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Chemical and Biological Technologies in Agriculture
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Polyphenol composition and antioxidant activity of fermentation combined with enzymatic hydrolysis modified Astragalus membranaceus stems

Chemical and Biological Technologies in Agriculture volume 11, Article number: 153 (2024) Cite this article

Abstract

Astragalus membranaceus (AM) roots are a well-known homologous medicine and food in China. AM stems, which are discarded and not used effectively, also contain many active compounds and exhibit beneficial effects. It has the potential to be explored as antibiotic alternative. Fermentation combined with enzymatic hydrolysis (FEH) is an effective strategy for extracting polyphenol and improving the usage of AM stems. Therefore, in this study, the conditions of FEH and extraction for polyphenol in AM stems were screened. The antioxidant activity of extract from AM stems without and with FEH (AMSE and FAMSE) was evaluated. The metabolite profiles of phenolic acids and flavonoids in AMSE and FAMSE were characterized by ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS). The results showed that the highest polyphenol content from AM stems was obtained with cellulase and pectinase (1:1, 2000 U/g), moisture content 43%, fermentation temperature 30 °C, and fermentation time 7 days. Selected extraction conditions of polyphenol were ethanol concentration 50%, ultrasonic power 500 W, extraction temperature 35 °C, and extraction time 40 min. On this condition, compared with AMSE, the polyphenol and flavonoid contents in FAMSE were significantly higher. FAMSE exhibited stronger DPPH, hydroxyl radical scavenging rate and reducing power than AMSE. The relative content of 3-(4-hydroxyphenyl)-propionic acid, dihydroferulic acid, isoferulic acid, 4-hydroxybenzoic acid, 4-hydroxyphenyllactic acid, ferulic acid, vanillic acid, syringic acid, gentisic acid, sinapic acid, apigenin, tricin, acacetin, daidzein, genistein, formononetin, prunetin, pratensein, rhamnocitrin and galangin were significantly upregulated in FAMSE.

Graphical Abstract

Introduction

Synthetic antibiotics have been used in farm animal production for decades to prevent and treat infectious diseases and improve animal productivity [1]. However, excessive use of antibiotics may lead to antibiotic residue in animal products and further hazards to human health [2, 3]. The need to have research on medicinal plants has been acknowledged worldwide by practitioners in order to find safe and effective antibiotic alternatives. Traditional Chinese Herbal Medicine plants, such as Astragalus membranaceus (AM), have been found that using as antibiotic replacements could increase growth performance, improve feed efficiency, and enhance antioxidant capacity and immunity of farm animals [4, 5]. AM is a perennial medicinal herb, whose roots is a well-known homologous medicine and food in China [6]. AM roots contains various active ingredients, such as polyphenol, flavonoids, polysaccharides, triterpenes, and saponins [7, 8], and has a broad range of bioactivities, including antioxidant, immune regulation, anti-inflammatory, anticancer and antivirus [9,10,11,12]. Several reports are available for antioxidant activities and immune functions of aerial parts of AM, such as stems and leaves [13,14,15,16]. Nayeem et al. found that methanolic extracts of AM stems exhibited analgesic and anti-inflammatory activity due to presence of polyphenol [17]. Thus, polyphenol of AM stems has the potential to be explored as a new antibiotic alternative.

Polyphenol are crucial secondary plant metabolites, which possess at least one aromatic ring with one or more hydroxyl substituents [18]. These compounds represent the most important group of natural antioxidants [19], which can reduce inflammation and ameliorate oxidative stress [20, 21]. Polyphenol can be categorized into two main subgroups: phenolic acids and flavonoids [22]. Cui et al. obtained two main flavonoids, isoquercitrin and astragalin, from AM stems with strong antioxidant activity [16]. However, due to plant cell wall recalcitrance, the extraction of AM stems polyphenol is limited [23]. It is suggested that fermentation is a useful strategy, which could effectively release the polyphenol from processing by-products of cereals, fruits, and vegetable oil [24]. Wang et al. observed an increase in polyphenol content of fermentation of rapeseed meals with mixed strains of Bacillus subtilis and Saccharomyces cerevisiae [25]. Qiao et al. reported that fermentation by Lactobacillus plantarum and/or Enterococcus faecium increased the production of polysaccharides, saponins, and flavonoid of AM roots [26]. During the fermentation, extracellular enzymes such as cellulase and pectinase were produced by microorganisms, which could rupture the herbal cell wall and release the bioactive ingredients inside [27]. Recently, fermentation combined with enzymatic hydrolysis (FEH) is regarded as a more promising method for the bioconversion of biomass or production active ingredients. Bei et al. found that compared to the microbial fermentation, the polyphenol contents of oats were increased by FEH [28]. Liu et al. proved that FEH enhanced the polyphenol content and antioxidant activities of aqueous solution of rice bran [29]. Moreover, our previous study also found that FEH with mix strain (S. cerevisiae, B. subtilis, and L. plantarum at a ratio of 1:1:1) and 500 U/g glucanase enhanced polyphenol contents and antioxidant activities of rice bran [30].

