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1. Introduction

Camellia oleifera seed cake meal, a rich source of bioactive compounds, including polysaccharides, saponins, flavonoids, and polyphenols, has been investigated as a potential source of bioactive compounds with therapeutic benefits [1,2,3,4]. Unfortunately, camellia seed cake meal is discarded as industrial waste and is underutilized. Camelliagenin B is a tea sapogenin derivative with the chemical formula C30H48O5, associated with various pharmacological benefits, including anti-inflammatory, antioxidant, analgesic, and antimicrobial properties [5,6,7,8,9]. It was first identified and reported in Camellia japonica in Japan. Estimated studies indicate its mechanism in suppressing inflammatory cytokines such as IL-1β, IL-6, and TNF-α, all known to be elevated in inflammatory diseases [10,11,12,13]. These bioactive compounds possess immense potential and are awaiting exploration and utilization. Traditionally, tea saponins have been extracted from C. oleifera through solvent-based methods, often resulting in low yields and impurities [14]. However, it has been observed that tea saponins from C. oleifera by microbial biotransformation have successfully been modified to obtain novel saponin derivatives [15].

This technique involves the chemical alteration of natural compounds to produce natural and biodegradable products using various microorganisms [16,17,18,19,20]. This can be attributed to the ability of microorganisms to employ diverse metabolic pathways for biotransforming compounds, primarily through enzymatic hydrolysis, oxidation, glycosylation, and deglycosylation. In case of biotransforming tea saponins into their more bioavailable forms, the enzyme, such as β-glucosidase, cleaves sugar moieties, modifying the aglycone’s structure, producing sapogenins [21]. Oxidative enzymes catalyze hydroxylation or ring-opening reactions, further diversifying saponin derivatives. Certain fungi (e.g., Aspergillus and Rhizopus) and bacteria (e.g., Bacillus and Streptomyces) can selectively perform deglycosylation of tea saponins into their metabolites with improved pharmacological properties [22]. Furthermore, microbial glycosyltransferases may introduce new sugar units, thereby altering solubility and bioavailability [23,24].

Due to its eco-friendly approach, the process has become more convenient, as it eliminates the need for harsh chemicals, improves the yield and purity of compounds, and offers scalability for isolating bioactive compounds [25,26,27]. Recently, it has garnered significant attention as a promising technology for producing various bio-products, including enzymes, organic acids, antibiotics, and bioactive compounds [28,29]. In line with our ongoing efforts to identify new pentacyclic triterpenoids (PTs), we have previously conducted biotransformation studies on PTs [30,31,32,33,34,35]. Therefore, this study explores the microbial biotransformation of Camelliagenin B obtained from Camellia oleifera seed cake meal, an underutilized industrial by-product. Using three microbial strains, Bacillus subtilis ATCC 6633, Bacillus megaterium CGMCC 1.1741, and Streptomyces gresius ATCC 13273, this study investigates multiple catalytic reactions and structural modifications of tea saponins with enhanced pharmacological properties. Through NMR-based structural analysis, ten novel derivatives were identified, demonstrating the potential of microbial fermentation as an alternative, sustainable, and efficient strategy to the conventional chemical synthesis method. This research work not only contributes to the valorization of agricultural waste but also advances the discovery of bioactive compounds with therapeutic applications. Also, this initiative reflects our ongoing efforts on the biotransformation of saponins in oil crops.

2. Materials and Methods

2.1. Chemicals and Materials

The C. oleifera seed cake was obtained from Ankang Tea Oil Co., Ltd.: 10 December 2021, in Ankang City, Shaanxi Province, China. The seed cake was defatted to remove the oil and then ground into a fine powder, which was sieved through an 80-mesh sieve to ensure uniformity. A tea saponin standard of at least 98% purity and AB-8 macroporous resin, utilized for purification steps, were acquired from Shanghai Yuanye Biological Technology Co. in Shanghai, China. The microbial strain, Bacillus megaterium CGMCC 1.1741, was purchased from the China General Microbiological Culture Collection, Institute of Microbiology, Chinese Academy of Sciences. Bacillus subtilis ATCC 6633 and Streptomyces griseus ATCC 13273 were acquired from John P. N. Rosazza at the University of Iowa in the USA. These strains were inoculated on potato dextrose agar (PDA) slant culture medium and then stored at 4 °C with fresh subcultures prepared as needed. For long-term preservation, microbial strains were maintained using cryopreservation at −80 °C in 20% glycerol or lyophilization for Streptomyces griseus ATCC 13273 and Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.174, respectively.

