Analysis of Functional Component Alterations and Antioxidant Response Mechanisms in Microbial-Enzymatic Co-Fermentation-Induced Quinoa Bran

Abstract

This study utilised Bacillus subtilis and cellulase combined with Bacillus subtilis to ferment quinoa bran. The effects of different fermentation methods on the functional components, antioxidant activity, and structural changes of quinoa bran were evaluated. Fermentation altered the functional components of quinoa bran and enhanced its antioxidant capacity. The phenolic acid and polysaccharide contents increased in BFQ (Bacillus subtilis-fermented quinoa bran) and BEFQ (bacterio-enzyme co-fermented quinoa bran), whereas the protein content decreased. After fermentation, the phenolic acid content in BEFQ increased by 81.68%, while the DPPH and ABTS radical-scavenging rates increased by 43.99% and 31.44%, respectively. The antioxidant capacity in BEFQ was ranked as follows: ferulic acid > p-coumaric acid > vanillic acid > 4-hydroxybenzoic acid. Thus, the antioxidant ability of quinoa bran phenolic acids was primarily dependent on hydroxycinnamic acid derivatives. The polysaccharide content in BEFQ increased by 80.73%. The DPPH and ABTS radical-scavenging rates increased by 52.59% and 50.48%, respectively, whereas the protein content decreased by 21.88%. Furthermore, the DPPH and ABTS radical-scavenging rates increased by 76% and 75.39%, respectively. These results indicate that fermentation using cellulase combined with Bacillus subtilis has the potential to enhance the antioxidant capacity and utilisation of quinoa bran.

1. Introduction

Quinoa bran, the primary by-product of quinoa processing, constitutes approximately 10% of the grain weight [1] and is rich in phenolic compounds, proteins, and polysaccharides. It is a potential source of bioactive components beneficial to human health [2]. However, in practical applications, the complex cell wall structure of bran hinders the release of its constituents. Phenolic acids, in particular, primarily occur in bound, esterified, or glycosylated forms within cell wall polysaccharides. This significantly limits their bio-accessibility and reduces their functional activity [3,4].

As a processing method, microbial fermentation offers advantages, such as being eco-friendly and having mild processing conditions. Fermentation can effectively reduce antinutritional factors in grains and improve their functional properties, thereby serving as an efficient means of releasing functional components from cereals [5]. Bacillus subtilis exhibits rapid proliferation and strong antifungal activity. It secretes various enzymes, including cellulases and proteases, which can degrade plant cell wall structures, liberate bound phenolic acids, and potentially alter the molecular weight of polysaccharides and composition of proteins [6]. Cellulases can directly and efficiently degrade structural polysaccharides, such as cellulose, which are abundant in quinoa bran, thereby disrupting the physical barrier of the cell wall. This creates favourable conditions for microorganisms and their endogenous enzymes to act more effectively on the embedded functional components. The fermentation of quinoa bran using cellulases leverages the targeted hydrolytic capacity of the enzymes and the metabolic regulatory effects of Bacillus subtilis, resulting in a synergistic effect. This synergy stems from the division of labour between the two: cellulase provides the crucial hydrolytic function, releasing the bound nutrients in the fibrous matrix by generating fermentable sugars, which in turn offer the best raw materials for the metabolism of Bacillus subtilis. Then, the bacteria subtilis effectively convert these sugars into a series of bioactive compounds, thereby enhancing the yield and value of the biological process, which no single component can achieve alone. This process further disrupts the cell wall structure [7], significantly enhancing the release of functional components and boosting antioxidant activity.

Srinivasu et al. [8] investigated the functional properties of quinoa bran protein. Ge [9] and Liu [10] studied the effects of quinoa bran insoluble and soluble dietary fibres on glucose metabolism and colitis, respectively, in experimental mice. Ding et al. [11] reported the inhibitory effects of quinoa bran terpenoids on colon cancer cells. Currently, there is a lack of comprehensive and quantitative comparative studies on the simultaneous changes in multiple key functional components as well as amino acid, free phenolic acid and bound phenolic acid conversion, and polysaccharide molecular weight changes during quinoa bran fermentation. Furthermore, their contributions to the antioxidant capacity of quinoa bran have not been systematically explored.

This study aimed to elucidate the differences between Bacillus subtilis fermentation and microbial-enzymatic co-fermentation in regulating the functional components of quinoa bran and enhancing its antioxidant activity. High-performance liquid chromatography (LC) coupled with mass spectrometry (MS) (HPLC-MS) was used to qualitatively and quantitatively analyse the phenolic acid, monosaccharide, and amino acid profiles in quinoa bran before and after fermentation. The study also aimed to clarify the structure-activity relationships between functional component monomers and antioxidant activity, thereby exploring the antioxidant response mechanisms of phenolic substances, polysaccharides, and proteins. This study aims to enhance the functional properties of quinoa bran through biotransformation technology. Squeo et al. [12] utilized by-products from durum wheat milling to produce low-fat, high-fibre biscuits, providing dietary options for specific nutritional needs groups, such as athletes. Anelise et al. [13] applied brewer’s yeast-fermented rice bran as a substitute for wheat flour in gluten-free biscuits, offering a choice for individuals with celiac disease. Applying fermented quinoa bran in the field of functional foods and nutraceuticals will increase the added value of quinoa, reduce waste emissions, and contribute to resource conservation and sustainable development.