Thus, this study was conducted to screen conditions of FEH and extraction for polyphenol in AM stems, evaluate the in vitro antioxidant activity as well as phenolic acids and flavonoids metabolite profile of polyphenol in AM stems extracts without and with FEH (AMSE and FAMSE) and provide a new antibiotic alternative rich in polyphenol from AM stems.

Materials and methods

Materials

AM stems, soybean meal, wheat bran, red dates powder, and molasses were purchased from the local market in Guyang, Baotou, Inner Mongolia, China. The stems of AM were air-dried, cut into slices, and used as the main substrate for FEH. Bacillus subtilis (CGMCC 1.0892), Lactobacillus plantarum (CGMCC No. 1.12934), and Saccharomyces cerevisiae (CGMCC No. 2.1190) were purchased from the China General Microbiological Culture Collection Centre (Beijing, China). Cellulase, pectinase, and xylanase were obtained from Beijing Solai Biotechnology Co., LTD. (Beijing, China). Other regents were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Screening of FEH conditions for AM stems

The AM stems is processed by FEH to release polyphenol. Based on previous studies, B. subtilis and S. cerevisiae were precultured in nutrient broth medium and malt extract medium at 35 °C and 30 °C in a rotary shaker (120 rpm) for 24 h, respectively. L. plantarum was precultured in MRS broth at 37 °C for 24 h. The compound probiotics inoculum was prepared by mixing the three inoculants (1.0 × 108 CFU/mL) at a ratio of 1:1:1. The AM stems (73%, w/w) were mixed with soybean meal (9%, w/w), wheat bran (9%, w/w), red dates powder (4.5%, w/w), and molasses (4.5%, w/w), and the mixture was inoculated with 1.0% of compound probiotics inoculum. After stirring evenly, a certain amount of distilled water was added. The effects of enzymatic type (cellulase, xylanase, pectinase, and cellulase + pectinase), enzyme amount (1000, 2000, 3000, and 4000 U/g), moisture content (20%, 33%, 43%, 50% and 55%, v/w), fermentation temperature (22 °C, 26 °C, 30 °C, and 32 °C) and fermentation time (3, 5, 7, and 9 d) on polyphenol content were investigated with a single-factor optimization experiments. The fermented products were dried at 45 °C and crushed to determine polyphenol content. And the polyphenol content of AM stems without fermentation or enzymatic hydrolysis (AMS) and AM stems with FEH under the optimum conditions (FAMS) were compared.

Screening of polyphenol extraction conditions from FAMS

The FAMS were prepared by the above optimum FEH conditions. Then, the ultrasonic-assisted ethanol extraction method was used to extract the polyphenol of FAMS. An ultrasonic cleaner (Branson 8510 Ultrasonic Cleaner, USA) was used. The ratio of ethanol to sample was 1:30. The effects of concentration of ethanol (40%, 50%, 60%, 70%, and 80%), ultrasonic power (200, 300, 400, and 500 w), ultrasonic extraction temperature (35 °C, 45 °C, 55 °C, 65 °C and 75 °C) and ultrasonic extraction time (20, 30, 40, 50 and 60 min) on polyphenol content were investigated with a single-factor optimization experiments. The extract was filtered, then the supernatant was centrifuged (3500 rpm, 15 min), concentrated and freeze-dried by a vacuum freeze-dryer (Scientz-100F). And the polyphenol and flavonoid contents of extracts of AMS and FAMS (AMSE and FAMSE) were determined.

The determination of polyphenol and flavonoid contents

The polyphenol contents were determined using previous methods [30] with gallic acid as the standard. The determination of flavonoid contents was conducted followed our previous method [31].

