Optimization of Polyphenols Release from Highland Barley Bran by Solid-State Fermentation and Antioxidant Activity Characterization

Abstract

Response surface tests were used to determine the optimal conditions for Bacillus subtilis solid-state fermentation of highland barley bran. The polyphenol composition and antioxidant activity of the fermented bran were also assessed. The results showed that the optimal fermentation conditions for highland barley bran were 10% inoculum, a liquid–feed ratio of 1.80, a fermentation temperature of 30 °C, and a fermentation time of 93.5 h. Under these conditions, the polyphenol content of highland barley bran was 12.43 mg/g. After fermentation, the ferulic acid content of the bran decreased, catechol and gallic acid contents increased significantly, and catechins shifted from the bound state to the free state. In addition, solid-state fermentation improved the antioxidant capacity of highland barley bran, and the DPPH• radical scavenging rate, Fe ion-reducing capacity, and hydroxyl radical scavenging rate of highland barley bran increased after fermentation.

1. Introduction

Highland barley is the most important grain crop in Tibetan areas of China and plays a vital role in the economic development of Tibetan areas. It is an edible, forage, brewing, and medicinal variety of barley [1,2] with high protein, high fiber, high vitamin, low fat, and low sugar contents [3,4,5,6]. Highland barley processing produces large amounts of by-products [7]. Research has found that highland barley bran is rich in dietary fiber and contains a large number of antioxidant phenolic compounds, which can effectively prevent high blood lipids, high blood sugar, cardiovascular diseases, and other diseases [7,8,9]. Currently, highland barley bran is mainly used as animal feed or is directly discarded, resulting in large amounts of waste [10]. The research and utilization of highland barley bran is of great significance to improving the economic value of highland barley and promoting the development of the highland barley industry.

Solid-state fermentation is a fermentation process in which one or more microorganisms in the medium are immobilized and, although rich in water, there is little or no free-flowing water [11]. Fermentation can utilize simple, readily available, inexpensive, and natural mixtures, such as cereal grains, legume seeds, and wood fiber materials, as substrates [12,13,14]. Solid-state fermentation offers higher fermentation productivity, lower catabolic deterrence, lower water activity, and lower aseptic operation requirements than other bioprocessing technologies [15]. Currently, solid-state fermentation technology is applied to microbial agents, production of fermented foods, organic waste conversion, and other related fields [11,16].

Solid-state fermentation has been used to increase the polyphenol content of grains. Ren et al. used a mixture of Bacillus subtilis and brewing yeast to ferment wheat bran and found that the water-soluble polyphenol content of wheat bran increased by 294.41% [17]. Pei et al. used Lactobacillus plantarum LB-1 and brewing yeast for the solid-state fermentation of wheat bran; the reducing power of the fermented bran increased by 0.88 times after 36 h of fermentation [18]. Liu et al. reported that fermentation with six strains of lactobacilli significantly increased the free phenol content of black rice [19].

Currently, there are relatively few studies on improving the functional antioxidant components of highland barley bran through fermentation. Here, solid-state fermentation of highland barley bran using B. subtilis was performed to analyze the effect of fermentation conditions on the polyphenol content of highland barley bran. We determined the optimal fermentation process using response surface methodology and studied the composition and antioxidant activity of polyphenols. This study aimed to provide insights and a theoretical basis for the development of the functional components of highland barley bran.

2. Materials and Methods

2.1. Materials

Highland barley bran was obtained from Jixiang Grain Agricultural Development Co., Ltd. (Lhasa, China). B. subtilis was purchased from Le Diagnosis Biotechnology Co., Ltd. (Nanjing, China). LB medium was used for strain activation and fermentation seed solution preparation. Catechol, catechin, caffeic acid, coumaric acid, gallic acid, and ferulic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl, sodium carbonate, potassium ferrocyanide, ethanol, potassium hydroxide, methanol, ethyl acetate, and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

2.2. Equipment

The following equipment was used: CJ-1D purification bench, Beijing Zhongxing Weiye Century Instrument Co., Ltd. (Beijing, China); HY-08 high-speed pulverizer, Beijing Huanya Tianyuan Machinery Technology Co., Ltd. (Beijing, China); YX-18HDD steam sterilizer, Jiangyin Binzhou Medical Equipments Co., Ltd. (Jiangyin, China); 303–3B Constant Temperature Incubator, Tianjin Tongli Xinda Instrument Factory (Tianjin, China); ZXY-48 Constant Temperature Shaker, Changzhou Runhua Electric Appliance Co., Ltd. (Changzhou, China); 16M Centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd. (Changsha, China); Agilent-1260 high-performance liquid chromatography system, Agilent Technologies Ltd. (Santa Clara, CA, USA).