Chemical reagents, including hydrochloric acid (HCl) and acetone (HPLC grade), were sourced from Sinopharm Chemical Reagent Co., Ltd. in Shanghai, China. GE Healthcare supplied chromatography media, such as Sephadex LH-20, in the USA. High-performance thin-layer chromatography (HPTLC) analyses were performed using precoated silica gel plates (plate number: GF254) and silica gel obtained from Qingdao Marine Chemical Group Co. in Qingdao city, Shandong province, China. Reverse-phase high-performance liquid chromatography (RP-HPLC) separations were carried out using an Agilent 1100 instrument equipped with an Alltech 3300 evaporative light scattering (ELSD) detector and an Ultimate® XB-C18 column (250 mm × 21.2 mm, 5 μm) with elution mixtures of CH3CN and water. Steriochemical analysis and structural verification of the compounds were elucidated using high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) collected on the Agilent 1260-6530 QTOF spectrometer. One- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR) based on heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY) were obtained with a Bruker DRX-600 spectrometer in chloroform-d and pyridine-d5 using tetramethylsilane (TMS) as the internal standard, and the chemical shifts are expressed in parts per million (δ) and coupling constants (J) in Hertz.

2.2. Preparation, Separation, and Purification of the Camelliagenin B (1)

Camelliagenin B was prepared by acid hydrolysis of tea saponin by accurately weighing 50 g of crude tea saponins dissolved in a 135 mL methanol–HCl solution (methanol: water = 3:1), with a HCl concentration of 2.5 mol/L, for 5.5 h, and the reaction was conducted in an oil bath at 80 °C with continuous stirring. The hydrolyzed sample was subsequently filtered, and the pH was adjusted to neutral using a 0.1 g/mL NaOH solution; a significant amount of light-grey precipitate was observed. The precipitate was extracted thrice with ethyl acetate, and the extracts were concentrated to obtain a mixture of aglycone extracts (13.3 g). The samples were separated and purified using silica gel (100 mesh to 200 mesh, 385 g; and 200 mesh to 300 mesh) column chromatography. The fraction eluted with ethanol was collected, concentrated, and dried for further analysis. The extracts were visualized and identified using TLC with dichloromethane (CH2Cl2) and methanol (CH3OH) as mobile phases at different ratios: 250:1, 100:1, 50:1, 30:1, 20:1, 10:1, and 5:1. A large yellow–brown substance was noted at a 50:1 ratio. A total of 14 fractions were obtained. After excluding three fractions with lower content, the remaining 11 were separated by liquid-phase preparation (flow rate 12 mL/min, 60% acetonitrile (CH3CN), 40% water, target substance was 18 min, a total of 2.13 g was obtained and identified as Camelliagenin B (1) (Figure 1), white powder with a molecular formula C30H48O5, in which HR-ESI-MS m/z = 511.3381 [M + Na]+ (calcd for C30H48O5Na, 511.3394)), as reported in the literature [36]. The 13C NMR data show five carbon signals: δC 207.61, δC 68.76, δC 70.59, δC 72.01, and δC 74.69. Consequently, the biotransformation of camelliagenin B (1) was carried out.

2.3. Biotransformation of Camelliagenin B (1)

Among 30 microbial strains screened, only B. subtilis ATCC 6633, B. megaterium CGMCC 1.1741, and S. griseus ATCC 13273 strains showed significant catalytic activity towards the substrate (Camelliagenin B), and were grown in potato dextrose (PD) and soybean meal (SM) medium in a two-stage fermentation protocol; the medium composition is given in Table 1. During the stage I fermentation period, the medium was divided into 250 mL round-bottomed flasks. Each flask was split into 50 mL, sterilized at 121 °C for 20 min, and then incubated at 180 rpm/min at 28 °C for 24 h. The seed solution (1.6 mL) from the 24 h stage I culture was transferred into fresh medium to initiate stage II fermentation. The mixture was then incubated under the same conditions, followed by the addition of 1 mL camelliagenin B (10 mg) dissolved in ethanol as a substrate separately into the stage-II culture medium. Control groups were prepared as substrate-free culture (microorganisms without camelliagenin B in a sterile culture medium) and strain-free culture (camelliagenin B without microorganisms in a sterile culture medium). The cultures were further incubated for 4 days, respectively, and then extracted three times with ethyl acetate. The organic layer was filtered and concentrated under reduced pressure using a rotary evaporator. The sample was spotted on a silica gel thin-layer chromatography (TLC) plate. The fermented extracts of Camelliagenin B (1) obtained after biotransformation were separated and purified using preparative RP-HPLC with a mobile phase comprising 36–45% acetonitrile (CH3CN) in 70–55% water at retention times ranging from 16–21 min. Significantly, 10 compounds, 1a and 1b, were yielded by B. subtilis ATCC 6633. Similarly, compounds 1c and 1d were biotransformed by S. griseus ATCC 13273, and finally, compounds 1e, 1f, 1g, 1h, 1i, and 1j were obtained by B. megaterium CGMCC 1.1714. The structures of these compounds were elucidated based on their 1D/2D NMR and HR-ESI-MS spectra.

