Method for increasing yield of monoterpene glycoside in endophytic fungus fermentation product separated from Chinese herbaceous peony

Abstract

The invention discloses a method for increasing the yield of monoterpene glycoside in an endophytic fungus fermentation product separated from paeonia lactiflora, and relates to the technical field of plant fermentation. The method comprises the following steps: material treatment; separating strains; identifying strains; preparing a strain and an elicitor: drying and crushing the overground part of the Chinese herbaceous peony by using the endophytic fungus ER6 separated and identified in the step S3, and sieving with a 80-mesh sieve to prepare powder as a key elicitor; a potato glucose liquid culture medium is used as a basic fermentation culture medium, an activated ER6 strain is inoculated into a fermentation container filled with a sterilized PDB culture medium, and dry powder of the overground part of paeonia lactiflora is added into the culture medium to serve as a biological elicitor to be cultured in a shaking table; and harvesting fermentation liquor. According to the method, a specific endophytic fungus ER6 is separated from paeonia lactiflora roots, and it is found that the yield of monoterpene glycoside in a fermentation product can be remarkably increased by using the overground part of paeonia lactiflora as a biological elicitor to ferment the endophytic fungus ER6.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant fermentation, and particularly relates to a method for improving the yield of monoterpenoid glycosides in a fermentation product of endophytic fungi isolated from Paeonia lactiflora. BACKGROUND

[0002] The livestock industry faces an increasing demand for safe and efficient growth promoters after the ban on antibiotics. Monoterpenoid glycosides (such as paeoniflorin) in the roots of Paeonia lactiflora have been proven to have the potential to increase muscle mass and reduce fat in healthy organisms, but their industrial application is limited by traditional plant extraction methods such as solvent extraction and reflux extraction. These methods have inherent drawbacks such as high cost, high resource consumption, and poor sustainability, which make it difficult to meet the demand for large-scale application in the livestock industry.

[0003] Combining the application requirements of the field of plant fermentation and the aforementioned industry pain points, the use of Paeonia lactiflora endophytic fungi to ferment monoterpenoid glycosides is a potential strategy to replace plant extraction and achieve sustainable supply. This approach not only aligns with the green production philosophy of plant fermentation technology, but also avoids the resource dependence and cost defects of plant extraction methods from the root. Currently, co-fermentation of Paeonia lactiflora endophytic fungi and aboveground parts has been explored, laying a foundation for subsequent construction of Paeonia lactiflora endophytic fungal microbial cell factories and optimization of monoterpenoid glycoside production efficiency. However, the endophytic fungi isolated in this field can produce monoterpenoid glycosides, and the synthesis level of the target product in the basic fermentation medium is extremely low, far from meeting the economic requirements of industrial production. The low production problem is due to the fact that under non-specific induction conditions, the monoterpenoid glycoside biosynthesis pathway of endophytic fungi is usually in a silent or low expression state, making it difficult to efficiently accumulate target products. This is also a typical problem in the directed synthesis of microbial secondary metabolites in plant fermentation technology.

[0004] Therefore, the core technical problem existing in the field is how to effectively activate and strengthen the monoterpenoid glycoside biosynthesis pathway in Paeonia lactiflora endophytic fungi to break through the fermentation yield bottleneck and meet the economic supply demand of monoterpenoid glycoside growth promoters for large-scale application in the livestock industry.

[0005] In view of the above core problem, the field has attempted conventional fermentation optimization strategies (such as carbon and nitrogen source optimization, pH control, etc.), but such strategies focus on optimizing the growth environment of microorganisms and are difficult to directly act on the silent monoterpenoid glycoside biosynthesis pathway, resulting in limited yield improvement. In contrast, the biological transformation strategy, i.e. by adding specific precursors or inducers exogenously, has been proven to be an efficient approach to activating microbial secondary metabolism and directing the improvement of target product yield. However, for the specific production system of Paeonia lactiflora endophytic fungi, what kind of substrate can serve as an efficient precursor or inducer to precisely activate its monoterpenoid glycoside synthesis pathway and improve product yield, there is currently no relevant research report, which is a key blank point restricting the industrialization of this technology.