2.3. Methods

2.3.1. Sample Preparation

Highland barley bran was crushed through a 60-mesh sieve; 20 g were weighed and sterilized, according to the required liquid–feed ratio of the test. B. subtilis was inoculated into LB medium and fermented at 32 °C, 100 rpm for 10 h. The activated B. subtilis suspension was then added, mixed well under the test conditions of solid fermentation, dried at 55 °C after fermentation, crushed, and preserved for further experiments.

2.3.2. Single-Factor and Response Surface Experimental Design

The main factors affecting the fermentation of highland barley bran are fermentation temperature, fermentation time, liquid–feed ratio, and inoculum amount. The polyphenol content of highland barley bran was used as an index to determine the optimal single-factor conditions. The design of the one-factor test for fermentation conditions is presented in

Table 1. One-factor test for highland barley bran fermentation.

Fermentation Conditions	Process Parameters
Fermentation temperature/(°C)	24	27	30	33	36
Fermentation time/(h)	24	48	72	96	120	144
Liquid–feed ratio	0.75	1	1.25	1.5	1.75	2
Vaccination load/(%)	2.5	5	7.5	10	12.5	15
Fermentation temperature/(°C)	24	27	30	33	36

.

Based on the experimental results, a response surface test was designed with highland barley bran polyphenol content as the response value and fermentation temperature, fermentation time, liquid–feed ratio, and inoculum amount as independent variables. The response surface factor levels are listed in

Table 2. Factor level table for the highland barley bran fermentation response surface test.

Level	Factors
	Fermentation Temperature/(°C)	Fermentation Temperature Time/(h)	Liquid–Feed Ratio	Vaccination Load/(%)
−1	30	72	1.5:1	7.5
0	33	96	1.75:1	10
1	36	120	2:1	12.5

.

2.3.3. Polyphenol Content Determination

Polyphenol extraction was performed as previously described [20]. The samples were weighed (0.5 g), and 50 mL of a 60% ethanol solution was added and placed in a water bath at 55 °C for extraction for 2 h. The mixture was centrifuged at 7000 rpm for 25 min, and the supernatant was recovered and filtered using a 0.45 μm organic filtration membrane.

Polyphenol content was determined as previously described [21]. The polyphenol extract (0.5 mL) was added to 0.5 mL of forint reagent and 6 mL of water and mixed well. After 6 min of reaction, 5 mL of 10% Na₂CO₃ was added, and the mixture was allowed to react for 1 h at room temperature (20–28 °C), protected from light. Absorbance was measured at 765 nm. A standard curve was plotted against gallic acid, and the phenol content was calculated as gallic acid equivalents per gram of sample (mg/g).

2.3.4. Phenolic Acid Content Determination

The content of six phenolic acids in highland barley bran was determined before and after fermentation by liquid chromatography [22,23]. Well-mixed samples were weighed in a centrifuge tube, and 45 mL of 95% ethanol was added and mixed by ultrasonic vortexing for 30 min. The mixture was centrifuged at 7000 rpm for 20 min, and the supernatant was recovered; the residue was re-extracted with 95% ethanol twice and combined with the three extracts of the supernatant. At 45 °C, using rotary evaporation, the extract was then dried and concentrated, and the residue was fixed in 70% methanol at 2 mL. The free phenol extract was filtered using a 0.22 μm microporous filter membrane.

Then, 30 mL of 2 M sodium hydroxide was added to the residue, and it was stirred thoroughly for 1 h. The pH of the solution was adjusted to neutral with HCl and extracted twice with 30 mL of ethyl acetate. The two extracts of ethyl acetate were combined. At 45 °C, using rotary evaporation, the extract was then dried and concentrated, and the residue was fixed in 70% methanol at 5 mL. The combined phenol extract was filtered using a 0.22 μm microporous filter membrane.

The analytes were determined using an Agilent C18 column (4.6 mm × 150 mm × 5 μm) with a DAD detector at 35 °C, injection volume of 10 μL, flow rate of 1.0 mL/min, detection wavelengths of 280 nm, a 1% aqueous acetic acid mobile phase A, and methanol mobile phase B. The analytes were analyzed on an Agilent-1260 LC column.

2.3.5. Determination of Antioxidant Activity

The extract was prepared by weighing 1 g of sample. Then, 10 mL of pure water was added, and the mixture was stirred at 55 °C for 1 h, cooled, and centrifuged.