The detailed structural elucidation and 13C NMR spectroscopic data of 1a-1j are given in the supplementary file (Table S1 and Figures S1–S42, respectively).

3. Results and Discussion

3.1. Identification of Compounds Biotransformed by B. Subtilis ATCC 6633

The biotransformation of Camelliagenin B (1) by B. subtilis ATCC 6633 produced two compounds (1a-1b, Figure 2A). Compound 1a was found to be a white powder, HR-ESI-MS, m/z = 673.3901 [M + Na]+, (calcd for C36H58O10Na, 673.3922), indicating an increase of 6 carbon atoms compared with compound 1. Compared with the increase of 162 amu in the substrate, the unsaturated unit also increased by 1 unit. In the 13C NMR spectroscopic data, a new set of six-carbon carbon signals, δC 104.85, 78.76, 78.69, 75.51, 71.69, and 63.00, was observed, of which the signal δC 104.85 indicates that the sugar end carbon is an ether bond signal (that is, the added sugar chain is connected to the hydroxyl signal on the pentacyclic triterpene). At the same time, the displacement of δC 68.38 (C-16), δC 74.33 (C-22), and δC 70.20 (C-28) in hydrocarbon did not change significantly. Compared with the substrate camelliagenin B (1), the δC 81.58 and δC 71.72 (C-3) displacements shifted to lower fields. It is speculated that hexacarbon sugar is connected to the C-3 position, and other signals have not changed significantly. Based on the above analysis, the structure of compound 1a was identified as 3-O-β-D-glucopyranosy-camelliagenin B.

Compound 1b was found to be a white powder, HR-ESI-MS, m/z = 673.3904 [M + Na]+ (calcd for C36H58O10Na, 673.3922). In the 13C NMR, a new set of six-carbon sugar-carbon signals δC 107.11, 78.99, 78.55, 75.81, 71.65, and 62.76 were added, of which the glycoterminal carbon signal δC 107.11 appeared. The sugar chain is connected to the hydroxyl group. At the same time, the displacement of the protohydroxycarbon signal δC 68.69 (C-16), δC 71.70 (C-3), and δC 69.49 (C-28) did not change significantly; only in the signal δC 86. 72 compared with the substrate δC 74.40 (C-22), the chemical displacement moves to the low field. The hexacarbon sugar is assumed to be connected to the C-22 position, and other signals have not changed significantly. In summary, the structure of compound 1b is 22-O-β-D-glucopyranosy-camelliagenin B.

3.2. Identification of Compounds Biotransformed by S. Gresius ATCC 13273

The biotransformation of Camelliagenin B (1) by S. gresius ATCC 13273 produced two compounds (1d-1c, Figure 2B). Compound 1c was found to be a white powder, HR-ESI-MS m/z = 519.3341 [M + H]+ calcd for C30H46O6, 519.3316, an increase of 30 amu compared with the substrate. In contrast, the unsaturation increased by 1 unit. First of all, in the 1H NMR spectroscopic data, only five methyl hydrogen signals appeared, and compared with the substrate, the two methyl hydrogen signals connected to the C-20 position were reduced by one. Only one other hydrogen signal was left, with δH 1.77 (s, 3H, C-29/30) methyl group. The NOESY (Figure 3) showed that the methyl hydrogen δH 1.77 (s, 3H, C-29/30) and two protons δH 4.8 (d, J = 5.8 Hz, β-H122) and δH 2.78 (d, J = 12.4 Hz, β-H1-18) were strongly correlated, indicating that the methyl group was in β-configuration, attributed to the C-30 bit. Later, a carbonyl carbon signal, δC 181.30, was added to the 13C NMR spectroscopic data, showing no other characteristic carbon signal changes. It is speculated that the increase of 1 unsaturation is due to the presence of this carbonyl group. The HMBC data showed that the carbonyl signal δC 181.30 was compared with δH 1.77 (s, 3H, H-30), δH 3.63 (m, H1-19), and δH 2.52 (d, J = 5.1 Hz, H1-21) via long-range correlation, combined with the disappearance of the methyl hydrogen signal at the C-29 position above, attributed the new carbonyl signal δC 181.30 to the C-29 bit. According to the calculated molecular formula, the oxygen atom is increased by two, and the new carbon signal δC 181.30 is a carboxyl signal. Therefore, the structure of compound 1c was determined as 3β, 16α, 22α, 28-tetrahydroxy-23-aldehyde-olean-12-en-29-oic acid, which was found to be similar to the sapogenin metabolite with the chemical formula C30H46O6 extracted from the stem bark of Kalopanax pictus, which can effectively reduce the activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a protein complex involved in inflammation in HepG2 cells when induced by tumor necrosis factor (TNF) [37]. This suggests that the compound has anti-inflammatory properties.

