phenolic concentration was 9.12 mg/g. Compared with the predicted values, the error values of SOD activity and TPC were 1.16% and 0.24%, respectively, indicating that the model was effective.

3.3. Fermentation Kinetics and Specific Rate of FBMJ

As can be seen from Figure 3A, after 16 h, due to the reduced sugar content, and the increased total acid inhibited the growth of the bacteria and the specific growth rate of Lactobacillus spp. decreased. The Lactobacillus bacteria entered the stable period after 18 h and the highest number of lactic acid bacteria reached 8.79 × 10⁸ CFU/mL. As can be seen from Figure 3B, the initial growth rate was slow, thereafter gradually becoming faster following the adaptation period. Then, the growth rate gradually declined to zero. As shown in the figure, the specific growth rate of the lactic acid bacteria exhibited a bell-shaped curve. The generation of total acid content in the FBMJ showed a strong correlation with bacterial body growth and presented a growth-coupling-type relationship. Combined with the reducing sugar consumption rate curve shown in Figure 3A, the specific consumption rate was inversely correlated with the specific growth rate of Lactobacillus spp.

3.4. Antioxidant Activity

3.4.1. Effects of BMJ and FBMJ on H₂O₂-Induced Cytotoxicity in C2C12 Cells

To further evaluate the antioxidant activity of BMJ and FBMJ, mouse C2C12 cells were used to establish a cell model of oxidative stress. Hydrogen peroxide (H₂O₂) is an unstable chemical with high oxidative capacity that induces intracellular redox interference [28].

In this study, we used H₂O₂-treated C2C12 cells to construct an in vitro oxidative stress model to evaluate the antioxidant function of FBMJ. After 24 h of treatment with either BMJ or FBMJ at concentrations of 0, 0.25, 0.5, 1, 2, 4, 8, and 10 mg/mL, C2C12 cell viability was determined using the CCK-8 method (Figure 4). As shown in Figure 4, FBMJ concentrations below 4 mg/mL had no apparent toxic effect on the cells, and instead showed a certain promoting effect on cell proliferation. When the concentration of FBMJ was 1 mg/mL, the cell viability increased by 12.96%. In the subsequent oxidative damage protection experiments, 0.5–2 mg/mL of BMJ and FBMJ were selected as the experimental concentrations with an administration time of 24 h.

Treatment of the model group with only 500 μmol/L H₂O₂ for 24 h resulted in a 24.85% decrease in cell viability (p < 0.001). In the drug group, the cells were exposed to different concentrations (0.5, 1, and 2 mg/mL) of either BMJ or FBMJ for 24 h and then treated with 500 μmol/L H₂O₂ for 24 h, whereby cell viability increased in a dose-dependent manner (Figure 5). Compared to the model group, the drug groups administered concentrations of 0.5 mg/mL BMJ and 1 mg/mL FBMJ exhibited the most significant protective effects against oxidative stress, whereby the cell viability of the two groups increased by 25.8% and 30.22%, respectively. (p < 0.001). These results suggested that both BMJ and FBMJ can protect C2C12 cells from oxidative stress.

3.4.2. Effect of BMJ and FBMJ on Intracellular ROS Levels in C2C12 Cells

The ROS that are generated in cells oxidize biomolecules such as lipids, proteins, and DNA, thereby inducing mitochondrial damage and cell death [29]. To investigate the cytoprotective effect of FBMJ against oxidative stress in more detail, we assessed intracellular ROS production using the fluorescent ROS indicator DCFH-DA.

Compared to the control group, 500 μmol/L H₂O₂ treatment increased the level of ROS; however, BMJ and FBMJ each reversed this trend in a dose-dependent manner. The results obtained by flow cytometry analysis showed that compared to the control group, the degree of ROS fluorescence in the H₂O₂ group increased by 2.44 times. Compared to the H₂O₂ group, the ROS fluorescence in the high-dose MBJ group decreased by 3.26 times, while that of the high-dose FBMJ group decreased by 14.78 times. The high-dose FBMJ group results were 6 times lower than those in the control group, demonstrating that FBMJ had a stronger antioxidant effect (Figure 6). Consistent with our results, another study showed that mulberries fermented by gut bacteria inhibited ROS in cells and alleviated ROS-related diseases [30].

3.4.3. Effects of FBMJ on SOD and GSH-Px Activities and MDA Content

When cells are oxidatively stimulated, intracellular GSH-Px and SOD maintain redox homeostasis by scavenging oxidized lipids and oxygen free radicals [31], thereby achieving the REDOX reaction to return the cell to homeostasis.

The previous ROS experiments showed that FBMJ had a preventative effect on the oxidative stress injury of cells (Figure 6). Through the detection of intracellular SOD activity, GSH-Px activity, and MDA levels, it was found that the activities of SOD and GSH-Px in the cells of the model group were significantly decreased (p < 0.0001), while the content of MDA was significantly increased (p < 0.0001).

Table 7. Effects of FBMJ on SOD activity, GSH-PX activity, and MDA content in C2C12 cells.

Group	SOD (U/mL)	MDA (nmol/mg Prot)	GSH-Px (U/mg Prot)
Control group	52.97 ± 0.32	4.57 ± 0.04	17.52 ± 0.68
Model group	44.65 ± 0.26 ####	5.31 ± 0.03 ####	9.08 ± 0.72 ###
BMJ(1mg/mL)	47.79 ± 0.11 ****	4.38 ± 0.03 ****	22.80 ± 0.46 ****
FBMJ(1mg/mL)	51.41 ± 0.34 ****	3.63 ± 0.06 ***	38.12 ± 0.39 ****

Note: Compared with the control group (### p < 0.001, #### p < 0.0001). Compared with the model group (*** p < 0.001, **** p < 0.0001).

shows that, compared to the model group, adding FBMJ to the cells in advance significantly increased the SOD activity to 6.76 U/mL and GSH-Px activity to 29.04 U/mg Prot in the cells, thereby reducing the intracellular MDA content (1.68 nmol/mg Prot). The effect of FBMJ was greater than that of BMJ.

It is worth mentioning that the effect of FBMJ on each index was higher than that of BMJ, indicating that fermentation improved the antioxidant capacity of BMJ.

4. Conclusions

Healthy food is in increasing demand by modern consumers. Probiotic fermentation can be used to improve the nutrition and flavor of food. Therefore, in this study, we evaluated the suitability of 20 strains of probiotics for the fermentation of black mulberries and selected 6 of these strains as the most suitable. Then, a uniform design method was used to determine the optimum inoculum concentration of each strain in the fermentation process. The inoculation ratios of each strain were 27.96% for L. paracasei, 15.37% for L. casei, 16.64% for L. plantarum, 5.12% for L. delbrueckii, 15.83% for B. animalis subsp. lactis, and 19.08% for L. fermentum. According to the SOD activity and TPC, the fermentation process of FBMJ was optimized using RSM. The results showed that the optimum conditions for FBMJ production were as follows: fermentation time of 16 h, fermentation temperature of 35 °C, total bacterial content of 8.2 × 10⁶ CFU/mL, and sugar content of 1.85 °Brix. Experiments investigating the antioxidant activity of FBMJ indicated that FBMJ can promote the proliferation of C2C12 cells and has a significant protective effect against H₂O₂-induced oxidative stress. Compared to the model group, high-dose FBMJ treatment reduced the ROS content. The mechanism underlying this outcome is that FBMJ reduces the MDA content of injured cells and increases the activities of GSH-Px and SOD.