2. Materials and Methods

2.1. Materials and Reagents

Quinoa bran was obtained from Shanxi Huaqishun Food Technology Co., Ltd. (Xinzhou, China). Bacillus subtilis (CMCC (B) 63501) was purchased from the Beijing Microbiological Culture Collection Center (Beijing, China). Cellulase and phenolic acid standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). The AB-8 macroporous adsorption resin was purchased from Tianjin Haoju Resin Technology Co., Ltd. (Tianjin, China). All other chemicals and reagents used in this study were of analytical or chromatographic grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Methods

2.2.1. Sample Preparation

First, 5 kg Quinoa bran (moisture content is 8.61%) was milled and sieved through a 60-mesh screen. High-pressure sterilisation was performed using an autoclave (YX-18HDD; Jiangyin Binzhou Medical Devices Co., Ltd., Jiangyin, China). Based on the optimization experiments before the research, the fermentation time, liquid-to-solid ratio, inoculation volume, and cellulase dosage were determined, as shown in

Table 1. Preparation conditions of the samples.

Name	Fermentation Time (h)	Liquid to Solid Ratio (mL/g)	Bacillus subtilis (OD600 = 0.8–0.9) Inoculation Amount (%)	Cellulase Dosage (u/g)
UFQ	-	-	-	-
BFQ	121	1.9	8	-
BEFQ	121	1.9	8	85

Note: “-” indicates no processing.

. The logarithmic phase of Bacillus subtilis (OD600 = 0.8–0.9) was selected for inoculation. According to the conditions in Table 1, unfermented quinoa bran (UFQ), Bacillus subtilis-fermented quinoa bran (BFQ), and bacterio-enzyme co-fermented quinoa bran (BEFQ) were prepared. All fermented products were dried in a 55 °C air-blowing oven (DGH-9140A; Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China), milled into powder, and stored for subsequent use.

2.2.2. Antioxidant Activity

The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity was determined according to the methods of Zhang et al. [14] and Kaced et al. [15]. The scavenging percentage against DPPH radicals and its half-maximal inhibitory concentration (IC₅₀) were calculated and compared with ascorbic acid.

The 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation-scavenging activity was determined according to the methods of Wu et al. [16] and Guo et al. [17]. The scavenging percentage against ABTS radicals and its IC₅₀ were calculated and compared with ascorbic acid.

2.2.3. Determination of Phenolic Acid Content in Quinoa Bran

For the preparation of free phenolic acid extract, phenolic acids from quinoa bran, both pre- and post-fermentation, were extracted using the method established by Zhang et al. [14].

For the preparation of bound phenolic acid extract, around 100 mg of frozen sample was accurately weighed into a centrifuge tube; 2 mL of 4 mol L⁻¹ aqueous sodium hydroxide was added, then hydrolysed at 40 °C for 2 h. The pH was adjusted to 2.0 by adding 4 mol L⁻¹ hydrochloric acid solution. Hexane (2 mL) was added to the mixture at room temperature, vortexed for 20 min, and the hexane layer was removed. The aqueous layer was extracted with ethyl acetate (2 × 2 mL), concentrated to dryness under reduced pressure using a rotary evaporator at 35 °C, and reconstituted in 0.2 mL of 50% aqueous methanol. Samples were diluted appropriately before instrumental analysis, as required.

Phenolic acid content was determined according to the method of Zhao et al. [18], with minor modifications. Gallic acid was used as the standard phenolic compound to prepare the calibration curve. Briefly, 0.5 mL of extract was mixed with an equal volume of Folin–Ciocalteu reagent and 4 mL of water. After incubation for 3 min, 3 mL of saturated sodium carbonate solution was added to neutralise the reaction mixture. The solution was incubated in the dark at room temperature for 2 h, and the absorbance was measured at 765 nm. Phenolic acid content was expressed as milligrams of gallic acid equivalents per gram of sample (mg g⁻¹).

2.2.4. Analysis of Phenolic Acid Components Using LC-MS

Analyses were performed using LC (Vanquish; Thermo Fisher Scientific, MA, USA) coupled with MS (Q Exactive; Thermo Fisher Scientific, Waltham, MA, USA).

Chromatographic conditions were as follows: separation was performed on a Waters HSS T3 column (Waters; Milford, MA, USA) (50 × 2.1 mm, 1.8 μm particle size). The mobile phase consisted of phase A (ultrapure water containing 0.1% formic acid, v/v) and phase B (acetonitrile containing 0.1% formic acid, v/v). The flow rate was 0.3 mL min⁻¹, the column temperature was maintained at 40 °C, and the injection volume was 2 μL. The gradient elution programme was as follows: 0–2 min, 90% A/10% B; 2–6 min, linear gradient to 40% A/60% B; 6–8 min, 40% A/60% B; 8.1–12 min, return to 90% A/10% B (equilibration).

MS conditions were as follows: Sheath gas, 40 arb; auxiliary gas, 10 arb; ion spray voltage, −2800 V; capillary temperature 350 °C; ion transfer tube temperature, 320 °C. The scanning mode was full scan-ddMS² in negative ion mode. The scan range for the first-stage mass spectrometry was m/z 80–900.

System reproducibility was monitored by injecting a QC sample every five injections. The relative standard deviation (RSD) of the retention times for the major analytes was less than 1.0%, demonstrating excellent chromatographic stability. Quantification of the target compounds was achieved using an external calibration curve, which exhibited good linearity (R² > 0.995).