In vitro antioxidant activity of AMSE and FAMSE

The in vitro antioxidant activities AMSE and FAMSE were estimated by DPPH and hydroxyl radical scavenging activities as well as reducing power. DPPH radical scavenging activity and reducing power were determined by the method of Liu et al. [31]. The hydroxyl radical scavenging activity was determined with the method of Yin et al. [32].

Phenolic acids and flavonoids metabolite profile of polyphenol in AMSE and FAMSE

The phenolic acids and flavonoids metabolite profile of polyphenol in AMSE and FAMSE was analyzed by a UPLC–ESI-MS/MS system. The samples were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. The 100 mg of sample was dissolved with 1.2 mL 70% methanol solution, vortexed 30 s every 30 min for 6 times, and placed in a refrigerator at 4 °C overnight. Following centrifugation at 12,000 rpm for 10 min, the solution were filtrated (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China) before UPLC–MS/MS analysis. The HPLC and mass spectrum conditions were conducted with previous method [33]. Based on the self-built database MWDB (Metware Biotechnology Co., Ltd. Wuhan, China) and public database of metabolite information, the qualitative analysis of substance was performed using secondary mass spectrometry data. The quantification of metabolites was performed using multiple reaction monitoring (MRM) mode analysis [33]. Three replicates for AMSE and FAMSE were performed.

Statistical analysis

All experiments were performed in triplicate. For screening conditions of FEH and extraction for polyphenol in AM stems, one-way ANOVA followed by Duncan’s multiple range tests in SAS were used. The differences were accepted as significant at P < 0.05. The data are expressed as the mean ± standard deviation.

Metabolite data were log2-transformed for statistical analysis to improve normality and were normalized. Principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) were used to analyze the multivariate differences of metabolites by R software. The metabolites with a fold change (FC) ≥ 2 or ≤ 0.5 and a variable importance in projection (VIP) ≥ 1 was considered as significantly differential.

Results and discussion

Screening of FEH conditions for AM stems

Microbial fermentation is a traditional process for modifying traditional Chinese medicine. During fermentation, cell wall hydrolyzing performed by enzymes, such as cellulases, amylase, hemicelluloses, and pectinase, play critical roles in releasing bound polyphenol [27]. Park et al. found that the phenolic contents of AM was significantly increased after Lactiplantibacillus plantarum fermentation [34]. Moreover, Wang et al. reported that complex enzymatic hydrolysis (cellulase, xylanase, hemicellulose and β-glucosidase) after fermentation increased the contents of phenolics, flavonoids, quercetin, and kaempferol in guava leaves tea [35]. Bei et al. found that the polyphenol contents of oats treated by FEH was higher than that by microbial fermentation [28]. It is indicated that FEH might be a more promising method for releasing bound polyphenol. Therefore, the effects of enzymatic type and enzyme amount were investigated. As presented in Fig. 1A, compared with xylanase, the usage of cellulase and pectinase alone significantly increased polyphenol content (P < 0.05). The highest polyphenol content was achieved using cellulase + pectinase (1:1). With increasing enzyme amount, the polyphenol content increased and then decreased, and the highest polyphenol content was reached with adding 2000 U/g enzymes (Fig. 1B). These results indicated that adding 2000 U/g cellulase + pectinase could hydrolyze the cell wall structure of AM stems and release polyphenol. Cellulases refer to a multi-enzyme mixture and have been reported as an effective tool for hydrolyzing plant cell walls [28]. Pectinase is also a cell wall degrading enzyme which degrades pectic polymers into galacturonic acid [36]. Consistent with our results, Siddiq et al. reported that pectinase and cellulase increased the phenolic contents by up to 6.4 times compared to the untreated blueberry juice [37].

Fig. 1
figure 1

Effects of FEH conditions on the polyphenol content of AM stems. A Enzyme type. B Enzyme amount. C Moisture content. D Fermentation temperature. E Fermentation time. Different lowercase letters in bars represent significant differences (P < 0.05)

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Moisture content, fermentation temperature, and fermentation time are essential factors for metabolite production during fermentation [31]. The suitable moisture content was found to be 43%, which yielded the highest polyphenol content (83.09 mg/g) (Fig. 1C). As shown in Fig. 1D, the polyphenol content increased with increasing fermentation temperature, and the maximum concentration was recorded at 30 °C. Then, at higher fermentation temperature, the polyphenol content decreased. The polyphenol contents significantly increased from 3 to 7 days, reached maximum values of 97.39 mg/g on the day 7, then declined in the subsequent culture (Fig. 1E). Our results suggested that the suitable FEH conditions of AM stems were as follows: enzyme type cellulase + pectinase (1:1), enzyme amount 2000 U/g, moisture content 43%, fermentation temperature 30 °C, and fermentation time 7 days. Under the suitable FEH conditions, the polyphenol content of AM stems was 85.48 mg/g, which was 2.43 times higher than that without FEH treatment (24.93 mg/g).

