In vitro gastrointestinal digestion and fermentation properties of Ganoderma lucidum spore powders and their extracts

In vitro gastrointestinal digestion and fermentation properties of Ganoderma lucidum spore powders and their extracts

LWT - Food Science and Technology 135 (2021) 110235 Contents lists available at ScienceDirect LWT journal homepage: www.elsevier.com/locate/lwt In ...

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LWT - Food Science and Technology 135 (2021) 110235

Contents lists available at ScienceDirect

LWT journal homepage: www.elsevier.com/locate/lwt

In vitro gastrointestinal digestion and fermentation properties of Ganoderma lucidum spore powders and their extracts Ming Cai a, *, Hua Mu a, Haoyong Xing a, Zhenhao Li b, Jing Xu b, Wei Liu c, Kai Yang a, **, Peilong Sun a a b c

Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou, Zhejiang, 310014, People’s Republic of China Longevity Valley Botanical Co., Ltd., Zhejiang, 321200, People’s Republic of China State Microbial Technology Zhejiang Province, Zhejiang Academic of Agricultural Science, Hangzhou, Zhejiang, 310021, People’s Republic of China

A R T I C L E I N F O

A B S T R A C T

Keywords: Ganoderma lucidum spores Sporoderm removal Polysaccharides Triterpenoids Gut microbiota

Ganoderma lucidum spores (GLS) constitute a healthy food for humans, but the hard spore shells are difficult to digest. In this study, digestive properties of GLS in different forms (sporoderm-unbroken, sporoderm-broken [SBGLS] and sporoderm-removed [SR-GLS]) were investigated in vitro digestion and fermentation. After simulated digestion, morphologies of all three spores changed slightly. After gastric and intestinal digestion, the amounts of polysaccharides in each digestive residue of GLS, SB-GLS and SR-GLS were greatly reduced, by approximately 90.3%, 96.5% and 99.2%, respectively. Triterpenoid quantities in the digestive residues were higher than those before digestion because the absolute weight of residue decreased. Total triterpenoid contents of GLS, SB-GLS and SR-GLS after digestion were 9.2 ± 0.8, 24.7 ± 1.3, and 32.5 ± 1.1 mg/g, respectively. The amounts of CO2, CH4 and H2 found in the water and alcohol extracts of SR-GLS were similar, but the amount of H2 in the alcohol extract was higher. In terms of short-chain fatty acids (SCFAs) production, propionic, butyric and valeric acids found in the water extracts were more abundant than those in alcohol extracts, which suggests that the polysaccharides were particularly important for SCFA production. This finding indicates that SR-GLS for the water extract has stronger metabolic promotion effects and better anti-inflammatory properties.

1. Introduction Ganoderma lucidum (Leyss. Ex Fr.) Karst (G. lucidum) is a famous food and medicinal mushroom that promotes human health (Zhu, Yao, Ahmad, & Chang, 2019). G. lucidum spores (GLS), extremely tiny 4- to 6-μm ovarian germ cells, are ejected from the pleats of the body during its growth (Zhao, Chang, Li, Suen, & Huang, 2014). GLS has antitumor, free radical scavenging, hepatoprotective, and immunity-improving ef­ fects (Na, Sang, Wu, Wang & Wang, 2017; Ko, Hung, Wang & Pin, 2008; Chen, Ke, et al., 2017). GLS contain a variety of active components, such as polysaccharides, triterpenoids, trace essential elements, sterols, nu­ cleosides and fatty acids. In particular, polysaccharides and triterpe­ noids are two important active components that are soluble in water and alcohol, respectively. They can be digested or absorbed in the human body to exert their bioactivities. The digestive characteristics of poly­ saccharides are affected by their sources and structures. Acidic poly­ saccharides, such as the polysaccharides in Fuzhuan brick tea (Chen,