Compound 1d is a white powder, HR-ESI-MS, m/z = 521.3459 [M + H]+ (calcd for C30H49O7, 521.3459), indicating an increase of 32 amu compared with the substrate, with no change in unsaturation. In the 13C NMR data, a new carbonyl signal at δC 181.01 and a characteristic carbon signal at δC 68.28 were observed, indicating the addition of an original C-23. The aldehyde group signal at the site disappears, with the presumptive disappearing aldehyde signal being converted into one of the two new carbon signals mentioned above. The carboxyl signal at δC 181.01 was identified at the C-29 position based on NMR data and interpretation similar to compound 1c. Two proton signals were added to the 1H NMR: δH 3.72 (d, J = 10.1 Hz, 1H) and δH 4.1 (d, J = 10.2 Hz, 1H). In the HMBC spectroscopic data, both protons are associated with δC 13.21 (C-24), δC 42.96 (C-4), δC 48.86 (C-5), and δC 73.69 (C-3). The remote correlation of 1H-13C, combined with the disappearance of the aldehyde signal at the original C-23 position, indicates that the protons δH 3.72 (d, J = 10.1 Hz, H1-23b) and δH 4.14 (d, J = 10.2 Hz, H1-23a) should be attributed to H-23 bit. Further analysis showed that the new carbon signal at δC 68.28 was associated with proton δH 3.7 (d, J = 10.1 Hz, H1-23b) and δH 4.14 (d, J = 10.2 Hz, H1-23a) in HSQC, directly correlating with 1H-13C. Therefore, δC 68.28 is attributed to the C-23 position, representing the oxymethylene carbon signal after the original C-23 aldehyde group has reacted. Adding one carboxyl group without a change in unsaturation further supports this conclusion. The molecular formula shows that the oxygen atom increases by only two, indicating that δC 68.28 is the methylene carbon signal of the hydroxyl group. Therefore, compound 1d has been identified as 3β, 16α, 22α, 23, 28-pentahydroxy-olean-12-en-29-oic acid. Certain compounds exhibit structural analogies or derivatization patterns similar to those of compound 1d, such as gymnemanol, oleanolic A [38], maslinic A, and erythrodiol [30]. These intricate molecules possess a multifaceted structure with numerous hydroxyl groups that play a significant role in their diverse biological activities, encompassing anti-inflammatory, analgesic, neuroprotective, and antioxidant effects, necessitating thorough exploration [39,40].

3.3. Identification of Compounds Biotransformed by B. Megaterium CGMCC 1.1741

The biotransformation of camelliagenin B (1) by B. megaterium CGMCC 1.1741 was elucidated to be 1e-1i (Figure 4). Compound 1e, white powder, and 10% ethanol sulfate reaction appear purple–red, HR-ESI-MS, m/z = 551.3588 [M + COOH]− (calcd for C30H50O6COOH, 551.3589), compared with the material, increasing the molecular weight by 18 amu and reducing the unsaturated degree by 1 unit. In the 13C NMR, the original C-23 aldehyde group signal disappeared, and two new characteristic carbon signals, δC 67.87 and 73.17, were added. It is speculated that the aldehyde group signal in the substrate was converted into one of the two new characteristic signals. First, the δC 67.87 signal is remotely related to δH 1.11 (s, 3H, H-24) 1H-13C, indicating that it may be attributed to C-5 or C-23. Combined with the loss of the original C-23 aldehyde group, δC 67.87 can be classified as C-23. Continuing to analyze two new protons, δH 3.73 (d, J = 9.5 Hz, H1-23b), δH 4.19 (m, H1-23a), and δC 13.43 (C-24), δC 46.18 (C-5), δC 42.84 (C-4), δC 73.34 (C-3) are remotely related, indicating that the two protons should be attributed to the H-23 bit. At the same time, δC 67.87 is associated with δH 3.73 (d, J = 9.5 Hz, H1-23b) and δH 4.1 in the HSQC. 9 (m, H1-23a) is directly related, proving that the oxymethylene signal δC 67.87 belongs to the C-23 position. Another new carbon signal, δC 73.17, is remotely related to δH 1.31 (s, 3H, H-26) in the HMBC spectroscopic data, which is speculated to be attributed to C-7 or C-9. Continuing to analyze this, the new proton signal δH 4.51 (m, H1-7) is related to δC 10.76 (C-26), δC 44.23 (C-14), and δC 46.14 (C-8) remotely, indicating that δH 4.51 (m, H1-7) is classified as H-7. The HSQC spectroscopic data show that δC 73.17 is directly related to δH 4.51 (m, H1-7) with 1H-13C, so the δC 73.17 signal directly related to it is attributed to C-7. The calculation for the molecular formula shows that only one oxygen atom is added, and there is no change in unsaturation, except for the reduction in the unsaturated degree of the loss of the aldehyde group, indicating that the C-7 bit is connected to the hydroxyl group. According to the NOESY spectroscopic data, the proton δH 4.51 (m, H1-7) strongly correlates to δH 2.02 (s, 3H, α-H-27). Therefore, the hydroxy group connected to the C-7 position is a β configuration. In summary, the structure of 1e is determined to be 3β, 7β, 16α, 22α, 23, 28-hexahydroxy-olean-12-ene, which was found to be remarkably similar to that of theasapogenol E and camelliagenin E, and is a well-documented compound in the scientific literature [41,42].