2.2.5. Extraction and Purification of Major Phenolic Acid Components

Quinoa bran was extracted with 70% ethanol (containing 0.5% acetic acid) in a 55 °C water bath. The extract was concentrated under reduced pressure to yield a crude phenolic acid extract. The crude extract was dissolved in water, adjusted to pH 3, and extracted three times with ethyl acetate to transfer phenolic acids into the organic phase, which was subsequently adjusted to pH 8. Phenolic acids were backextracted into the aqueous phase. The aqueous phase was acidified to pH 3 to precipitate phenolic acids, which were filtered and collected. The phenolic acid precipitate was dissolved in 70% ethanol and loaded onto an AB-8 macroporous resin column. Gradient elution was performed using ethanol concentrations of 30%, 40%, 50%, and 60%. The eluates were collected, vacuum dried, and stored until further use.

Purification of phenolic acids by HPLC was as follows: Column, C18 (4.6 × 250 mm, 5 μm); mobile phase, methanol—0.1% phosphoric acid in water (30:70, v/v); flow rate, 1 mL min⁻¹; detection wavelengths: 310 nm (p-coumaric acid), 320 nm (ferulic acid), 254 nm (4-hydroxybenzoic acid), and 260 nm (vanillic acid). The target peaks were collected, concentrated under reduced pressure, dried, and stored for subsequent use.

Recrystallisation occurred as follows: each purified phenolic acid was dissolved in 75% hot ethanol, filtered while hot to remove insoluble impurities, and gradually cooled to 4 °C to induce crystallisation. The resulting white crystals (p-coumaric, ferulic, 4-hydroxybenzoic, and vanillic acids) were filtered, washed, and dried to obtain the pure compounds.

2.2.6. Quinoa Bran Polysaccharide Extraction and Content Determination

Quinoa bran polysaccharides were prepared by hot-water extraction according to the method of Benjarat et al. [19].

2.2.7. Molecular Weight Determination of Polysaccharides

A desalted polysaccharide sample (5 mg) was accurately weighed and dissolved in 5 mL of the mobile phase, dimethyl sulfoxide (DMSO), by heating at 80 °C for 3 h. The solution was then injected for chromatographic analysis.

Analysis was performed using a gel-permeation chromatography system coupled with refractive index (RI) and multi-angle laser light scattering (MALLS) detectors. The system comprised the following: liquid chromatography system, Ultimate 3000 (Thermo Scientific, Waltham, MA, USA); RI detector, Optilab T-rEX (Wyatt Technology, Santa Barbara, CA, USA); MALLS detector, DAWN HELEOS II (Wyatt Technology, Santa Barbara, CA, USA) (λ = 663.7 nm). Chromatographic conditions were as follows: column: Ohpak SB-803 HQ gel-filtration column (300 × 8 mm); column temperature, 60 °C; injection volume, 200 μL; mobile phase A, 0.5% (w/v) LiBr in DMSO; flow rate, 0.3 mL min⁻¹, elution mode, isocratic elution over 120 min; dn/dc value for DMSO solutions, 0.07 mL g⁻¹.

2.2.8. Determination of Monosaccharide Composition

Analyses were performed using a Thermo ICS 5000+ ion chromatography system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an electrochemical detector.

Chromatographic conditions were as follows: column: Dionex™ CarboPac™ PA20 (150 × 3.0 mm, 10 μm); injection volume, 5 μL. Mobile phases: A, H₂O; B, 0.1 M NaOH; C, 0.1 M NaOH + 0.2 M NaAc; flow rate, 0.5 mL min⁻¹; column temperature, 30 °C. Elution gradient: 0 min, 95% A/5% B/0% C (v/v); 26 min, 85% A/5% B/10% C (v/v); 42 min, 85% A/5% B/10% C (v/v); 42.1 min, 60% A/0% B/40% C (v/v); 52 min, 60% A/40% B/0% C (v/v); 52.1 min, 95% A/5% B/0% C (v/v); 60 min, 95% A/5% B/0% C (v/v).

2.2.9. Protein Extraction and Quantification

Quinoa bran protein was extracted according to the method of Mir et al. [20]. Nitrogen content was determined using a micro-Kjeldahl apparatus, and crude protein was calculated using a conversion factor of 6.25 to convert nitrogen content to protein content.

2.2.10. Free Amino Acid Composition Analysis

Each sample (0.1 g) was accurately weighed into a centrifuge tube. An M hydrochloric acid (HCl) solution was added, vortex-mixed thoroughly, and extracted at room temperature for 1 h. The mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected for dilution. Ten microlitres of the diluted sample were transferred to a derivatisation vial, followed by the addition 70 μL of AccQ•Tag Ultra Borate Buffer and 20 μL of AccQ•Tag Ultra Reagent. The mixture was vortex-mixed thoroughly and heated at 55 °C for 10 min. Samples were cooled to room temperature prior to instrumental analysis. Instrumental analysis was conducted as follows: Detection system identical to that used for phenolic acid analysis (Section 2.2.4). Column, Waters BEH C18 (Waters; Milford, MA, USA) (50 × 2.1 mm, 1.7 μm); mobile phase flow rate, 0.5 mL min⁻¹; column temperature, 55 °C; injection volume, 1 μL. Mass spectrometry conditions were as follows: ionisation mode, electrospray ionisation (ESI⁺); spray voltage, +3000 V; scan mode, positive ion; full scan range, m/z 150–700. The content of amino acids was determined by the internal standard method.

2.2.11. Data Processing and Analysis

All experiments were performed in triplicate, and results are expressed as mean ± standard deviation (SD). Data were subjected to analysis of variance (ANOVA) using IBM SPSS Statistics 26.0 (SPSS Inc., Chicago, IL, USA). Statistical analysis and data visualisation were conducted using Origin 2019 (OriginLab, Northampton, MA, USA), SIMCA 14.1 (Umetrics, Malmö, SWE) and the Chiplot cloud platform (https://www.chiplot.online/ accessed on 8 September 2025). The chemical structures of the phenolic acids were drawn using InDraw software 6.2.3 (Integle, Shanghai, China).