Xie, et al., 2017), are highly resistant to digestion. In contrast, some polysaccharides show gradual molecular weight reductions with suc­ cessive digestive reactions (Chen, Zhang, et al., 2016). Accordingly, the bioactivities of polysaccharides and triterpenoids are related to their digestive properties. In recent years, some studies have shown that GLS are superior to fruiting bodies for health (Sliva et al., 2003; Ma, Fu, Ma, Su, & Zhang, 2007). However, their hard spore wall, composed mainly of chitin, hinders the absorption and utilization of their active components during digestion. Breaking the spore’s wall can render GLS more suitable for direct absorption of their components by humans. Sporoderm broken GLS (SB-GLS) show more significant anti-human immunodeficiency virus-1 protease activity, which closely reflects sporophytes’ properties (Min, Nakamura, Miyashiro, Bae, & Hattori, 1998). Disruption of the spore wall could improve the release of active components, while a weak active effect is observed when the wall is intact (Huang et al., 2019). Currently, a sporoderm-removed GLS (SR-GLS) with a higher

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected], [email protected] (M. Cai), [email protected] (K. Yang). https://doi.org/10.1016/j.lwt.2020.110235 Received 28 May 2020; Received in revised form 11 September 2020; Accepted 14 September 2020 Available online 18 September 2020 0023-6438/© 2020 Elsevier Ltd. All rights reserved.

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concentration of active components have been manufactured to improve their bioactivity. To our knowledge, the digestive properties of different sporophytes of G. lucidum have not been investigated or compared. In vitro digestion and fermentation are two effective methods to evaluate the bioavailability of compounds during digestion. The char­ acteristics of polysaccharides from litchi pulp after intestinal digestion and fermentation have been demonstrated through these two modalities (Huang et al., 2019). Studies have shown that polysaccharide metabo­ lites exert regulatory effects on intestinal bacteria (Xu et al., 2017). Studies have also investigated the effects of some active constituents in vivo, especially those of the polysaccharides from G. lucidum on intes­ tinal microorganisms. Polysaccharides in GLS are thought to regulate the function of the intestinal immune barrier by increasing the variety of intestinal microorganisms (Jin et al., 2017). Additionally, ethanol ex­ tracts of G. lucidum were shown to play a positive roles in preventing the malnutrition caused by a high-fat diet and maintaining the microbial ecological balance (Hu et al., 2018). In the present study, simulated digestion models were used to explore the digestion properties of different forms of GLS. Accordingly, the effects of water and ethanol extracts from SR-GLS on the production of SCFAs by gut microbiota were evaluated using in vitro fermentation experiments. These results provide information for understanding the digestive properties of different forms of GLS.

2.4. Simulated gastrointestinal digestion Solutions of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to previously described methods (Lee, Cho, Lee, Kim, & Shim, 2014; Sarkar, Goh, & Singh, 2009) with some modifications. SGF, pH 1.5, was prepared by dissolving 2.00 g of NaCl, 3.20 g of pepsin and 7 mL of HCl in water to a total volume of 1 L. Ten grams of spores were added to a 250-mL SGF solution. The mixture was adjusted to pH 1.8 with 0.2 mol/L of HCl and shaken continuously at 100 rpm by magnetic stirring in water bath at 37 ◦ C for 2 h. SIF at pH 7.0, was prepared by dissolving 6.80 g of KH2PO4 and 10.00 g of pancreatin in water to a total volume of 1 L. After digestion in gastric juice, 2 g of sample GLS were added to 100 mL of the SIF solution. The mixture was adjusted to pH 6.8 using a 0.2 mol/L NaOH and shaken continuously at 100 rpm for 2 h. 2.5. In vitro fermentation Water and alcohol extracts were fermented in vitro with fresh feces, and the vegetative growth medium was modified several times accord­ ing to a reported method (Wang et al., 2019). Ten volunteers (five women and five men, aged 20–30 years) avoided taking antibiotics for at least 3 months prior to providing samples. Fresh stool samples were mixed into equal amounts and diluted with sterile phosphate-buffered solution (0.1 mol/L, pH 7.2) to obtain a 10% (w/v) stool slurry, which was then passed through a double gauze homogenizer and filter. Then, 0.5 mL of the 10% fecal slurry was added to 5 mL of culture medium, and 0.8 g of an extract were added to a 25 mL vial and cultured under constant stirring at 37 ◦ C. Glucose was used as a positive control, and after 24 h of fermentation, all samples were collected for further analysis.