The DPPH• free radical scavenging rate was determined according to the method described by Hong-Fei et al. [24]. Two milliliters of the extract was mixed with 4 mL of DPPH-ethanol solution (0.1 mmol/L), the reaction was carried out for 30 min in the dark at room temperature, and the absorbance was measured at 517 nm (A₁). The extract was replaced with distilled water to determine the absorbance A₂, and the DPPH- ethanol solution was replaced with 95% ethanol to determine the absorbance A₀.The scavenging rate was calculated as follows:

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\mathrm{\%}\right)=\left[1-{\displaystyle \frac{{\mathrm{A}}_1-{\mathrm{A}}_2}{{\mathrm{A}}_0}}\right]\times 100\mathrm{\%}$$

(1)

The Fe ion-reducing ability was determined referring to the method described by Musa et al. [25]. Two milliliters of extract was added to 0.2 mol/L phosphate buffer and 2 mL of 1% potassium ferricyanide solution, mixed well, and allowed to react at 55 °C in a water bath for 20 min. Two milliliters of 10% trichloroacetic acid was added to terminate the reaction. After resting for 10 min, 2 mL purified water and 0.4 mL of 0.1% ferric chloride solution were added and allowed to react at room temperature for 20 min. The absorbance was then determined at 700 nm. The magnitude of the absorbance value reflects the FRAP of bran.

The hydroxyl radical-scavenging rate was determined as described by Rajauria et al. [26]. Two mL of the extract was mixed well with 0.6 mL of 6 mmol/L FeSO₄ solution and 0.6 mL of 6 mmol H₂O₂, and the mixed solution was allowed to react for 10 min protected from light. A salicylic acid ethanol solution was added (0.6 mL of 6 mmol/L), mixed well, and allowed to stand for 20 min. The absorbance was measured at 510 nm (A₁). The extract was replaced with purified water to determine the absorbance A₀, and salicylic acid ethanol solution was replaced with anhydrous ethanol to determine the absorbance A₂. The scavenging rate was calculated as follows:

$$\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{y}\mathrm{l} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\mathrm{\%}\right)=\left[1-{\displaystyle \frac{{\mathrm{A}}_1-{\mathrm{A}}_2}{{\mathrm{A}}_0}}\right]\times 100\mathrm{\%}$$

(2)

2.3.6. Data Analyses and Processing

Each group of experiments was repeated three times, and the results are expressed as a mean ± standard deviation. Design Expert 8.0.6 and Origin 9.0 were used for the response surface test design, data analysis, and graphing.

3. Results and Discussion

3.1. Results of One-Factor Test on Fermentation of Highland Barley Bran

3.1.1. Effect of Fermentation Temperature on Polyphenol Content of Highland Barley Bran

The effect of fermentation temperature on the polyphenol content of highland barley bran was investigated using a liquid–feed ratio of 1.75:1, an inoculum amount of 10%, and a fermentation time of 96 h. The results are shown in Figure 1.

Temperature is a crucial factor during the fermentation process and can affect the fluidity of the plasma membrane [27,28]. When the fermentation temperature was lower than 33 °C, the polyphenol content of highland barley bran increased with increasing temperature, and the polyphenol content decreased significantly when the temperature was higher than 33 °C. This can be explained by the increase in fermentation temperature, which increases the metabolic rate of bacteria and accelerates enzyme production, which is favorable for the release of free phenols from the bran. When the temperature was too high, the growth of the bacteria was actually inhibited, which subsequently affected the polyphenol content of the bran. This is consistent with the research results of Ren et al. [17]. They fermented wheat bran with Saccharomyces cerevisiae and B. subtilis. The content of polyphenols gradually increased with the increase in fermentation temperature at 31–37 °C and decreased when the fermentation temperature continued to rise. A moderately increased fermentation temperature is conducive to the release of polyphenols.

3.1.2. Effect of Fermentation Time on Polyphenol Content of Highland Barley Bran

The effect of fermentation time on the polyphenol content of highland barley bran was investigated at a fermentation temperature of 33 °C, liquid–feed ratio of 1.75:1, and inoculum amount of 10%; the results are shown in Figure 2.