3. Results

3.1. Phenolic Acid Content and Antioxidant Capacity of Quinoa Bran

Phenolic acid compounds in cereal bran predominantly occur bound to cell wall polysaccharides via ester or glycosidic linkages [3]. As Figure 1 shows, fermentation significantly increased the phenolic acid content of quinoa bran, with BEFQ showing the highest level (13.39 mg g⁻¹), an 81.68% increase compared with UFQ. Organic acids produced during Bacillus subtilis metabolism can reduce the pH of the fermentation medium and increase cell wall permeability. Cellulase is a multi-enzyme complex. Under the combined action of esterases and β-glucosidase secreted by Bacillus subtilis, the plant cell wall can be effectively hydrolysed, releasing phenolic compounds encapsulated within the wall matrix [21,22]. This process promotes the hydrolysis of chemical bonds between phenolic acids and other compounds [23], thereby releasing phenolic acids. Similar findings were reported by Abduh in a study fermenting rice bran using Aspergillus niger [24].

Both antioxidant assays demonstrated that quinoa bran phenolic acids possessed strong radical-scavenging activity. The clearance abilities of DPPH and ABTS were significantly correlated with the content of phenolic acids (p < 0.05, Pearson Correlation Coefficients were 0.994 and 0.990, respectively). BEFQ exhibited the highest antioxidant capacity, with DPPH radical-scavenging rate (79.53%) being 43.99% higher than UFQ and 31.44% higher than BFQ. The ABTS radical-scavenging rate (77.25%) was 55.21% higher than UFQ and 36.30% higher than BFQ, consistent with elevated phenolic acid content. As shown above, cellulase combined with Bacillus subtilis increased the phenolic acid content of quinoa bran more effectively than Bacillus subtilis fermentation alone, thereby enhancing antioxidant capacity.

3.2. Characterization of Phenolic Acid Composition

Fermentation significantly increased the phenolic acid content of quinoa bran, consistent with the findings of Byanju [25]. To clarify the effects of the two distinct fermentation methods on the individual phenolic acid levels, LC-MS was employed, leading to the identification of 18 free and 16 bound phenolic acids, as Figure 2 and

Table 2. Free and bound phenolic acid contents in quinoa bran.

	Free Phenolic Acid			Bound Phenolic Acid
Sample (ng/mg)	UFQ	BFQ	BEFQ	UFQ	BFQ	BEFQ
Gallic acid	0.40 ± 0.02 c	0.68 ± 0.06 b	1.87 ± 0.02 a	0.26 ± 0.05 b	0.29 ± 0.08 b	0.59 ± 0.01 a
3,4-Dihydroxybenzoic acid	30.22 ± 0.25 a	6.57 ± 0.44 c	19.00 ± 0.87 b	4.87 ± 0.12 b	1.84 ± 0.51 c	9.82 ± 0.51 a
Protocatechualdehyde	0.24 ± 0.03 b	1.96 ± 0.18 a	2.29 ± 0.35 a	0.31 ± 0.02 a	1.29 ± 0.62 a	1.46 ± 0.17 a
4-Hydroxybenzoic acid	12.21 ± 0.14 b	15.82 ± 1.34 b	21.97 ± 1.26 a	18.52 ± 0.21 b	21.18 ± 1.23 ab	24.96 ± 1.43 a
Catechin	0.02 ± 0.00 a	0.06 ± 0.00 a	0.02 ± 0.00 a	NF	NF	NF
Vanillic acid	2.38 ± 0.18 c	16.08 ± 1.02 b	28.50 ± 1.69 a	12.91 ± 0.13 b	22.51 ± 1.06 a	26.91 ± 2.57 a
Caffeic acid	0.48 ± 0.04 b	1.90 ± 0.10 b	6.87 ± 0.87 a	0.94 ± 0.04 b	0.88 ± 0.06 b	2.47 ± 0.35 a
Syringic acid	0.16 ± 0.03 b	0.92 ± 0.05 ab	1.10 ± 0.32 a	0.73 ± 0.08 b	1.27 ± 0.17 ab	1.44 ± 0.11 a
L-Epicatechin	0.02 ± 0.00 a	0.01 ± 0.00 a	0.03 ± 0.00 a	NF	NF	NF
Vanillin	1.17 ± 0.02 b	15.99 ± 1.78 a	15.63 ± 1.64 a	11.97 ± 0.09 b	25.14 ± 2.05 a	22.04 ± 0.19 a
p-Hydroxycinnamic Acid	166.05 ± 4.19 b	188.45 ± 3.12 a	185.97 ± 2.98 a	145.40 ± 1.31 b	168.52 ± 3.77 a	177.46 ± 3.16 a
Syringaldehyde	0.12 ± 0.01 a	0.65 ± 0.02 a	0.75 ± 0.00 a	1.38 ± 0.05 a	1.24 ± 0.13 ab	0.90 ± 0.00 b
Ferulic acid	3.30 ± 0.08 b	8.85 ± 1.63 b	69.33 ± 2.33 a	60.54 ± 2.11 b	73.56 ± 2.16 a	82.83 ± 3.18 a
Sinapic Acid	0.50 ± 0.03 b	0.08 ± 0.00 c	0.76 ± 0.01 a	0.93 ± 0.04 a	1.02 ± 0.09 a	0.76 ± 0.02 a
Salicylic acid	5.05 ± 0.16 a	3.61 ± 0.22 a	4.25 ± 0.52 a	3.73 ± 0.07 a	2.94 ± 0.23 a	3.45 ± 0.27 a
Benzoic acid	5.82 ± 0.35 a	2.05 ± 0.11 b	2.71 ± 0.17 b	7.34 ± 0.21 a	3.42 ± 0.12 b	3.56 ± 0.13 b
Hydrocinnamic acid	0.04 ± 0.00 b	0.03 ± 0.00 b	0.08 ± 0.01 a	0.03 ± 0.00 a	0.02 ± 0.00 a	0.03 ± 0.00 a
Trans-Cinnamic acid	0.18 ± 0.01 a	0.17 ± 0.03 a	0.22 ± 0.02 a	0.08 ± 0.02 b	0.14 ± 0.00 a	0.11 ± 0.00 ab