2. Materials and methods 2.1. Chemicals Pepsin (≥2500 units/mg), hydrochloric acid, trypsin (USP grade), ursolic acid (98%) and potassium dihydrogen phosphate were pur­ chased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Ethanol and ethyl acetate were purchased from Lingfeng Chemical Re­ agent Co., Ltd. (Shanghai, China). Methyl red, methylene blue, boric acid (H3BO3), cupric sulfate (CuSO4), sulfuric acid (H2SO4) and other chemicals were of analytical grade.

2.6. Scanning electron microscopy Dried GLS was placed on a sample stage and coated by an ultrathin layer of gold using a sputter coating device. The morphologies of the different GLS after digestion were characterized by scanning electron microscopy (SEM) at a voltage of 15 kV and magnification of 2.0 k x. (JSM-6380, Japan Electronics Co., Ltd., Japan).

2.2. Materials G. lucidum spore powders in the forms of SR-GLS, SB-GLS and sporoderm-unbroken GLS were provided by Longevity Valley Botanical Co., Ltd (Wuyi, Zhejiang, China). Sporoderm-unbroken GLS is the original spore ejected from the fungal tube after the ganoderma fruit body grows to maturity. SB-GLS are obtained by breaking the spore walls with supersonification and low-temperature airflow to generate G. lucidum powder. SR-GLS are also produced by the supersonification and low-temperature airflow, which breaks the cell wall, followed by extraction, concentration and drying operations to completely remove the walls. All the samples were dried at 45 ◦ C in an oven to a constant weight for further experiments.

2.7. Determination of the main components Determination of total polysaccharide content. Anhydrous glucose was used to prepare a reference solution at 0.12 mg/mL according to a re­ ported method (Vazirian et al., 2014). Stoppered tubes were filled with 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of reference solution to 10 mL. Water was added to each tube to a volume of 2.0 mL, and then 6 mL of 1 mg/mL sulfonate solution was quickly added, and each closed tube was shaken immediately. After standing for 15 min, the solutions were cooled in an ice bath for 15 min, and their absorbances were measured at 625 nm in a UV spectrophotometer (UV-2450, Shimadzu, Japan). The total polysaccharide content in the digestive residue were also deter­ mined. One gram of residue was subjected twice to reflux extraction with 60 mL of water at 100 ◦ C for 4 h. All the extracts were filtered and concentrated. The residue was dissolved in 5 mL water, 75 mL of ethanol was slowly added, and the mixture was shaken at 4 ◦ C for 12 h. The precipitate was dissolved in water in a 50-mL flask. Content de­ terminations were made with 5-mL samples of the solution. The results are expressed as ratios (concentration of total polysaccharides [mg]/digestive residue [g]). Determination of total triterpenoid content. According to Chang’s method (Chang, Lin, & Lai, 2012), ursolic acid was used to prepare a stock solution at a concentration of 0.1 mg/mL. Stock solution samples of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mL in 10-mL tubes were blown dried at 60 ◦ C. Then, 0.4 mL of 5% vanillin glacial acetic acid and 1.0 mL perchloric acid were added to each tube. The mixtures were maintained

2.3. Preparation of water and alcohol extracts Approximately 10.00 g of SR-GLS was extracted three times with 200 mL of 95% ethanol in processes that lasted 12 h at 20 ◦ C. All the extracts were combined and concentrated to approximately 10 mL before they were freeze-dried. The residues were air-dried, and 200 mL of water was added for extraction at 95 ◦ C, which proceeded for 2 h, in triplicate. The extracts were collected, centrifuged, concentrated and freeze-dried. Finally, approximately 1.00 g of water extract and 1.80 g of alcohol extract with SR-GLS were obtained. The levels of total poly­ saccharides in the water extract was approximately 12.13 ± 0.20%, and that of total triterpenoids in the alcohol extract was approximately 2.56 ± 0.04%.