Compound 1f was found to be a white powder that reacts purple with 10% ethanol sulfate, HR-ESI-MS m/z = 549.3434 [M + COOH]−, (calcd for C30H48O6COOH), Compared with the substrate, only one oxygen atom is added, and there is no change in unsaturation. The 13C NMR spectroscopic data reveal the sole set of ene-carbon signals, indicating emission of the double-bond signal, leading to a migration instead of a new addition. Upon further analysis of the HMBC spectroscopic data, it is observed that the olefin proton δH 6.09 (d, J = 10.4 Hz, 1H) is remotely related to δC 52.80 (C-9), δC 47.86 (C-8), and δC 36.77 (C-10), while the olefin proton δH 5.81 (d, J = 11.3 Hz, 1H) is remotely related to δC 52.80 (C-9), δC 45.77 (C-14). According to the principle of olefin signal neighbor, δH 6.09 (d, J = 10.4 Hz, H1-11) and δH 5.81 (d, J = 11.3 Hz, H1-12) are attributed to H-11 and H-12 bits, respectively. Consequently, the carbon signals δC 131.46 and 132.49 directly related to them are classified as C-11 and C-12 bits, respectively. These findings indicate that the carbon–carbon double bond originally at the C-12 (13) position migrated to the C-11 (12) position. Additionally, the last new carbon signal δC 85.55 is observed in the HMBC spectroscopic data, with δH 1.86 (s, 3H, H-27), δH 2.48 (d, J = 15.0 Hz, H1-18), δH 6.09 (d, J = 10.4 Hz, H1-11), and δH 5.81 (d, J = 11.3 Hz, H1-12) being remotely related. Therefore, δC 85.55 is classified as C-13 bit. Subsequent analysis reveals that the aldehyde group is converted into a hydroxyl group without a change in the overall unsaturated degree. Moreover, none of the mentioned functional group changes have led to a change in unsaturation, implying that only an increase in unsaturation occurs at C-13, and δC 85.55 cannot be an alkyl signal. Further analysis of the calculated molecular formula shows no other heteroatoms, indicating that δC 85.55 is a carbon–oxygen signal. However, it is noted that compound 1g only adds one oxygen atom, occurring at the C-7 position. Considering the double bond migration to the C-11 (12) and the C-28 signal to the low field displacement of 8 ppm, an epoxy structure may exist between the C-28 and C-13. The proton signals directly connected to the C-28 δH 4.09 (d, J = 6.3 Hz, H1-28a) and δH 3.72 (d, J = 7.6 Hz, H1-2) can also be remotely related to δC 85.55 (C-13), further supporting this conclusion. In conclusion, compound 1f with the chemical formula C30H48O6 is determined to be 3β, 7β, 16α, 22α, 23-pentahydroxy-13, 28-epoxy-olean-11-ene, found to be similar to another sapogenin known as barringenol R1 from the root barks of Pittosporum verticillatum, which has potent cytotoxicity against human cancer cell line (SW480) and one rat cardio myoblast cell line (H9c2), indicating anti-cancer properties [43].

Compound 1g is a white powder with a purple–red color when reacting with 10% ethanol sulfate, HR-ESI-MS m/z = 531.3328 [M + COOH]− (calcd for C30H46O5COOH). A reduction of 2 amu compared with the substrate indicates an increase in unsaturation of one. In the 13C NMR spectroscopic data, new carbon signals are observed at δC 216.72, 85.11, and 67.96, while the original signals for the C-3 hydroxyl and C-23 aldehyde groups disappear. Based on nuclear magnetic data and analysis, like compound 1g, the signal at δC 85.11 is attributed to C-13, and the signals at δC 132.32 and 131.71 are attributed to C-11 and C-12, respectively, indicating the presence of C-13 in 1h. An epoxy structure is observed between C-28 and another new carbonyl carbon signal at δC 216.72, increasing unsaturation by one at the epoxy point, suggesting that C-3 is a ketone group. A new carbon signal at δC 67.96 is related to the disappearance of the C-23 aldehyde group. Furthermore, remote-phase relationships are observed for the proton δH 3.71 (d, J = 10.3 Hz, H1-23b) and the carbons at δC 52.99 (C-4), δC 46.65 (C-5), δC 17.42 (C-24), and δC 216.72 (C-3). In the HSQC spectroscopic data, the carbon at δC 67.96 and the protons at δH 3.71 (d, J = 10.3 Hz, H1-23b) and δH 4.05 (d, J = 10.3 Hz, H1-23a) are directly correlated, providing further evidence that the carbon at δC 67.96 corresponds to C-23. Hence, the chemical compound 1g has been identified as 3-oxo-16α, 22α, 23-trihydroxy-13, 28-epoxy-olean-11-ene, discovered to be similar to saikogenin, which has been widely reported to decrease nitric oxide (NO) production, and decrease prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) release in lipopolysaccharide (LPS)-activated macrophage raw 264.7 cells significantly [44,45,46].