With prolonged fermentation time, the polyphenol content of highland barley bran increased gradually, the polyphenol content reached its highest at 96 h of fermentation and then began to decrease slowly. During the pre-fermentation period, bacteria grew and were metabolically active, polyphenol gradually accumulated, and the free phenol content increased. B. subtilis growth tended to stabilize with an increase in fermentation time, and the polyphenol content reached its maximum, while fermentation continued. The bacteria then entered a decline period, and the ability to extract polyphenols was weakened. Part of the phenolic acids were degraded; polyphenol content gradually decreased.

The growth and metabolism of microorganisms adhere to a specific cycle. In the early stage of fermentation, B. subtilis acts mainly to promote its own growth and the synthesis of primary metabolites [29]. As the fermentation progresses, the bacteria begin to accumulate secondary metabolites and promote the biotransformation of related substances. In the late fermentation stage, the depletion of nutrients in the medium and the accumulation of toxic metabolites will lead to the inhibition of cell growth and the decomposition of related metabolites.

3.1.3. Effect of Liquid–Feed Ratio on the Polyphenol Content of Highland Barley Bran

Figure 3 shows the effect of the liquid–feed ratio on the polyphenol content of highland barley bran at a fermentation temperature of 33 °C, inoculum amount of 10%, and fermentation time of 96 h. When the moisture was too low, B. subtilis growth was limited; with an increase in moisture, bacterial motility increased and the substrate contact was more favorable, leading to an increase in polyphenol, reaching its maximum value when the liquid–feed ratio was 1.75:1. As the moisture continued to increase, the nutrients in the medium were diluted, culture oxygenation decreased, and B. subtilis respiration was inhibited, which affected polyphenol accumulation.

The water content of the substrate is one of the key factors related to the success of SSF. The characteristics of microorganisms, the properties of raw materials, culture conditions, and other factors jointly determine the water content of the substrate [30]. It has been reported that the total phenolic content (TPC) of fermented corn bran extract reached a maximum at 60% moisture content, and too high or too low a moisture content would lead to a decrease in TPC content [31].

3.1.4. Effect of Inoculum Amount on Polyphenol Content of Highland Barley Bran

Figure 4 shows the effect of the inoculum amount on the polyphenol content of highland barley bran at a fermentation temperature of 33 °C, liquid–feed ratio of 1.75:1, and fermentation time of 96 h. With the increase in inoculum amount, the polyphenol content of highland barley bran first increased and then gradually decreased. When the inoculum amount was lower than 10%, the substrate in the fermentation medium was sufficient to support adequate enzyme production in B. subtilis, which promoted the synthesis and release of polyphenols. As the amount of inoculum increased, the growth of the bacteria was hindered, and the release of free phenols was inhibited. Therefore, 10 % was considered the optimal inoculum amount.

The amount of inoculum determines the strength of bacterial growth and the rate of substrate metabolism. A low inoculum amount will result in slow growth of bacterial cells and the slow formation of metabolites. Conversely, when the inoculum amount is excessive, the bacterial cells overgrow in the early stage of fermentation and degrade phenolic substances in the later stage of fermentation to meet the growth demands. Additionally, an overly high inoculation quantity will also cause a decline in the oxygen permeability of the substrate and an insufficient supply of dissolved oxygen per unit volume, thereby influencing the accumulation of metabolites and the transformation of nutrients [32,33].

3.2. Optimization of Highland Barley Bran Fermentation Process by Response Surface Methodology

The experimental design and results for the response surface are presented in

Table 3. Response surface test design and results.

Test No.	A Inoculum Amount (%)	B Liquid- Feed Ratio	C Fermentation Temperature (°C)	D Fermentation Time (h)	Y Polyphenol Content (mg/g)
1	0	0	1	1	10.86 ± 0.21
2	−1	0	−1	0	10.60 ± 0.17
3	0	0	0	0	12.02 ± 0.05
4	0	1	0	1	8.95 ± 0.09
5	0	0	1	−1	7.06 ± 0.05
6	1	0	0	−1	7.03 ± 0.11
7	1	1	0	0	8.81 ± 0.20
8	0	0	0	0	12.27 ± 0.27
9	0	0	0	0	12.14 ± 0.21
10	0	0	0	0	12.02 ± 0.21
11	0	−1	1	0	9.99 ± 0.18
12	0	0	−1	1	9.81 ± 0.09
13	0	0	0	0	12.36 ± 0.29
14	0	−1	0	1	9.97 ± 0.22
15	1	−1	0	0	8.23 ± 0.21
16	−1	0	0	−1	7.77 ± 0.10
17	0	1	−1	0	11.88 ± 0.19
18	0	−1	0	−1	7.39 ± 0.11
19	1	0	−1	0	10.32 ± 0.09
20	−1	1	0	0	8.86 ± 0.18
21	1	0	1	0	8.86 ± 0.23
22	−1	0	−1	1	9.12 ± 0.19
23	−1	0	1	0	8.92 ± 0.07
24	0	0	−1	−1	10.16 ± 0.08
25	1	0	0	1	8.62 ± 0.21
26	0	1	0	−1	9.16 ± 0.25
27	0	−1	−1	0	10.86 ± 0.21
28	0	1	1	0	9.62 ± 0.09
29	−1	−1	0	0	8.64 ± 0.16