Screening of polyphenol extraction conditions from FAMS

Ultrasonic-assisted extraction technique is a proven green bio-refining technology [38], which can destroy cell walls to promote solute diffusion and increase the extraction efficiency of target compounds [39, 40]. Iftikhar et al. found that ultrasonic-assisted extraction resulted in a higher yield of rye bran's phenolic and flavonoid compounds than the conventional extraction technique [41]. Therefore, the ultrasonic-assisted extraction was used in this study to extract polyphenol from FAMS. Ethanol is generally recognized as a safe solvent and has been used to extract polyphenol. The ethanol concentration affects the target components' solubility and extraction yield [16]. As shown in Fig. 2A, the polyphenol content was significantly higher when the ethanol concentration was 50% and 60% (P < 0.05), however, there was no remarkable difference between 50 and 60%. Therefore, ethanol concentration of 50% was screened for the subsequent extraction test. With ultrasonic power increasing from 200 to 500 W, the polyphenol content increased (Fig. 2B), which was agreed with Ma et al., who confirmed the positive effects of increasing ultrasonic power on polyphenol content [42]. Thus, the ultrasonic power 500 W was chosen to extract polyphenol from FAMS. It is found that appropriate heat treatment increases the solubility of polyphenol in the solution [43]. The effects of ultrasonic extraction temperature on the polyphenol content are presented in Fig. 2C. It was found that highest polyphenol content was achieved at 35 °C. Furthermore, longer extraction time may accelerate the absorption of solvent, soften the plant tissues, weaken the cell wall integrity, and thus enhanced ingredient solubility [43]. However, excessive extraction time may oxidize polyphenol, reduce extraction efficiency, and waste time [16]. In this study, the polyphenol content fluctuated between 62.03 and 132.80 mg/g with 20–60 min extraction time (Fig. 2D). It can be seen that extraction time had a significant effect on polyphenol content of FAMS (P < 0.05). The polyphenol content was gradually increased up to 40 min of extraction and then started falling. Our results indicated that the suitable extraction conditions of polyphenol from FAMS were as follows: ethanol concentration 50%, ultrasonic power 500 W, extraction temperature 35 °C, and extraction time 40 min.

Fig. 2
figure 2

Effects of ultrasonic-assisted ethanol extraction conditions on the polyphenol content of FAMS. A Concentration of ethanol. B Ultrasonic power. C Ultrasonic extraction temperature. D Ultrasonic extraction time

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Polyphenol and flavonoid contents in AMSE and FAMSE

Figure 3 shows the polyphenol and flavonoid contents in AMSE and FAMSE. Under the optimal FEH and extraction conditions, the polyphenol content in AMSE and FAMSE were 66.22 mg/g and 129.34 mg/g, respectively. And the flavonoid contents in AMSE and FAMSE were 1.35 mg/g and 5.72 mg/g, respectively. Compared with AMSE, polyphenol and flavonoid contents in FAMSE was significantly higher by 92.46% and 323.70%, respectively (P < 0.05). A study investigated the effects of fermentation by Lactiplantibacillus plantarum on AM, and found that fermented AM had higher phenolic content [34]. It was also reported that hydrolysis with carbohydrase enzymes, such as pectinases and cellulases, at suitable conditions effectively released the insoluble phenolics [44]. More importantly, it was found that the flavonoid content in AM was significantly increased with Lactobacillus pentosus Stm solid-state fermentation combined with the addition of xylanase, cellulase, and pectinase [45]. Consistent with previous research findings, FSH treatment increased polyphenol and flavonoid contents in AM stems in this study. The release of polyphenol and flavonoid contents may be due to both the utilization of cell wall polysaccharides by probiotics and hydrolyzation of ester bond between phenolics and cell wall components by carbohydrase [29, 44].