[0006] In summary, there are two major bottlenecks in the prior art: on the one hand, the traditional method of extracting monoterpenoid glycosides from paeonia root relies too much on plant resources, has high extraction cost and low yield, and is difficult to meet the large-scale demand of the livestock industry; on the other hand, there is a lack of efficient elicitors to activate the biosynthesis of monoterpenoid glycosides, and the fermentation efficiency is low, which cannot meet the industrial demand. The present application aims to provide a method for improving the yield of monoterpenoid glycosides without genetic modification, only by simple process optimization. Developing a method for significantly improving the yield of monoterpenoid glycosides of paeonia endophytic fungi through specific biotransformation strategy is of great significance to solve the resource bottleneck and unlock the application potential of paeonia in the field of livestock industry. As a traditional Chinese medicinal material, the underground part (root) of paeonia is widely used for medicinal purposes, while the aboveground part (stem, leaf, flower) is discarded as agricultural waste, with very low resource utilization rate. Considering the similarity of secondary metabolite components between the aboveground and underground parts of paeonia (such as both containing phenolic and terpenoid precursor substances), and the easy availability and low cost of resources, combined with the symbiotic characteristics of endophytic fungi and host plants, it is speculated that adding the aboveground part of paeonia as an elicitor to the fermentation system can specifically activate the biosynthesis pathway of monoterpenoid glycosides of paeonia endophytic fungi, and fundamentally solve the problem of low yield in fermentation, and realize the high-value utilization of agricultural waste. SUMMARY

[0007] To solve the core problems in the prior art that the extraction of paeonia monoterpenoid glycosides relies on plant root resources, the fermentation of endophytic fungi has low yield due to the silence of the synthesis pathway, and the aboveground part of paeonia is wasted as waste, the present application provides a method for improving the yield of monoterpenoid glycosides in the fermentation product of an endophytic fungus isolated from paeonia (Paeonia lactiflora Pall.) produced in Heilongjiang Province. The method isolates a specific endophytic fungus Hymenula cereal ER6 from paeonia root, and finds that using the aboveground part of paeonia as a biological elicitor for fermentation can significantly improve the yield of monoterpenoid glycosides in the fermentation product.

[0008] The present application provides a method for improving the yield of monoterpenoid glycosides in the fermentation product of an endophytic fungus isolated from paeonia produced in Heilongjiang Province, characterized by comprising the following steps:

[0009] S1, material treatment: fresh paeonia root is washed with running water and sterilized;

[0010] S2, strain isolation: the root tissue after surface sterilization is cut into 1 cm small pieces, placed on PDA plate, and cultured in a 28℃ constant temperature incubator, and when the colonies grow around the tissue blocks, they are immediately transferred to new PDA plates for purification until pure endophytic fungal culture is obtained;

[0011] S3, strain identification: observe the colony morphology and microstructure of the colonies cultured on PDA plates for 7-15 days, extract the genomic DNA of the strains using a kit, perform PCR amplification using ITS1 and ITS4 as primers, sequence the PCR products, perform BLAST comparison of the sequences in the NCBI database, and construct a phylogenetic tree for identification, and one of the strains is identified as the endophytic fungus Hymenula cereal ER6 by the above method;

[0012] S4, strain and inducer preparation: the endophytic fungus ER6 separated and identified in step S3 is used as the strain, the aboveground part of Paeonia lactiflora is dried, crushed, and sieved through an 80-mesh sieve to make a powder, and the powder is sterilized and used as the key inducer;

[0013] S5, fermentation culture: using PDB liquid medium as the basic fermentation medium, the activated ER6 strain is inoculated into a fermentation container containing sterilized PDB medium, and the dry powder of the aboveground part of Paeonia lactiflora is added as a biological inducer; shake culture.

[0014] S6, fermentation broth harvesting: after the fermentation is completed, the fermentation broth is harvested.

[0015] Further, the mass percentage concentration of the dry powder of the aboveground part of Paeonia lactiflora added in step S5 is 0.5-1.5%.

[0016] Further, the fermentation conditions in step S5 are that the temperature is 28 DEG C, the rotation speed is 120 rpm, and the fermentation time is 7-28 days.

[0017] Further, in step S5, the mass percentage concentration of the dry powder of the aboveground part of Paeonia lactiflora added is 1%.