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Fig. 1. SEM images of GLS, SB-GLS and SR-GLS at different digestion stages. A: original GLS; B: GLS after gastric digestion; C: GLS after intestinal digestion; D: original SB-GLS; E: SB-GLS after gastric digestion; F: SB-GLS after intestinal digestion; G: original SR-GLS; H: SR-GLS after gastric digestion; I: SR-GLS after intes­ tinal digestion.

at 60 ◦ C in a water bath for 15 min, transferred to an ice bath, and then mixed with 5 mL of glacial acetic acid. The absorbances were measured at 548 nm within 15–30 min, and a regression equation was obtained: y = 9.9169x-0.0312, R2 = 0.9978. Total triterpenoids in the digestive residue were measured according to a published method (Ramakrishna, Babu, Veena, Pandey, & Rao, 2017) with some modifications. Then, 0.5 g of each residue was placed into individual tubes, 20 mL ethyl acetate was added, and ultrasonic extractions were performed twice for 30 min each time. The supernatants were all combined in a 50-mL flask with ethyl acetate as a stock solution. A 0.3 mL aliquot of the stock solution was placed into a 10 mL-colorimetric tube and dried with nitrogen at 60 ◦ C. The absorbance value was measured in the same manner as described above. The results are expressed as ratio concentrations of total triterpenoids (mg)/digestive residue (g).

2.8. Determination of triterpenoids by HPLC The triterpenoid profile before and after digestion was determined by HPLC (Chen, Xie, et al., 2017) with some modifications. The process was carried out on a Waters 1525 and UV detector 2487 (Waters, USA) with an Agilent C18 column (4.6 × 250 mm, 5 μm) with the following settings: mobile phase A, 0.1% glacial acetic acid solution; B, acetoni­ trile; gradient elution conditions, 0–5 min, 90% A; 5–10 min, 90 -85% A; 10–30 min, 85-70% A; 30–40 min, 70-60% A; 40–55 min, 60-55% A; and 55–70 min, 55–90% A; flow rate, 1 mL/min; and injection volume, 10 μL. The main differences in triterpenoids were identified on a Waters UPLC-Synapt G2 (Waters, USA) machine with ESI. Total ion chromato­ grams were acquired using the following operating parameters in negative mode: capillary voltage, − 2.5 kV; sample cone voltage, 40 V; 3

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Fig. 2. Effects of in vitro simulated digestion processes on polysaccharides and triterpenoids in different spores. A: total polysaccharides in residue; B: total poly­ saccharides in juice; C: total triterpenoids in residue; D: total triterpenoids in juice. GLS, Ganoderma lucidum spores; SB-GLS: sporoderm-broken GLS; SR-GLS: sporoderm-removal GLS.

sample extraction voltage, 5.0 V; ion source temperature, 120 ◦ C; des­ olvent gas temperature, 350 ◦ C. The spray gas was highly pure nitrogen (N2), the collision gas was highly pure argon (Ar), the backflush gas flow rate was set to 80 L/h, and the solvent gas flow rate was set to 800 L/h. The mass spectrum scan range was 100–1200 Da, and the scan time was 0.3 s.

compare the differences among various groups, and differences were considered statistically significant when P < 0.05.

2.9. Determination of main gas changes during fermentation

The spore morphologies of SR-GLS, SB-GLS and GLS before and after digestion are shown in Fig. 1. This results show that none of the spores were significantly damaged after digestion in vitro, but we found some slight differences among them. After digestion in simulated gastric and intestinal juices, the walls of the GLS wall retained their shape. SB-GLS retained broken walls and some soluble solutes were found. This finding indicates that the spore wall made from chitin was difficult to digest with the simulation juice. Chitin has been shown to have low biode­ gradability during digestion (Deepthi, Venkatesan, Kim, Bumgardener, & Jayakumar, 2016; Xiao et al., 2018). However, solutes such as poly­ saccharides and triterpenoids were partially digested in these juices (Yun, Li, Yang, & Zhang, 2018). When comparing the three spores, the active compounds in the GLS were not readily released through the wall, while those of the other spore forms were more easily released. Accordingly, the bioavailability of the polysaccharides or triterpenoids in spores during digestion can be increased by breaking or removing their walls.

3. Results and discussion 3.1. Morphological spore changes

The gas pressures and contents of CO2, CH4 and H2 in the fermen­ tation vials were directly tested by gas detectors (HT-1895, Dongguan Hongtai Instrument Technology, Dongguan, China). 2.10. Determination of short-chain fatty acids (SCFAs) SCFAs were identified using a GC-MS (GC-2010 plus, Shimadzu, Japan) system equipped with a DB-FFAP column (30 m × 0.32 mm × 0.5 μm, Agilent, USA) according to a described method (Chen, Huang, Fu & Liu, 2016) with some modifications. The contents of SCFAs were calculated based on standard calibration curves with crotonic acid. 2.11. Statistical analysis All experiments were performed in triplicate, and the results are expressed as means ± standard deviation. Statistical analysis was per­ formed using Origin version 8.5 (OriginLab, Microcal, USA). One-way analysis of variance with a Tukey’s multiple range test was used to 4

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Fig. 3. Analysis of triterpenoids in SR-GLS during simulated digestion. A: initial amount; B: amount after SGF digestion; C: amount after SIF digestion; D: MSidentified peaks observed between 32 and 60 min.