Compound 1h, which appeared as a white powder, exhibited a purple–red reaction in the 10% ethanol sulfate reaction, HR-ESI-MS m/z = 549.3433 [M + COOH]− (calcd for C30H48O6COOH, 549.3433). In the 13C NMR data, new characteristic carbon signals at δC 216.93, 72.79, and 68.51 were observed, while the original C-3 hydroxyl and C-23 aldehyde signals disappeared. The nuclear magnetic and HSQC spectroscopic data indicated that the unsaturated degree of the functional groups remained unchanged. Furthermore, the NOESY showed that the hydroxyl group connected to the C-7 position is in a β configuration. Therefore, 1h is identified as 3-oxo-7β, 16α, 22α, 23, 28-pentahydroxy-olean-12-ene. Additionally, this compound exhibited structural similarity to compound 1e, resembling theasapogenol E and camelliagenin E.

Compound 1i was obtained as a white powder, HR-ESI-MS m/z = 521.3490 [M + COOH]− (calcd for C29H46O5COOH) suggesting that there is one fewer double bond or ring structure in compound 1j compared with camelliagenin B. In the 13C NMR data of compound 1j, the original C-23 aldehyde group signal disappears, and only a new characteristic carbon signal δC 72.97 is added. It is speculated that the aldehyde group signal is converted into the characteristic signal δC 72.97, but according to the nuclear magnetic information similar to compound 1i, δC 72.97 is attributed to the C-7 position, and the hydroxyl group connected to it is a β configuration. In addition, in the case of a new C-7 oxygen atom, the molecular formula shows the change in the number of oxygen atoms, and the unsaturation is reduced by one. It is speculated that the oxygen atom in the original C-23 aldehyde group is lost, and the original aldehyde group may be converted into methyl or directly removed. In the 1H NMR data, the original 24-bit methyl single peak disappears, and a δH 1.24 (d, J = 6.3Hz, 3H) methyl split peak signal appears. In HMBC data, δH 1.24 (d, J = 6.3 Hz, 3H) is closely related to δC 38.96 (C-4), δC 49.79 (C-5) and δC 75.80 (C-3), indicating that the methyl is connected to the C-4 position. In addition, the coupling constant and peak split of the δH 1.24 (d, J = 6.3 Hz, 3H) signal suggest that there is also a proton connected to the C-4 position. In HSQC spectroscopic data at this time, the proton signal δH 1.59 (m, H1-4) was found to be directly related to C-4 (δC 38.96), further proving that there is a proton connected to the C-4 position. The above evidence shows that the C-4 position of 1j is connected to a methyl monohydrogen atom. In contrast, the substrate C-4 position is connected to a methyl monoaldehyde group, so the aldehyde group in the substrate is directly removed to form hydrogen atoms. The reduction of a carbon atom in the molecular formula proves this conclusion one step further. In the NOESY, it is found that δH 1.59 (m, H1-4) is strongly related to δH 0.99 (s, β-H3-25), proving that the methyl is in a β configuration. Compounds with the same A-ring structure have been reported57. They use X-ray diffraction technology to determine the configuration, consistent with our 13C NMR data. Thus, 1i was identified as 3β, 7β, 16α, 22α, 28-pentahydroxy-24-norolean-12-ene.