[0018] Further, in step S5, the mass percentage concentration of the dry powder of the aboveground part of Paeonia lactiflora added is 0.83%.

[0019] Further, in step S5, the fermentation time is 7 days.

[0020] Further, the fermentation broth obtained in step S6 is dried at 50 DEG C to obtain a solid extract.

[0021] The application further provides a method for identifying the endophytic fungus Hymenula cereal ER6 of Paeonia lactiflora:

[0022] The fungal genomic DNA extraction kit is used to extract the DNA of each strain;

[0023] PCR amplification was performed using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3').

[0024] The PCR reaction volume was 50 μL, with 1.0 μL of genomic DNA, 5.0 μL of 10×Buffer (containing Mg²⁺), 1.0 μL of Taq polymerase, 1.0 μL of dNTPs, 1.5 μL each of forward and reverse primers, and ddH₂O added to a final volume of 50 μL.

[0025] The PCR reaction conditions were as follows: pre-denaturation at 95 °C for 5 min, followed by the following cycle: denaturation: 95 °C for 30 s, annealing: 58 °C for 30 s, extension: 72 °C for 1 min, for a total of 35 cycles. After the cycle, the extension was performed at 72 °C for 7 min.

[0026] After the PCR products were detected by agarose gel electrophoresis, they were sequenced, and the obtained sequences were compared with the NCBI database by BLAST. A phylogenetic tree was then constructed using MEGA 11.0 software.

[0027] The present invention also provides a fermentation broth obtained by the preparation method described above.

[0028] The present invention also provides the application of the fermentation broth in the preparation of natural growth-promoting feed additives.

[0029] Compared with existing technologies, the beneficial effects of this invention are as follows: 1. This invention is the first to discover, using the above-ground parts of peony as a biological inducer, combined with a specific endophytic fungal strain ER6, to specifically and efficiently activate the metabolic pathway for monoterpene glycoside synthesis in the fungus. Experimental data confirm that this combination is the optimal combination for achieving monoterpene glycoside biosynthesis, with a yield significantly higher than the control group using other peony tissues (roots, cork) or exogenously added chemical precursors (such as pyruvate, naphthaleneacetic acid). This confirms the superior efficacy of this strategy in targeted enhancement of the synthesis of target secondary metabolites.

[0030] 2. This process achieves peak synthesis of monoterpenoid glycosides within a short period of 7 days, demonstrating high production efficiency and space-time yield. Based on conventional PDB medium and utilizing low-cost, easily prepared peony aerial part powder as an inducer, the entire process is cost-effective, simple to operate, and possesses good industrial scale-up potential and economic feasibility.

[0031] 3. Pharmacodynamic experiments showed that the fermentation broth obtained by this method exhibited statistically significant enhancements in in vitro antioxidant activity (including total reducing power, DPPH free radical scavenging rate, and ABTS⁺· scavenging rate) compared to the basal fermentation broth. This indicates that this technology not only quantitatively increases the yield of a single indicator component (monoterpene glycoside) but also simultaneously enhances the bioactivity spectrum of the fermentation product, demonstrating a synergistic effect among its components. This lays the foundation for developing highly active natural product formulations.

[0032] 4. This invention transforms the aerial parts of peony, which are traditionally discarded or devalued, into a highly efficient bioprocess inducer. This achieves the "whole plant" and "high-value" utilization of peony medicinal resources, breaking through the traditional model that relies on root extraction, and providing a brand-new technical path for the sustainable development of Chinese medicinal resources. Attached Figure Description

[0033] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0034] Figure 1 The diagram shows endophytic fungi on the roots of peony, where A) ER3, B) ER5, C) ER6, D) ER1, E) ER2, F) ER4, and G) ER7.

[0035] Figure 2 Micrographs of endophytic fungi in peony roots (10×40), where A) ER3, B) ER5, C) ER6, D) ER1, E) ER2, F) ER4, G) ER7, with red arrows pointing to spores;

[0036] Figure 3 A phylogenetic tree diagram;

[0037] Figure 4 This is a graph showing the yield of monoterpenoid glycosides in endophytic fungi after biotransformation.

[0038] Figure 5 The graph shows the effect of substrate addition on the production of monoterpene glycosides by endophytic fungi.