3.2. Main compound effects after digestion

contents in the SGF digestion juice of GLS, SB-GLS and SR-GLS were 1.0 ± 0.1, 1.6 ± 0.2, and 2.5 ± 0.2 mg/g, respectively; after SIF digestion, they were 4.6 ± 0.3, 5.8 ± 0.3, and 8.3 ± 0.4 mg/g, respectively. Accordingly, SR-GLS can release more triterpenoids in the juice than the other GLS.

After digestion, the total residue sizes in both the gastric and intes­ tinal stages were decreased, and the amounts of the residues were decreased after intestinal digestion. As shown in Fig. 2, the poly­ saccharide contents in the GLS were greatly reduced in all the digestive residues, resulting in greater contents in juices. As shown in Fig. 2A and B, the total polysaccharide contents of GLS, SB-GLS and SR-GLS were 3.1 ± 0.2, 11.5 ± 0.6, and 121.2 ± 1.1 mg/g, respectively; but, they were 1.7 ± 0.1, 1.0 ± 0.1, and 2.5 ± 0.2 mg/g and 0.3 ± 0.1, 0.4 ± 0.1, and 1.0 ± 0.1 mg/g after gastric and intestinal digestion, respectively. These findings indicate that some polysaccharides can be released and digested by the juices. Among the three spores, SR-GLS produced the highest total polysaccharide concentration in the juice, reaching approximately 35.6 ± 1.0 mg/g and 20.0 ± 0.7 mg/g in the two digestions. Some studies have demonstrated that the polysaccharides from G. lucidum exhibit small Mw changes after digestion in the stomach, but that the Mw continuously decreased gradually during intestinal digestion (Ding et al., 2017). The Mw of soluble polysaccharides in kiwifruit was found to decrease during simulated gastrointestinal tract digestion (Carna­ chan, Bootten, Mishra, Monro, & Sims, 2012). Therefore, this Mw decrease may be due to the destruction of agglomerates rather than the disruption of glycosidic bonds. As shown in Fig. 2C and D, the total triterpenoids in the digestive residue increased gradually after simulated gastric and intestinal di­ gestions, as indicated by the weight of the residues being significantly reduced. The total triterpenoid concentrations of GLS, SB-GLS and SRGLS after intestinal digestion were 9.2 ± 0.8, 24.7 ± 1.3, and 32.5 ± 1.1 mg/g, respectively. These findings indicate that the triterpenoids may remain mostly attached to the solids during digestion, and that only a small portion was dissolved in the supernatant. The total triterpenoid

3.3. Profile changes in triterpenoids Fig. 3 shows that the profiles and properties of triterpenoids in SRGLS changed significantly during digestion in both SGF and SIF. The concentrations of triterpenoids in the juices decreased. Ganoderic acid B and A, which are two important triterpenoids compounds in G. lucidum, were identified as peaks at 40.55 and 43.11 min (Fig. 3D). These tri­ terpenoids changed due to their digestive properties. 3.4. Effect of the three main gases on fermentation The high levels of prebiotics in some foods can lead to increased intestinal gas production, which can cause bloating (Beards, Tuohy, Gibson & Bacterial, 2010). We found the air pressures of the three tested samples (water and alcohol extracts of SR-GLS and the glucose positive control), were similar after the in vitro fermentation (P > 0.05), as shown in Fig. 4A. Accordingly, this finding suggests that human spore con­ sumption does not create additional gas production or lead to bloating. As shown in Fig. 4B and C, similar levels CO2 and CH4 were produced in the samples and the control (P > 0.05). The alcohol extracts produced much less H2 than the others (P < 0.05), as shown in Fig. 4D. H2 is a main fermentation byproduct of hydrogen-producing bacteria, mostly Firmicutes including sulfate-reducing bacteria, methanogenic archaea and acetic acid bacteria (Carbonero, Benefiel, & Gaskins, 2012; Saha­ kian, Jee, & Pimentel, 2010). However, intestinal hydrogen production 5

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Fig. 4. Comparison of air pressure and content differences after 24 h of fermentation. A: air pressure; B: CO2; C: CH4; D: H2; ▴: significant at P < 0.05.

increases represent proportional increases in these flora or their activities.

acids were in greater abundance in the water extracts. Additionally, the water extract of SR-GLS presented better intestinal anti-inflammatory properties than SR-GLS in the alcohol extract.