Fig. 3
figure 3

Polyphenol and flavonoid contents in AMSE and FAMSE

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In vitro antioxidant activities of AMSE and FAMSE

Reactive oxygen (ROS) and reactive nitrogen (RNS) species are produced during the metabolism in the animal body. Over production of ROS and RNS may lead to physiological imbalances, which result in chronic inflammation and numerous diseases [46]. Natural antioxidants from plants can protect animal body from adverse free radicals and prevent many diseases. Previous studies have reported that polyphenol are natural antioxidants against ROS and RNS species [47]. The improvement of antioxidant capacity was strongly associated with increased polyphenol content [44]. It was found in this study that FSH treatment increased polyphenol and flavonoid contents in AM stems. Therefore, the DPPH and hydroxyl radical scavenging rate as well as reducing power of AMSE and FAMSE were tested (Fig. 4). As shown in Fig. 4A–C, both AMSE and FAMSE exhibited antioxidant activity in the test concentration. Notably, the DPPH and hydroxyl radical scavenging rate as well as reducing power of FAMSE were significantly higher than those of AMSE (P < 0.05). Consistently, Kuo et al. revealed that fermentation improved the phenolic content and scavenging activity against DPPH and ABTS of Chenopodium formosanum Koidz [48]. Zhang et al. found FEH enhanced phenolic compound content and DPPH radical scavenging rate of quinoa [49].

Fig. 4
figure 4

Antioxidant activities of AMSE and FAMSE. A DPPH radical scavenging rate. B Hydroxyl radical scavenging rate. C Reducing power. Different lowercase letters in bars represent significant differences between AMSE and FAMSE (P < 0.05)

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Phenolic acids metabolite profile of polyphenol in AMSE and FAMSE

Phenolic acids are hydroxyl derivatives of aromatic carboxylic acids, which are powerful natural antioxidants [49]. A total of 179 phenolic acids were identified in AMSE and FAMSE. In the PCA score plot (Fig. S1A), three principal components (PC1, PC2, and PC3) were 77.94%, 6.98%, and 5.93%, respectively. In verification chart of OPLS-DA model (Fig. S1B), The R2X, R2Y and Q2 were 0.862, 1 and 0.995, respectively. It is demonstrated that the model was stable and reliable by the Q2 values exceeded 0.9. The PCA and OPLS-DA analysis showed that the two groups were well-separated, suggesting that FSH significantly affected phenolic acids metabolite profile of AM stems. There were 119 significantly different phenolic acids metabolites between AMSE and FAMSE, among which 39 were upregulated and 80 were down-regulated (Fig. S1C).

The differential phenolic acids metabolites between AMSE and FAMSE are shown in Table 1. In comparison with AMSE, the relative contents of 3-(4-hydroxyphenyl)-propionic acid, dihydroferulic acid (DHFA), isoferulic acid (IFA), 4-hydroxybenzoic acid, 2-hydroxy-3-phenylpropanoic acid, moracin Y, 4-hydroxyphenyllactic acid, ferulic acid (FA), vanillic acid, syringic acid, 2,3-dihydroxybenzoic acid, gentisic acid, benzoic acid and sinapic acid in FAMSE increased, while the relative contents of 4-O-glucosyl-4-hydroxybenzoic acid, glucosyloxybenzoic acid, salidroside, 1-O-salicyloyl-β-d-glucose, 1-(4-hydroxybenzoyl)glucose, 6ʹ-O-feruloyl-d-sucrose, 5-hydroxyisovanillic acid, 4-hydroxybenzoic acid glucosyl xyloside, 3,4-dihydroxybenzaldehyde-xylose-glucoside, benzoylmalic acid, dihydroferulic acid glucoside and glucosyringic acid decreased. It is reported that 4-O-glucosyl-4-hydroxybenzoic acid, glucosyloxybenzoic acid, 1-O-salicyloyl-β-d-glucose, 1-(4-hydroxybenzoyl)glucose, 5-hydroxyisovanillic acid, 4-hydroxybenzoic acid glucosyl xyloside, 3,4-dihydroxybenzaldehyde-xylose-glucoside, benzoylmalic acid and glucosyringic acid could be degraded into other compounds, such as 4-hydroxybenzoic acid, vanillic acid, syringic acid, 2,3-dihydroxybenzoic acid, gentisic acid and benzoic acid, by enzymes (cellulase, pectinase et al.) produced by microbial metabolism or added exogenous. 6ʹ-O-feruloyl-d-sucrose, dihydroferulic acid glucoside also could be metabolized into other compounds, such as DHFA, IFA and FA. And salidroside could probably degraded into tyrosol and glucose by β-d-glucosidase [49].