Note: NF indicates not found; different letters indicate significant differences at p < 0.05.

illustrate.

The enzymes produced by Bacillus subtilis disrupt the structural integrity of quinoa bran cell walls and hydrolyse glycosidic, ester, and ether bonds, thereby releasing bound phenolic acids from the fibrous matrix into their free forms [3]. In addition, cellulase catalyses the separation of bound phenolics from macromolecular fibres, further enhancing their release. Most free phenolic acids followed the trend BEFQ > BFQ > UFQ, with ferulic acid showing the most significant increase, 20.00-fold in BEFQ and 1.68-fold in BFQ. Similar results were reported by Chen D. [5]. The synergistic action of cellulase substantially increased the ferulic acid content in quinoa bran. Vanillic acid levels increased 5.76-fold in BFQ and 10.97-fold in BEFQ. Compared with that in UFQ, vanillin content increased by over 12-fold in both BFQ and BEFQ. However, cellulase supplementation had no significant effect on free vanillin, indicating that its release was primarily attributable to Bacillus subtilis fermentation.

Fermentation also reduces the levels of certain phenolic acids. The decline in benzoic acid may result from its conversion to benzoyl-CoA via the enzymatic system in Bacillus subtilis, which combines with Oxaloacetate to form Citrate and enters the TCA cycle for further metabolism to produce energy [26]. Chlorogenic acid, formed by the esterification of caffeic acid with other moieties, is hydrolysed by Bacillus subtilis-secreted esterases, leading to reduced chlorogenic acid and elevated caffeic acid levels post-fermentation [27].

Bound phenolic acids, which are typically covalently linked to cellulose, proteins, or polysaccharides, are liberated by the combined action of Bacillus subtilis extracellular enzymes and cellulase. These enzymes cleave the chemical bonds between phenolic compounds and biomacromolecules, yielding low-molecular-weight phenolic esters with enhanced bioactivity and extractability [21]. Consequently, BFQ and BEFQ showed increased bound phenolic acid content, which plays an important role in enhancing the antioxidant capacity of quinoa bran. Syringaldehyde and benzoic acid levels decreased, whereas catechin and L-epicatechin were undetectable in quinoa bran.

3.3. Principal Component Analysis (PCA) of Phenolic Acids in Quinoa Bran

Phenolic acid data from the three quinoa bran types were normalised and subjected to PCA to evaluate differences in phenolic acid composition and identify the key phenolic acids responsible for these variations. The results are shown in Figure 3. For both free and bound phenolic acids, the first principal component (PC1) accounted for 96.88% and 99.79% of the total variance, respectively, serving as the dominant factor in explaining variability and distinguishing phenolic acid profiles among the quinoa bran samples. Notably, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, and vanillic acid were the predominant contributors to both free and bound phenolic acid fractions. Similar conclusions were reported by Skrajda-Brdak for fermented dough [28].

3.4. Antioxidant Capacity of Phenolic Acids

The primary phenolic acids in quinoa bran were p-coumaric acid and ferulic acid, consistent with the findings of Lei et al. [29]. These two compounds belong to the hydroxycinnamic acid class [22], whereas 4-hydroxybenzoic acid and vanillic acid are categorised as hydroxybenzoic acids [30]. Purified forms of the four major phenolic acids from BEFQ were subjected to antioxidant capacity assays (

Table 3. Antioxidant capacity of major phenolic acids in quinoa bran.

Name	Category	Structural Formula	Molecular Formula	Molecular Weight	DPPH (IC50 µg/mL)	ABTS (IC50 µg/mL)
Ferulic acid	Hydroxycinnamic acid		C10H10O4	194.18	15.19 ± 0.62 c	5.19 ± 0.56 b
p-Hydroxycinnamic Acid			C9H8O3	164.16	15.86 ± 0.38 c	4.84 ± 0.31 b
4-Hydroxybenzoic acid	Hydroxybenzoic acid		C7H6O3	138.12	25.86 ± 0.18 a	7.27 ± 0.29 a
Vanillic acid			C8H8O4	168.14	20.82 ± 0.67 b	6.32 ± 0.21 a
Ascorbic acid	Polyhydroxyl compound		C6H8O6	176.12	5.19 ± 0.08 d	2.92 ± 0.06 c

Note: different letters indicate significant differences at p < 0.05.

). Hydroxycinnamic acids exhibited stronger radical-scavenging activity than hydroxybenzoic acids, and similar conclusions were reported by Călinoiu F. L. [31].

The antioxidant capacity of phenolic acids is determined by their chemical structure. Phenolic hydroxyl groups are critical for antioxidant activity as they neutralise free radicals by donating hydrogen atoms; however, differences in molecular weight and additional functional groups (e.g., methoxy or carboxyl groups) influence efficacy. Generally, higher molecular weight correlates with greater antioxidant activity [6]. As shown in Table 3, ferulic acid demonstrated the strongest DPPH and ABTS radical-scavenging activities among the four phenolic acids, with the lowest IC₅₀ values.