3.5. Effect of fermentation on SCFA production

4. Conclusions

Fig. 5 shows the effects of different extracts on SCFA production of acetic, propionic, butyric, valeric, isobutyric and isovaleric acids during fermentation by metabolites in the human intestine. We found similar amounts of acetic and isobutyric acids produced from the extracts (P > 0.05), but the amounts of propionic (P < 0.01), butyric (P < 0.01) and valeric acids (P < 0.05) were significantly lower in the alcohol extracts than in the water extracts. This results was due to the lower concen­ tration of carbohydrates available in the alcohol extracts than in the water extracts. On the other hand, the yield of isovaleric acid from the water extracts was significantly higher than that from the glucose pos­ itive control sample (P < 0.05). During fermentation, polysaccharides can promote the production of SCFAs, which can be used as special components to regulate the composition of the intestinal flora and promote the growth of beneficial bacteria such as Bifidobacterium (Ding et al., 2018). Accordingly, the water extracts yielded more SCFAs pro­ duced by the intestinal flora than the alcohol extracts (P < 0.05). Tjellstrom et al. (2012) proposed two observation indicators to facilitate the analysis of major fatty acids and determine their functions, namely, indicator A (acetic acid – propionic acid – butyric acid)/total short-chain fatty acids and indicator B (isobutyric acid + isovaleric acid), which may be used to assess the activities of carbohydrates. In­ dicator A reflects the proinflammatory properties of SCFAs, and indi­ cator B reflects the anti-inflammatory properties of SCFAs. As shown in Fig. 6, the indicators A for alcohol extracts and B for water extracts were higher than those for the glucose control (P < 0.05). These findings indicate that the amounts of propionate and butyric acids produced in the alcohol extracts were low, while those of isobutyric and isovaleric

This study demonstrates the digestive characteristics of different forms of GLS and the effects of SR-GLS in water and alcohol extracts on in vitro colon fermentation. The morphologies and main compounds (such as total contents of polysaccharides and triterpenoids and tri­ terpenoids) of the three spores were compared. Accordingly, SR-GLS can promote active component release more easily than other forms of GLS, and those active components are absorbed during simulated digestion. Additionally, water and alcohol extracts of SR-GLS, which are rich in polysaccharides and triterpenoids, respectively, were compared to assess the proliferation and metabolism of intestinal microorganisms. We found that water extract with SR-GLS has stronger metabolismpromoting effects and better anti-inflammatory properties. This study provides a basis for further demonstrating GLS effects, especially those of its polysaccharides and triterpenoids, as functional foods that can affect digestive mechanisms. CRediT authorship contribution statement Ming Cai: Conceptualization, Methodology, Validation, Supervision. Hua Mu: Methodology, Data curation, Writing - original draft. Haoyong Xing: Methodology, Software. Zhenhao Li: Resources. Jing Xu: Re­ sources. Wei Liu: Methodology, Data curation. Kai Yang: Supervision, Writing - review & editing. Peilong Sun: Supervision, Writing - review & editing.

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Fig. 5. Metabolism of six SCFAs after 24 h of fermentation compared with initial extracts. A: acetic acid; B: propionic acid; C: butyric acid; D: valeric acid; E: isobutyric acid; F: isovaleric acid; ▴: significant at P < 0.05; ▴▴: significant at P < 0.01.

Fig. 6. Indicators A and B of extracts from SR-GLS during fermentation. A=(acetic acid-propionic acid-butyric acid)/total short-chain fatty acids, B = isobutyric acid + isovaleric acid; ▴: significant at P < 0.05; ▴▴: significant at P < 0.01.

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Declaration of competing interest

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