Table 1 Relative content of part of phenolic acids differential metabolites in AMSE and FAMSE
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Phenolic acids can be further divided into two distinctive groups: hydroxycinnamic (basic skeleton C6-C3) acids and hydroxybenzoic (basic skeleton C6-C1) [50]. Ferulic, caffeic, p-coumaric, and sinapic acids are major representative substances of hydroxycinnamic acids [51]. As the major hydroxycinnamic acid, FA is widely known for its antioxidant, antimicrobial, and anti-inflammatory properties [52]. In this study, the relative content of FA (3.93-fold) in FAMSE was higher compared to AMSE (Table 1). FA can be metabolized to DHFA and IFA [53]. It is reported that DHFA is a better antioxidant and anti-inflammatory than FA [54, 55]. A study by Lee et al. revealed that DHFA obtained from fermented rice bran extract reversed the reduction of viability of PC12 cells caused by H2O2 and enhanced the transcription levels of antioxidant genes [56]. The IFA could be used for treating several inflammatory diseases, clearance of ROS, elimination of viral infections, and alleviation of metabolic diseases and specific cancers [57]. Furthermore, esters form of FA, methyl ferulate, and ethyl ferulate, were proven to more efficiently prevent lipid oxidation [58, 59]. As shown in Table 1, the relative contents of DHFA, IFA, ethyl ferulate and ferulic acid methyl ester in FAMSE were upregulated by 104.45-, 3.34-, 13.65- and 6.21-fold, respectively. Additionally, the relative contents of sinapic acid were increased by 1.34-fold. Sinapic acid has been reported against various pathological conditions, such as oxidative stress, inflammation, infections, and cancer [60].

The family of hydroxybenzoic acids occurs naturally in plants and is the simplest class of natural antioxidants [21]. As a type of hydroxybenzoic acids, 4-hydroxybenzoic acid is a valuable aromatic compound used as raw material for producing liquid crystal polymers and paraben, which shows the higher antioxidant activity compared with its esters [61]. Vanillic acid is an oxidized form of vanillin with pleasant creamy odor and antioxidative activity [62]. Syringic acid is a high bioavailability and low toxicity polyphenol, which can be administered as a preventative measure against a range of diseases due to its antioxidant activity [63]. Similarly, many studies have been published about the antioxidant activity of gentisic acid, which have proposed its potential use in treating diseases [64]. As shown in Table 1, the relative contents of 4-hydroxybenzoic acid (3.70-fold), vanillic acid (2.51-fold), syringic acid (4.45-fold), 2,3-dihydroxybenzoic acid (26.93-fold), gentisic acid (2.53-fold) and benzoic acid (3.00-fold) in FAMSE significantly accumulated. Consistent with our results, Degrain et al. revealed that 2,5-dihydroxybenzoic acid was the predominant phenolic acid in the L. plantarum-fermented nightshade leaves, and L. plantarum fermentation improved the bioavailability of vanillic acid of nightshade leaves [65]. Darwesh et al. demonstrated that contents of p-hydroxybenzoic acid, gentisic acid, and syringic acid increased in Saccharomyces cerevisiae-fermented cinnamon [66]. Azkia et al. found that fermentation enhanced vanillic acid and vanillin levels in black glutinous rice [67]. In addition, 3-(4-hydroxyphenyl)-propionic acid (HPPA) and 4-hydroxyphenyllactic acid increased in FAMSE in this study. It is reported that 4-hydroxyphenyllactic acid can be produced by many lactic acid bacteria [68]. HPPA was a major microbial metabolite of A-type procyanidin, and inhibited suppressing effects on cellular oxidative stress and inflammation [69, 70].

Our results revealed that FEH changed phenolic acids metabolite profile of AM stems. The relative content of HPPA, DHFA, IFA, 4-hydroxybenzoic acid, 4-hydroxyphenyllactic acid, FA, vanillic acid, syringic acid, gentisic acid, and sinapic acid were significantly increased in FAMSE, which may be the reason for its enhanced antioxidant activity.