Ferulic acid contains two substituents: a 3-OCH₃ (methoxy group) at the ortho position and a 4-OH (hydroxyl group) at the para position. Its conjugated structure stabilises the phenoxy radical (ArO·) formed after hydrogen donation, thereby enhancing free radical scavenging [32]. p-Coumaric acid has a single para-hydroxyl group (4-OH) and an acrylic acid side chain that moderately stabilises the radical intermediate [23]. During oxidation, 4-hydroxybenzoic acid donates a hydrogen atom from its hydroxyl group. The resulting phenoxy radical (ArO·) stabilises the benzene ring’s conjugated system to some extent, but its antioxidant activity is weaker than that of p-coumaric acid (which contains an acrylic acid side chain) [7] and ferulic acid (which bears a methoxy substituent). Vanillic acid, which contains a methoxy group, exhibited lower IC₅₀ values for DPPH and ABTS radical-scavenging than 4-hydroxybenzoic acid. However, despite sharing a methoxy group with ferulic acid, the absence of an acrylic acid side chain in vanillic acid results in weaker antioxidant activity than that of ferulic acid [33].

Phenolic acids inhibit oxidation by chelating metal ions via their carboxyl and hydroxyl groups, thereby disrupting free radical chain reactions [34]. However, structural variations (e.g., substituent type and position) dictate specific antioxidant mechanisms and potency [35]. For example, the methoxy group of ferulic acid enhances electron delocalisation, whereas the absence of such a group in 4-hydroxybenzoic acid limits its efficacy. These structural nuances explain why ferulic acid outperforms other phenolic acids in quinoa bran as a radical scavenger.

3.5. Polysaccharide Content and Antioxidant Capacity of Quinoa Bran

After fermentation, both the polysaccharide content and antioxidant activity of quinoa bran increased, consistent with the findings of Chen [36]. The polysaccharide content of BEFQ reached 13.88%, representing an 80.73% increase compared with UFQ (7.68%) and an 18.13% increase compared with BFQ (11.75%), with significant differences between the groups. This indicates that Bacillus subtilis fermentation was the primary driver of polysaccharide release, with cellulase supplementation further enhancing this effect. It indicates that it is feasible to increase the polysaccharide content by co-fermentation. The scavenging rate of DPPH and ABTS was significantly correlated with the content of polysaccharide (p < 0.05, Pearson Correlation Coefficient were 0.978 and 0.994, respectively). Figure 4 shows the antioxidant capacity at a polysaccharide concentration of 0.5 mg mL⁻¹. Quinoa bran polysaccharides exhibited higher DPPH radical-scavenging capacity than ABTS radical-scavenging capacity, suggesting that their functional groups preferentially interact with DPPH radicals. BEFQ polysaccharides demonstrated the strongest antioxidant activity, with DPPH and ABTS scavenging capacities of 61.37% and 50.39%, respectively. These values represent increases of 52.59% and 50.48%, respectively, compared with UFQ. However, their antioxidant capacity remained lower than that of phenolic acids.

3.6. Molecular Weight of Quinoa Bran Polysaccharides

The combined action of microorganisms and cellulase during fermentation disrupts the quinoa bran cell wall, reduces mass transfer resistance [37], and promotes the release of antioxidant-active polysaccharides. The antioxidant potential of polysaccharides is closely associated with their molecular weight [38]. Figure 5 visually shows the decrease in the absolute molecular weight of quinoa bran polysaccharidesand the weight-averaged molecular weight (Mw) and number-averaged molecular weight (Mn) of polysaccharides from UFQ, BFQ, and BEFQ are shown in

Table 4. Influence of fermentation methods on the molecular weight of quinoa bran polysaccharides.

Name	Mn (×10 5Da)	Mw (×10 5Da)	Mw/Mn
UFQ	4.124 ± 0.396 a	4.700 ± 0.325 a	1.140 ± 0.31 a
BFQ	2.821 ± 0.297 ab	2.863 ± 0.240 b	1.015 ± 0.021 a
BEFQ	1.844 ± 0.185 b	2.243 ± 0.156 b	1.216 ± 0.037 a

Note: different letters indicate significant differences at p < 0.05.

. Fermentation reduced the molecular weight of the polysaccharides. BEFQ polysaccharides exhibited an Mw nearly half that of UFQ. This reduction is likely due to fermentation-induced acidification, which disrupts polysaccharide molecular chains, degrades complex macromolecules into smaller fragments, and exposes reducing ends. This process enhances polysaccharide antioxidant activity. Lower molecular weight polysaccharides also exhibit reduced viscosity and enhanced mass transfer, contributing to improved bioavailability [36]. The presence of cellulase further reduced the average molecular weight of the polysaccharides. This suggests that β-glycosidic bonds within quinoa bran polysaccharides serve as cellulase cleavage sites, explaining the lower average molecular weight of BEFQ polysaccharides. The polydispersity index (Mw/Mn) of the three quinoa bran polysaccharides showed no significant differences, with all values below 2. This indicates a relatively narrow molecular weight distribution and good dispersity of quinoa bran polysaccharides [17].

3.7. Monosaccharide Composition of Quinoa Bran Polysaccharides

The monosaccharide composition of quinoa bran polysaccharides obtained by different fermentation methods is presented in

Table 5. Influence of fermentation methods on the monosaccharide composition of quinoa bran polysaccharides.