[0039] Figure 6 Figure showing the effect of fermentation time on the production of monoterpene glycosides by endophytic fungi;

[0040] Figure 7 The graph shows the total reducing power measurement.

[0041] Figure 8 The graph shows the results of the DPPH· scavenging rate determination.

[0042] Figure 9 For ABTS + • Graph showing the results of the clearance rate measurement. Detailed Implementation Specific implementation method one:

[0044] Materials and reagents: Peony (Paeonia lactiflora Pall.) roots were collected from Jiamusi City, Heilongjiang Province. The roots, cork, and aerial parts of the peony used for biotransformation were dried, pulverized, and passed through an 80-mesh sieve for later use. Benzoic acid standard, methanol, isopropanol, acetonitrile, and formic acid (chromatographic grade); pyruvic acid, naphthaleneacetic acid, potassium dihydrogen phosphate, acetic acid, DPPH, ABTS, penicillin sodium, streptomycin sulfate, lactophenol cotton blue, potato extract, mercuric chloride, and glucose (analytical grade). Laboratory-prepared triple-distilled water was used.

[0045] Example 1:

[0046] S1. Fungal Isolation: Endophytic fungi were isolated using the tissue block isolation method. Fresh peony roots were rinsed with running water and then disinfected in a clean bench with 0.1% HgCl2 solution for 60 seconds (rinsed three times with sterile water) and 75% ethanol for 20 seconds (rinsed three times with sterile water, 30 seconds each time).

[0047] S2. Cut the surface-sterilized root tissue into 1 cm pieces. 3 Small segments were placed in a container containing penicillin sodium and streptomycin sulfate (45 μg / mL each). -1 The culture was incubated upside down on PDA plates at 28 ℃, with colony growth observed daily. Once colonies appeared around the tissue block, it was immediately transferred to a new PDA plate for purification until a pure culture was obtained. Simultaneously, 100 μL of the final rinse water was spread onto a blank plate and incubated under the same conditions for 7 days. No colonies grew on the plate, indicating thorough surface disinfection and no exogenous bacterial contamination.

[0048] S3. Preliminary morphological identification was performed by observing the colony morphology (size, color, texture, etc.) after culturing on PDA plates for 7-15 days, combined with microscopic structure (hyphae and spore morphology observed under an optical microscope after lactophenol cotton blue staining). DNA was extracted from each strain using a fungal genomic DNA extraction kit (Kangwei Century). PCR amplification was performed using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGC TTATTGATATGC-3'). The PCR reaction volume was 50 μL, containing 1.0 μL of genomic DNA, 5.0 μL of 10×Buffer (containing Mg²⁺), 1.0 μL of Taq polymerase, 1.0 μL of dNTPs, 1.5 μL each of forward and reverse primers, and ddH₂O to a final volume of 50 μL. The PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 5 min, followed by the following cycling steps: denaturation: 95 ℃ for 30 s, annealing: 58 ℃ for 30 s, extension: 72 ℃ for 1 min, for a total of 35 cycles. After each cycle, an extension was performed at 72 ℃ for 7 min. The PCR products were analyzed by agarose gel electrophoresis and then sent to Shanghai Paisennong Biotechnology Co., Ltd. for sequencing. The obtained sequences were BLAST compared with the NCBI database, and a phylogenetic tree was constructed using MEGA 11.0 software. The ITS sequence of this strain showed more than 95% similarity to *Hymenulacerealis* (GenBank accession number: MF574268.1), thus identifying it as a new strain and naming it *Hymenula cerealis* ER6. Subsequently, it was inoculated onto PDA slant medium and cultured at 28 ℃. After the mycelial growth stabilized, it was stored at 4 ℃.

[0049] S4. The endophytic fungi isolated and identified using the above steps are dried, pulverized, and passed through an 80-mesh sieve to make powder as a key inducer.

[0050] S5. Fermentation culture: PDB (potato dextrose liquid) was used as the basic fermentation medium. The activated ER6 strain was inoculated into a fermentation vessel containing PDB medium. Dried powder of peony aerial parts was added to the medium as a biological inducer. The mass-volume percentage concentration of the dried powder of peony aerial parts was 0.5%. The fermentation conditions were: temperature 28 ℃, rotation speed 120 rpm, and fermentation time 7 days.