Flavonoids metabolite profile of polyphenol in AMSE and FAMSE

Flavonoids is an essential group of naturally occurring polyphenol compounds with basic structures consist of C6-C3-C6 rings. According to the substitution patterns, flavonoids could be categorized into a series of subclass, isoflavones, flavones, flavonols, flavanones, flavanols, and anthocyanins [71]. In this study, a total number of 463 flavonoids were identified in AMSE and FAMSE. Isoflavones, flavones, and flavonols were the main subclasses of flavonoids, which account for more than 70.84% of flavonoids in AMSE and FAMSE (Fig. 5). AMS and FAMS were separated in Fig. S2A, indicating that FEH had a substantial effect on flavonoids metabolite profile of AM stems. As shown in Fig. S2B, the R2X, R2Y and Q2 were 0.845, 1 and 0.994, respectively, demonstrating that the OPLS-DA model was stable and reliable and could be applied to further screen differential flavonoids metabolites. There were 245 significantly differential flavonoids metabolites between AMSE and FAMSE (77 upregulated, 168 down-regulated) (Fig.S2C).

Fig. 5
figure 5

Flavonoids classification chart according to subgroups

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The differential flavonoids metabolites between AMSE and FAMSE were mainly classified into three subgroups, isoflavones, flavones, and flavonols, which account for 75.51% of the total differential flavonoids metabolites (Fig. 6A). As shown in Fig. 6B, most of the down-regulated flavonoids metabolites in FAMSE have molecular weights greater than 400 Da. In contrast, the molecular weights of upregulated flavonoids metabolites ranged in 200–400 Da and 500–600 Da.

Fig. 6
figure 6

Classification (A) and molecular weight range (B) of differential flavonoids metabolites between AMSE and FAMSE

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Flavones is the most common flavonoids subclass, found in herbs and vegetables [21]. Apigenin, tricin and acacetin are the representative compounds of flavones. Apigenin is a polyphenolic flavone with a low molecular weight, responsible for antioxidant potential and chelating redox-active metals [72]. Tricin typically accumulates in the leaves and stems of herbaceous and cereal plants. It exists as free and derivative form, showing potential pharmaceutical applications due to its low toxicity and antioxidative activity [73]. Moreover, acacetin and diosmin have been proven to exhibit extensive biological capabilities, including antioxidative and anti-inflammatory properties [74, 75]. In this study, higher relatively contents of apigenin, tricin, acacetin, diosmin, isoschaftoside were observed in FAMSE (Table 2). Consistent with our findings, a previous study reported that the concentration of apigenin and tricin in brown juice from alfalfa were increased after lactic acid bacteria-fermentation [76]. It is revealed that the hydroxylation of the A-ring of flavonoids, especially for 5- and 7-hydroxylations, is beneficial for enhancement of antioxidant activity [77]. In this study, relatively contents of 6,7-dihydroxyflavone and 3ʹ,4ʹ,7-trihydroxyflavone were higher in in FAMSE. Moreover, relatively contents of 5,6,7-tetrahydroxy-8-methoxyflavone, 5,7,3ʹ,5ʹ-tetrahydroxy-6-methylfavanone, apigenin-6-C-(2ʺ-glucosyl) arabinoside and tricin-7-O-(2ʺ-O-rhamnosyl) galacturonide were increased. As the major bioactive constituents of AM, methoxylated flavonoids and their glycosides have been proven to have superior antioxidant activity [78].

Table 2 Relative content of part of differential flavones metabolites in AMSE and FAMSE
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Isoflavones are flavones with a B ring attached to C3 instead of C2, commonly found in leguminous plants [79]. Daidzein and genistein are the most omnipresent isoflavones, which have similar biological activity and redox behavior [79]. The antioxidative activity of formononetin [80], prunetin [81, 82] and pratensein [83] had been confirmed. As shown in Table 3, the relative contents of daidzein (105.46-fold), genistein (29.38-fold), formononetin (2.03-fold), prunetin (2.80-fold), pratensein (2.96-fold), 4ʹ,6,7-trihydroxyisoflavone (42.69-fold) and isoluteolin (25.40-fold) in FAMSE were significantly increased compared to AMSE. A report confirmed that isoflavonoid glycosides were degraded into aglycones when the AM extract was fermented, for instance, formononetin-glycosides into formononetin [84].

Table 3 Relative content of part of differential isoflavones metabolites in AMSE and FAMSE
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Flavonols, the most commonly occurring group of flavonoids, are flavones hydroxylated in C3, including kaempferol, quercetin, and isorhamnetin [21]. In this study, most relative contents of kaempferol glycosides were down-regulated in FAMSE, while the relative contents of rhamnocitrin and galangin were significantly upregulated (Table 4). It is suggested that during the FEH process, glycosidic bonds in AM stems were presumably hydrolyzed, thus leading to a decline in kaempferol glycosides. Rhamnocitrin has been reported to have antioxidant, anti-inflammatory, and antitumor activities [85]. Galangin is a potent antioxidant primarily derived from different medicinal herbs, which could alleviate oxidative stress and increase immune function [86].