Compound 1j, a white powder, exhibits a distinct color change to purple when reacted with 10% sulfuric acid in ethanol. HR-ESI-MS m/z = 503.3382 [M + COOH]−, (calcd for C29H46O4COOH, 503.3378). Notably, the degree of unsaturation remains unchanged when the substrate is reduced by 30 amu. A closer examination of the 13C NMR data of 1j reveals the disappearance of the aldehyde signal at position C-23, replaced by a characteristic carbon signal at δC 85.25. Similarly, the 1H NMR data show the disappearance of the original 24-position methyl mono-signal, replaced by a methyl doublet signal at δH 1.25 (d, J = 6.4 Hz, 3H). These spectral changes indicate that the original C-23 aldehyde group has been reduced, leaving a single hydrogen atom, and the methyl group signal at δH 1.25 (d, J = 6.4 Hz, 3H) adopts an α configuration. The NMR data reveal that the signal at δC 85.25 is assigned to the C-13 position, while the signals at δC 132.25 and 131.95 are assigned to C-11 and C-12, respectively. This suggests the presence of an epoxy structure between C-13 and C-28 in 1j. Hence, structure 1j is confirmed and defined as 3β, 16α, 22α-trihydroxy-13, 28-epoxy-24-norolean-11-ene, which was found to share structure similarity with the different chemical compounds obtained from the Paeonia genus, and all the isolated compounds were studied further for their anti-inflammatory, antioxidant, and anti-cancer effects against various cell lines and other biological activities [47].

The biocatalysis of camelliagenin B (1) with B. subtilis was characterized by glycosylation at C-3 and C-22, likely to be mediated by cytochrome P450 catalytic reactions, particularly through enzymes such as CYP102A1, which is known for its broad substrate specificity in B. megaterium [48,49,50,51]. S. gresius was observed to catalyze camelliagenin B (1), generating compounds 1c and 1d with stable C-29 carboxylation, probably through CYP125-mediated sterol side-chain degradation pathways [52,53,54]. Comparing the two catalytic reactions, interestingly, B. subtilis yielded monolithic products with two glycosylated compounds (C-3/C-22), instead of C-28, which was observed for the first time. In contrast to the chemicalization results, S. gresius exhibited strong oxidation at the specific site of the C-29 methyl group. Intriguingly, B. megaterium was found to biotransform camelliagenin B into six new compounds 1e-1j, likely to exhibit exceptional catalytic versatility, possibly through UDP–glucose-dependent mechanisms similar to those reported in other Bacillus spp [55,56]. Moreover, it showed carbonylation at C-3, hydroxylation at C-7 and C-11, hydroxylation and rearrangement reaction at C-23 position, and removal of the aldehyde group at the C-23 position. The hydroxylation of C-11 was considered allylic and unstable, leading to rearrangement reactions under different compounds and external conditions. All compounds except for one were rearranged into an △11-13,28-cyclic ether structure. To investigate whether the rearrangement reaction occurs spontaneously, we re-transformed the microorganisms under the same conditions and performed TLC analysis directly after transformation. The isolated metabolites were used as control spot plates, and the results indicate that this rearrangement reaction occurs during substrate transformation.

The C-23 aldehyde reaction of camelliagenin B reveals a connection between eukaryotes’ saikosaponin biosynthesis, suggesting evolutionary conservation of these biochemical pathways between eukaryotes and prokaryotes [57]. Our research has made significant progress by eliminating the C-23 aldehyde group, a critical step in the sterol synthesis pathway. This process involves the oxidation of the C-23 methyl group, forming a methylene hydroxyl, aldehyde group, and, ultimately, a carboxyl group [58,59,60,61]. Additionally, the newly discovered catalytic reaction provides crucial insights into the biosynthesis of certain pentacyclic triterpenes in plants, such as glycyrrhizic acid and saponins [62,63]. Moreover, this research reveals a specific biochemical reaction prevalent in eukaryotic organisms that had not been previously documented in bacteria [64].

Remarkably, the above-isolated compounds share a foundation with other known saponins, which have been reported for their varied biological properties [65,66]. The similarity extends beyond their chemical composition and can be modified by adjusting specific functional groups, which have the potential to generate different metabolites with unique biological activities [64,65,66,67]. For example, glycosylation at C-3/C-22 is likely to exhibit improved bioavailability, antifungal, and immunomodulatory effects. When both are glycosylated in saponins, they may exhibit balanced hydrophilicity and reduced toxicity, as reported in ginsenosides and soyasaponins [24,67]. Site-selective oxidation by S. gresius at C-29 has been observed in various oleanane triterpenoids having anti-inflammatory actions [31,68].

Overall, from a broader perspective, these findings highlight several key advantages of microbial biotransformation over conventional chemical synthesis. The process exhibits exceptionally high regioselectivity, strong stereoselectivity, and a range of reaction types, particularly in handling complex natural compounds with multiple reactive sites [69]. Compared with chemical methods, microbial biotransformation offers a more environmentally friendly approach, minimizing issues of molecular rearrangements and isomerization [70,71]. Therefore, the biotransformation of camelliagnin B has proven to be a highly productive method for expanding the structural diversity of this compound, offering unique reactions and metabolites with vast potential applications.