	Ara (mg/g)	Rha (mg/g)	Gal (mg/g)	Glc (mg/g)	Gal-UA (mg/g)	Glc-UA (mg/g)
UFQ	16.17 ± 0.36 c	3.72 ± 0.16 a	13.05 ± 0.73 a	163.25 ± 1.01 c	13.20 ± 1.06 a	16.24 ± 1.13 a
BFQ	19.46 ± 1.02 b	3.90 ± 0.21 a	12.75 ± 0.88 a	209.93 ± 1.23 b	15.70 ± 0.97 a	13.88 ± 0.61 a
BEFQ	22.46 ± 0.64 a	3.64 ± 0.33 a	12.14 ± 0.69 a	243.97 ± 1.14 a	11.82 ± 0.65 a	13.24 ± 0.99 a

Note: different letters indicate significant differences at p < 0.05.

. Quinoa bran polysaccharides were composed of glucose, arabinose, galactose, rhamnose, galacturonic acid, and glucuronic acid. Among these, glucose, arabinose, and galactose were the predominant monosaccharides. Cellulase primarily targets β-1,4-glycosidic linkages [39]. Co-fermentation with microorganisms and cellulase increased glucose content by 49.08%. This suggests that the main chain of quinoa bran polysaccharides comprised glucose and arabinose residues linked via β-1,4-glycosidic bonds. Galactose and rhamnose contents showed no significant differences between BFQ and BEFQ groups. This suggests that these monosaccharides were located on the periphery of the polysaccharide chains or on shorter side chains, potentially connected by α-1,4-glycosidic linkages, which are not cleaved by cellulase.

3.8. Protein Content and Antioxidant Capacity of Quinoa Bran

Fermentation reduced the protein content of quinoa bran, with BFQ showing a 13.67% decrease as Figure 6 shows. This reduction is attributable to the high protease-producing capacity of Bacillus subtilis. During fermentation, substantial amounts of extracellular proteases are secreted. These enzymes hydrolyse structurally complex, high-molecular-weight quinoa bran proteins into low-molecular-weight peptides and free amino acids. In synergistic fermentation, cellulase effectively hydrolyses structural polysaccharides, such as cellulose and hemicellulose, in quinoa bran, disrupting the dense bran cell wall and exposing otherwise entrapped proteins [40]. This disruption increases the accessible surface area for enzyme-protein interactions, thereby promoting the conversion of quinoa bran proteins into peptides and free amino acids. Consequently, BEFQ exhibited a 21.88% reduction in protein content.

Antioxidant-active sites in proteins are often embedded within complex tertiary structures. Hydrolysis into peptides exposes amino acid residues with reducing capacity, facilitating their interaction with and neutralisation of free radicals. Additionally, low-molecular-weight peptides are more readily absorbed and can reach the target sites to exert antioxidant effects more efficiently [41]. The antioxidant capacity of quinoa bran protein before and after fermentation was evaluated using DPPH and ABTS radical-scavenging assays, respectively. It was found that the differences between them were significant. The DPPH scavenging rate of BFQ (36.04%) was 2.12-fold higher than that of UFQ (17.00%). Similarly, the ABTS scavenging rate of BFQ (43.55%) was 2.06-fold higher than that of UFQ (21.09%). In contrast, BEFQ proteins showed DPPH and ABTS scavenging rates of 29.92% and 36.99%, respectively, slightly lower than those of BFQ. These results demonstrated that fermentation significantly reduced the protein content of quinoa bran while enhancing its antioxidant capacity. However, microbial-enzyme co-fermentation slightly reduced antioxidant capacity relative to microbial fermentation alone. Overall, the antioxidant capacity of quinoa bran proteins was lower than that of both phenolic acids and polysaccharides.

3.9. Amino Acid Composition of Quinoa Bran

Amino acids are the fundamental building blocks of proteins, and their composition and concentration play critical roles in determining antioxidant activity. As

Table 6. Effects of fermentation methods on the composition of free amino acids in quinoa bran.

Name	UFQ	BFQ	BEFQ
EAA/(ug/g)
Phenylalanine (Phe) h	22.02 ± 1.03 c	854.87 ± 6.31 a	231.80 ± 5.22 b
Methionine (Met) h	1.59 ± 0.05 c	244.86 ± 10.13 a	49.87 ± 2.15 b
Lysine (Lys)	34.55 ± 2.35 c	463.62 ± 16.77 a	171.11 ± 8.23 b
Leucine (Leu) h	25.53 ± 0.13 c	153.83 ± 6.44 b	315.56 ± 10.19 a
Tryptophan (Trp)	12.86 ± 0.83 c	178.50 ± 8.04 a	71.29 ± 6.33 b
Threonine (Thr)	33.02 ± 0.97 c	69.78 ± 1.05 b	88.57 ± 1.85 a
Valine (Val) h	40.31 ± 0.55 c	126.28 ± 1.52 b	136.83 ± 1.37 a
Isoleucine (Ile) h	20.83 ± 0.05 b	88.30 ± 2.33 a	86.53 ± 2.39 a
Histidine (His)	9.09 ± 0.09 b	8.73 ± 0.12 b	208.48 ± 4.37 a
NEAA/(ug/g)
Alanine (Ala) h	90.54 ± 2.13 c	201.28 ± 3.10 b	268.48 ± 3.64 a
Glycine (Gly)	50.73 ± 1.59 b	96.53 ± 2.13 a	102.73 ± 4.16 a
Glutamic acid (Glu)	319.35 ± 5.78 b	345.42 ± 6.11 b	455.95 ± 5.69 a
Arginine (Arg)	251.50 ± 8.83 b	102.65 ± 6.12 c	999.70 ± 12.14 a
Tyrosine (Tyr)	35.08 ± 0.12 c	917.16 ± 14.16 a	216.62 ± 7.21 b
Proline (Pro) h	115.54 ± 4.67 b	69.14 ± 5.26 c	351.70 ± 7.34 a
Serine (Ser)	50.55 ± 0.56 b	49.13 ± 0.69 b	137.06 ± 3.44 a
Aspartic acid (Asp)	108.35 ± 3.99 b	67.55 ± 1.14 c	216.31 ± 4.09 a
Ornithine (Orn)	5.70 ± 0.05 b	34.82 ± 0.34 a	1.32 ± 0.00 c
Citrulline (Cit)	2.45 ± 0.01 b	16.98 ± 0.58 a	3.61 ± 0.10 b
Total EAA/(ug/g)	199.80 ± 2.59 c	2188.77 ± 20.17 a	1360.04 ± 10.21 b
HAA/(ug/g)	329.22 ± 6.12 c	1917.06 ± 13.33 a	1512.06 ± 12.35 b
Total AA/(ug/g)	1229.59 ± 10.79 b	4089.43 ± 15.32 a	4113.52 ± 22.28 a
EAA/AA	0.16	0.54	0.33
HAA/AA	0.27	0.47	0.37