Table 4 Relative content of part of flavonols differential metabolites in AMSE and FAMSE
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Altogether, the flavonoids metabolite profile of AM stems was altered after FEH, and the relative content of apigenin, tricin, acacetin, daidzein, genistein, formononetin, prunetin, pratensein, rhamnocitrin and galangin were upregulated in FAMSE. Flavonoids are mostly glycosides and not as aglycones in plants [87]. Microbial fermentation could promote flavonoids glycosides converted to aglycones, which are more bioactive, easily absorbed in the small intestine, and have more potent antioxidant activity [88, 89]. The combination with enzymatic hydrolysis with microbial fermentation could boost the hydrolysis of sugar moieties, expedite the extraction of flavonoids, thus improve the antioxidant activities [87].

Conclusion

The suitable FEH conditions for polyphenol from AM stems were enzyme type cellulase and pectinase, enzyme amount 2000 U/g, moisture content 43%, fermentation temperature 30 °C, and fermentation time 7 days. Selected extraction conditions of polyphenol were ethanol concentration 50%, ultrasonic power 500 W, extraction temperature 35 °C, and extraction time 40 min. With these above conditions, the polyphenol and flavonoid contents as well as antioxidant activity of AM stems were increased. The relative contents of phenolic acids and flavonoid metabolites, DHFA, IFA, FA, apigenin, tricin, daidzein, et al., were significantly upregulated, which may be the reason for enhanced antioxidant activity.

Availability of data and materials

No datasets were generated or analyzed during the current study.

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Acknowledgements

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Funding

This work was financially supported by the Outstanding Youth Science Foundation of Inner Mongolia Agricultural University (Grant no. BR230403), Major Science and Technology Program of Inner Mongolia Autonomous Region (Grant no. 2021ZD0023-3), National Center of Technology Innovation for Dairy Program (Grant no. 2022-scientific research-2, Grant no. 2023-QNRC-10) and Inner Mongolia Education Department Special Research Project for First Class Disciplines (grant number YLXKZX-NND-007).

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Authors and Affiliations

  1. College of Animal Science, Inner Mongolia Agricultural University, Hohhot, 010018, China

    Na Liu, Xiaoping An, Yuan Wang, Jingwei Qi, Yang Jia, Xia Li, Zhalaga Zhang & Xinnan Chen

  2. National Center of Technology Innovation for Dairy-Breeding and Production Research Subcenter, Hohhot, 010018, China

    Na Liu, Xiaoping An, Yuan Wang, Jingwei Qi, Xia Li, Zhalaga Zhang & Xinnan Chen

  3. Key Laboratory of Smart Animal Husbandry at Universities of Inner Mongolia Autonomous Region, Integrated Research Platform of Smart Animal Husbandry at Universities of Inner Mongolia, Inner Mongolia Herbivorous Livestock Feed Engineering Technology Research Center, Hohhot, 010018, China

    Na Liu, Xiaoping An, Yuan Wang, Jingwei Qi, Yang Jia, Xia Li, Zhalaga Zhang & Xinnan Chen

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Contributions

N.L. methodology, investigation, formal analysis, writing—original draft, and writing—review and editing. XP. A. conceptualization, methodology, data curation and project funding acquisition. Y. W. methodology, validation and formal analysis. JW. Q. supervision, project administration and funding acquisition. Y. J. methodology and formal analysis. X. L. methodology. ZLG. Z. methodology. XN. C. methodology. All authors reviewed the manuscript.

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Supplementary Information

Additional file 1: Fig. S1.

Phenolic acids metabolites identified in AMSE and FAMSE. (A) The three-dimensional scatter plot of the PCA model. (B) The verification chart of the OPLS-DA model. (C) Volcano plot of the differential phenolic acids metabolites. Fig. S2. Flavonoids metabolites identified in AMSE and FAMSE. (A) The two-dimensional scatter plot of the PCA model. (B) Permutation test of the OPLS-DA model. (C) Volcano plot of the differential flavonoids metabolites.

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Liu, N., An, X., Wang, Y. et al. Polyphenol composition and antioxidant activity of fermentation combined with enzymatic hydrolysis modified Astragalus membranaceus stems. Chem. Biol. Technol. Agric. 11, 153 (2024). https://doi.org/10.1186/s40538-024-00674-x

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