. Regression analysis was performed on the experimental results to establish a regression model of polyphenol content (Y) on inoculum amount (A), liquid–feed ratio (B), fermentation temperature (C), and fermentation time (D), and the following multiple quadratic regression simulation equation was obtained:

Y = 12.16 − 0.17A + 0.18B − 0.69C + 0.73D + 0.09AB + 0.055AC + 0.06AD − 0.35BC − 0.7BD + 1.04CD − 2.09A² − 1.26B² − 0.44B² − 2.07D²

(3)

The analysis of variance (ANOVA) of the regression model is presented in

Table 4. ANOVA of the regression model.

Source	Square Sum	Degrees of Freedom	Mean Square	Value of F	Value of p	Significance
Model	70.05	14	5.00	73.27	<0.0001	**
A	0.35	1	0.35	5.08	0.0408	*
B	0.40	1	0.40	5.91	0.0291	*
C	5.77	1	5.77	84.47	<0.0001	**
D	6.39	1	6.39	93.64	<0.0001	**
AB	0.032	1	0.032	0.47	0.5022
AC	0.012	1	0.012	0.18	0.6802
AD	0.014	1	0.014	0.21	0.6531
BC	0.48	1	0.48	7.07	0.0187	*
BD	1.95	1	1.95	28.50	0.0001	**
CD	4.31	1	4.31	63.05	<0.0001	**
A2	28.26	1	28.26	413.81	<0.0001	**
B2	10.38	1	10.38	151.94	<0.0001	**
C2	1.27	1	1.27	18.58	0.0007	**
D2	27.85	1	27.85	407.88	<0.0001	**
Residuals	0.96	14	0.068
Lost proposal	0.86	10	0.086	3.77	0.1063	insignificant
Pure error	0.092	4	0.023
Sum	71.00	28

“*” indicates significant differences; “**” indicates highly significant differences.

. The model used in the experiment was as follows: p < 0.0001, excellent significance; coefficient of determination R² = 0.9865; and adjusted coefficient of determination R²Adj = 0.9731. The loss of fit term was not significant, which indicates that the model is reliable and can be used to analyze and predict the effect of fermentation conditions on the polyphenol content of highland barley bran. From the p-value, it can be seen that the one-time C, D, and all secondary terms had highly significant effects on the results, and the primary terms A and B had significant effects on the results. The order of the effect of each factor on the polyphenol content of highland barley bran was fermentation time > fermentation temperature > liquid feed ratio > inoculum amount.

The effect of the interaction of these factors on the polyphenol content of highland barley bran is shown in Figure 5. In the response surface plot, the steeper the slope and the denser the contour line, the greater the effect of the factor on the response value and the more significant the interaction. The interaction terms BD and CD had highly significant effects on the results (p < 0.01), and the interaction term BC also had a significant effect (p < 0.05), which was consistent with the ANOVA results.

According to the regression equation, the optimal process conditions for highland barley bran fermentation were as follows: inoculum amount, 9.87%; liquid–feed ratio, 1.81; fermentation temperature, 30 °C; and fermentation time, 93.26 h. Considering practical operation, the process conditions were adjusted to an inoculum amount of 10%, liquid–feed ratio of 1.80, fermentation temperature of 30 °C, and fermentation time of 93.5 h. In the validation test, the polyphenol content of highland barley bran was 12.43 ± 0.19 mg/g, which was similar to the predicted value of 12.5001 mg/g, indicating reliable results. Compared to the pre-fermentation period, the polyphenol content of highland barley bran increased by 203.17%.

3.3. Determination of Phenolic Acid Content of Highland Barley Bran before and after Fermentation

Six phenolic acids were detected in the free phenol and bound phenol extracts of highland barley bran before and after fermentation, and the results are shown in Figure 6 and

Table 5. Polyphenols content in highland barley bran.