Note: EAA, essential amino acid; NEAA, non-essential amino acid; AA, amino acids; HAA(h), hydrophobic amino acids. Different letters indicate significant differences at p < 0.05.

shows, a total of 19 free amino acids, including nine essential amino acids, were detected in quinoa bran. Fermentation significantly increased the concentrations of both free and essential amino acids. This increase resulted from the efficient hydrolysis of quinoa bran proteins by proteases secreted by Bacillus subtilis. Similar findings were reported by Aboobacker [42] in a study on Spirulina fermentation. The essential amino acid ratio in BFQ reached the WHO/FAO recommended value of 0.40. Prior to fermentation, glutamic acid (Glu, 319.35 ± 5.78 μg/g) was the most abundant free amino acid in quinoa bran. Following Bacillus subtilis fermentation, the concentrations of 14 free amino acids, including eight essential amino acids, increased significantly. Tyrosine (917.16 ± 14.16 μg/g) was the most abundant amino acid. Following microbial–cellulase co-fermentation, the concentrations of all 18 detected free amino acids increased. Arginine (Arg, 999.70 ± 12.14 μg/g) was the most abundant amino acid in BEFQ. Although the total free amino acid concentration in BEFQ was similar to that in BFQ, the essential amino acid concentration decreased by 21.13%. This decrease is likely attributable to cellulase hydrolysing recalcitrant cellulose and hemicellulose in quinoa bran into monosaccharides, thereby providing Bacillus subtilis with abundant utilisable carbon sources. This promoted rapid microbial growth and proliferation. Consequently, Bacillus subtilis utilised the liberated free amino acids for microbial protein synthesis, reducing the essential amino acid concentration in BEFQ. In contrast, slower microbial growth during Bacillus subtilis-only fermentation resulted in lower amino acid consumption.

Hydrophobic amino acids have been reported to confer higher antioxidant activity to proteins [43]. This may be due to hydrophobic side chains facilitating the enrichment of antioxidant amino acids, thereby enhancing free radical scavenging efficiency. After fermentation, the hydrophobic amino acid ratio increased to 0.47 in BFQ and 0.37 in BEFQ, enhancing the bioactivity of fermented products. Xin Cui [44] demonstrated the critical role of sulphur-containing amino acids in the antioxidant activity of Tenebrio Molitor proteins. Methionine (Met) was the only sulphur-containing amino acid detected in quinoa bran. The Met concentration in BFQ (244.86 ± 10.13 μg/g) was 154.00-fold higher than that in UFQ (1.59 ± 0.05 μg/g) and 4.91 times than that in BEFQ (49.87 ± 2.15 μg/g). In contrast, Fu [39] observed a decrease in free sulfhydryl group content, derived from sulphur-containing amino acids, during cellulase-synergistic fermentation of sourdough. Furthermore, functionally active amino acids such as histidine (His) and tyrosine (Tyr) were present in fermented bran. Amino acids may contribute to antioxidant processes through specific bioactivities, including potential metal chelation or radical scavenging.

4. Conclusions

Quinoa bran, a by-product of quinoa processing, is rich in bioactive components, such as phenolics, proteins, and polysaccharides. In industrial practice, it is often discarded as waste. This study systematically investigated the effects of cellulase-assisted Bacillus subtilis fermentation on the functional components of quinoa bran and compared the content, composition, and antioxidant activity of phenolic acids, polysaccharides, and proteins in UFQ, BFQ, and BEFQ. The results demonstrated that fermentation, particularly in combination with cellulase, significantly altered the biochemical profile of quinoa bran, promoting the release of phenolic acids, polysaccharides, and amino acids, thereby enhancing the antioxidant activity of these functional components. Among the bioactive constituents, phenolic acids, particularly hydroxycinnamic acids, exhibited the highest antioxidant capacity in fermented quinoa bran. Hydroxycinnamic acids, the primary antioxidant constituents within phenolic acids, made a major contribution to the enhanced antioxidant capacity of quinoa bran. In conclusion, cellulase-assisted Bacillus subtilis fermentation proved highly effective in improving the bioactive composition and antioxidant activity of quinoa bran. These findings provide new insights into the high-value utilization of quinoa bran as a functional food ingredient and support its potential application in the development of food additives for antioxidant-rich products, functional beverages, and nutritional foods.