4. Conclusions

This study highlights the potential of microbial transformation of camelliagenin B from Camellia oleifera seed cake meal into novel bioactive metabolites. By employing Bacillus subtilis ATCC 6633, Bacillus megaterium CGMCC 1.1741, and Streptomyces griseus ATCC 13273, we successfully isolated ten unique derivatives of camelliagenin B. These findings not only underscore the capacity of microbial strains to produce diverse and potent compounds but also provide a sustainable approach to valorizing tea saponins as a valuable resource. The resulting compounds exhibited unique structural modifications, including glycosylation, hydroxylation, and oxidation reactions. These novel compounds open avenues for developing new pharmaceuticals, functional foods, and natural preservatives, leveraging the underutilized Camellia oleifera seed cake meal. Moreover, this research underscores the importance of green chemistry and biologically friendly methods in producing value-added waste products, aligning with the principles of sustainability and environmental stewardship.

Author Contributions

Conceptualization, R.R. and J.Z. (Jian Zhang); Formal analysis, J.Z. (Jingling Zhang) and Y.M.; Funding acquisition, J.Z. (Jian Zhang); Investigation, X.J. and W.W.; Supervision, J.Z. (Jian Zhang); Writing—original draft, R.R.; Writing—review and editing, J.Z. (Jingling Zhang), X.J. and B.Y. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

 Informed Consent Statement

Not applicable.

 Data Availability Statement

The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author(s).

 Acknowledgments

During the preparation of this manuscript/study, the author (s) used Grammarly to check and improve grammatical errors or mistakes. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

 Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Table

Figure 1 Schematic illustration: The extraction and preparation of camelliagenin B (1).

Figure 2 Biotransformation of compounds from Camelliagenin B (1). (A) 1a, 1b by B. subtilis ATCC 6633 and (B) 1c, 1d by S. gresius ATCC 13273.

Figure 3 Key HMBC and NOESY correlations of compounds 1c and 1d.

Figure 4 Biotransformation of compounds 1e, 1f, 1g, 1h, 1i, and 1j from Camelliagenin B (1) by B. megaterium CGM 1.1741.

Composition of culture medium.

Potato Dextrose (PD) Soybean Meal (SM)
Peeled potatoes 200 g Glucose 20 g
Glucose 20 g Yeast powder 5 g
KH2PO4 3 g NaCl 5 g
MgSO4.7H2O 1.5 g K2HPO4 5 g
Distilled water 1 L Soybean meal 5 g
Distilled water 1 L

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11070407/s1. The detailed structure elucidation and 13C NMR spectroscopic data of 1a-1j are given in the supplementary file section S1 (Table S1 and Figures S1–S42, respectively).

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© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

This study investigates the potential for the microbial transformation of camelliagenin B, a saponin derived from Camellia oleifera seed cake meal, to develop novel metabolites. We employed three microbial strains, specifically Bacillus subtilis ATCC 6633, Bacillus megaterium CGMCC 1.1741, and Streptomyces griseus ATCC 13273, to biotransform camelliagenin B into its derivatives. The compounds were purified and separated using chromatographic techniques, such as high-performance liquid chromatography (HPLC). Structural identification was carried out using spectroscopic methods, including nuclear magnetic resonance (NMR) and mass spectrometry (MS). Ten bioactive compounds were obtained (1a-1j), of which nine were novel with multiple tailoring reactions, such as allyl oxidation, C-C double-bond rearrangement, hydroxylation, dehydrogenation, and glycosylation, observed in camelliagenin B analogs. The structures of these compounds were determined by 1D/2D NMR and HR-ESI-MS analysis. Therefore, this study showcases the capacity of microbial transformation as a sustainable and environmentally friendly method for generating bioactive compounds from C. oleifera seed cake meals. The individual chemicals can potentially facilitate the design of novel medicinal agents, functional foods, and natural preservatives.

Details

Title
Assisted Isolation of Camelliagenin B from Camellia oliefera Seed Cake Meal and Microbial Transformation by Bacillus subtilis ATCC 6633, Bacillus megaterium CGMCC 1.1741, and Streptomyces gresius ATCC 13273
Author
Raj Richa 1 ; Zhang, Jingling 1 ; Meng Yanyan 1 ; Jiang Xuewa 1 ; Wang, Wei 1 ; Zhang, Jian 2   VIAFID ORCID Logo  ; Yu Boyang 3 

1  Department of Traditional Chinese Medicine Resources, School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198, China; [email protected] (R.R.); [email protected] (J.Z.); [email protected] (Y.M.); [email protected] (X.J.); [email protected] (W.W.) 
2  Department of Traditional Chinese Medicine Resources, School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198, China; [email protected] (R.R.); [email protected] (J.Z.); [email protected] (Y.M.); [email protected] (X.J.); [email protected] (W.W.), Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing 211198, China; [email protected] 
3  Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing 211198, China; [email protected] 
First page
407
Publication year
2025
Publication date
2025
Publisher
MDPI AG
e-ISSN
23115637
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.3390/fermentation11070407
ProQuest document ID
3233183668
Copyright
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.