Items/(mg/kg)	Free Phenol Pre-Fermentation	Free Phenol after Fermentation	Bound Phenol Pre-Fermentation	Bound Phenol after Fermentation
gallic acid	0.36 ± 0.07	205.12 ± 0.15	5.93 ± 0.09	68.19 ± 0.22
coumaric acid	ND	ND	1.57 ± 0.09	1.29 ± 0.11
catechin	64.42 ± 0.16	122.22 ± 0.33	266.39 ± 0.13	194.05 ± 0.24
catechol	0.93 ± 0.06	17.82 ± 0.08	ND	6.07 ± 0.19
caffeic acid	ND	0.07 ± 0.01	1.92 ± 0.03	2.28 ± 0.03
ferulic acid	2.83 ± 0.07	0.90 ± 0.03	187.78 ± 0.11	158.32 ± 0.15

“ND” indicates not detected.

. Catechin was the compound with the highest content in highland barley bran, followed by ferulic acid. Comparing before and after fermentation, the content of catechin in the free phenol increased, whereas that in the bound phenol decreased, and catechin transitioned from the bound state to the free state. After fermentation, catechin and gallic acid contents in the free and bound phenol significantly increased, indicating that catechin and gallic acid were newly produced during the fermentation process of B. subtilis. Ferulic, caffeic, and coumaric acids exist mainly in the form of bound phenols in highland barley bran, and the ferulic acid content decreased after fermentation, which may have been degraded by the phenolic acid decarboxylase produced during the fermentation process.

Microorganisms contain a diverse array of enzymes that can break down grain cell walls and facilitate the release of polyphenolic compounds during fermentation. There have been reports on the use of solid-state fermentation to concentrate phenolic compounds in grains. Xu et al. [34] fermented defatted barley bran with Rhizopus oryzae, resulting in a significant increase in the content of syringic acid and chlorogenic acid post-fermentation, while the content of bound ferulic acid decreased by 22%. Nie et al. [35] discovered that fermentation of rye by Lactobacillus plantarum significantly increased the content of catechin, caffeic acid, ferulic acid, and coumaric acid. The increase in ferulic acid differed from our research findings, possibly due to the use of different fermentation strains.

3.4. Analysis of the Antioxidant Activity of Highland Barley Bran before and after Fermentation

The DPPH• radical scavenging rate, Fe ion-reducing capacity, and hydroxyl radical scavenging rate of highland barley bran were determined before and after fermentation (Figure 7). After fermentation, the reducing capacity and antioxidant activity of highland barley bran were enhanced, the DPPH• radical scavenging rate increased by 12.32%, which was higher than that of the hydroxyl radical scavenging rate (8.73%), and the Fe ion-reducing capacity A700 increased from 0.215 to 0.349. Similarly, Lactobacillus fermentum NB02 was used to ferment oat bran, and it was found that its scavenging of DPPH radicals, scavenging of ABTS+ radicals, and ·OH scavenging activity significantly increased [36].

Phenolic compounds in highland barley bran are mainly present in the cell walls of the cortex, where they are usually combined with sugars and proteins [37]. Typically, when phenolic compounds are combined with sugars and proteins through glycosidic and ester bonds, it is difficult for these compounds to exert their antioxidant properties [38]. Microbial solid-state fermentation loosened the bran cell wall, which allowed for the release of phenolic substances and a significant increase in free phenol content, leading to an increase in antioxidant activity in vitro. In conclusion, fermentation treatment can effectively improve the antioxidant capacity of bran by enhancing the release of phenolic compounds.

4. Conclusions

In this study, solid-state fermentation of highland barley bran using B. subtilis was carried out. The optimal fermentation conditions of highland barley bran were determined using a one-way test and response surface test and were as follows: 10% inoculum, liquid–feed ratio of 1.80, fermentation temperature of 30 °C, and fermentation time of 93.5 h. Under these conditions, the polyphenol content of fermented highland barley bran was 12.43 mg/g, which was 203.17% higher than that of pre-fermentation, and the solid-state release of polyphenols from highland barley bran by fermentation was significant.

Microbial fermentation changed the composition ratio of highland barley bran polyphenols. After fermentation, the free phenol and ferulic acid contents of bound phenol decreased, catechol and gallic acid contents increased significantly, and catechins changed from the bound state to the free state. Solid-state fermentation increases the antioxidant capacity of highland barley bran, with increased DPPH• radical scavenging, Fe ion-reducing capacity, and hydroxyl radical scavenging. This research offers a theoretical foundation for the development and utilization of polyphenols in highland barley bran. But the correlation between antioxidant activity and the proportion of phenolic acids is unclear, and further studies are required.