[0051] S6. Fermentation broth harvest: After fermentation is completed, the fermentation broth is harvested.

[0052] The fermentation broth obtained in step S6 was dried at 50 °C to obtain a solid extract.

[0053] Example 2:

[0054] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 7 days.

[0055] Example 3:

[0056] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1.25%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 7 days.

[0057] Example 4:

[0058] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1.5%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 7 days.

[0059] Example 5:

[0060] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 0.5%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 14 days.

[0061] Example 6:

[0062] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 14 days.

[0063] Example 7:

[0064] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added was 1.25%, the fermentation conditions were a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 14 days.

[0065] Example 8:

[0066] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1.5%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 14 days.

[0067] Example 9:

[0068] The difference from Example 1 is that the mass-volume percentage concentration of the dried peony aerial parts added in step 5 is 0.5%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 21 days.

[0069] Example 10:

[0070] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 21 days.

[0071] Example 11:

[0072] The difference from Example 1 is that the mass-volume percentage concentration of the dried peony aerial parts added in step 5 is 1.25%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 21 days.

[0073] Example 12:

[0074] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1.5%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 21 days.

[0075] Example 13:

[0076] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 0.5%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 28 days.

[0077] Example 14:

[0078] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 28 days.

[0079] Example 15:

[0080] The difference from Example 1 is that the mass-volume percentage concentration of the dried peony aerial parts added in step 5 is 1.25%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 28 days.

[0081] Example 16:

[0082] The difference from Example 1 is that in step 5, the mass-volume percentage concentration of the dried powder of the above-ground parts of peony added is 1.5%, the fermentation conditions are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 28 days.

[0083] Example 17:

[0084] The difference from Example 1 is that in step 5, the endophytic fungi are inoculated into a 150 mL Erlenmeyer flask containing PDB medium, and 1.25 g of peony aerial parts are added to each 150 mL fermentation system, that is, the mass-volume percentage concentration of the dried peony aerial parts is 0.83%, and the fermentation time is 7 days.

[0085] Comparative example:

[0086] Step S3: Biotransformation substrate screening. Seven endophytic fungi (ER1-ER7) were inoculated into PDB medium (150 mL / 250 mL Erlenmeyer flasks) containing different substrates. Six treatment groups were set up: control group: PDB medium only; plant tissue group: 1% (w / v) of peony root, cork, or aerial parts (stem and leaf mixture) powder were added; precursor compound group: pyruvate (final concentration 1 mL·L⁻¹) was added. -1 ) or naphthaleneacetic acid (final concentration 1 mg·L -1 Each group was repeated three times, and cultured in a shaker at 28 ℃ and 120 rpm for 7 days.

[0087] Based on the optimal strain-substrate combination obtained through screening, the process was further optimized: Substrate addition levels were increased to 0.5%, 0.75%, 1%, 1.25%, and 1.5% (w / v) and cultured for 7 days. Fermentation time was increased to 7, 14, 21, and 28 days at the optimal substrate addition levels. After fermentation, the fermentation broth was harvested for subsequent analysis.

[0088] Determination of monoterpene glycoside content

[0089] The fermentation broth was dried in a 50 ℃ oven and then extracted with 60% ethanol (solid-to-liquid ratio 1:10) by ultrasonication (300 W) for 60 min. The extract was concentrated by rotary evaporation and then treated with alkaline hydrolysis-benzoic acid. Brief procedure: The extract was dissolved in 1% NaOH, 0.5 mL was transferred to a 10 mL volumetric flask, 8 mL of mobile phase was added, and the solution was diluted with 1 mol·L⁻¹. -1 Adjust the pH to 3-6 with HCl, and bring the mobile phase to a final volume. Benzoic acid content was determined by HPLC (Agilent 1260 Infinity II) and converted to total monoterpene glycoside content. Chromatographic conditions: Column: Elite C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: Phase A (0.05 mol·L⁻¹).-1 Phase B (potassium dihydrogen phosphate - acetic acid = 350:8): Phase B (methanol - isopropanol = 100:6) = 70:30; Flow rate: 1.0 mL / min -1 Column temperature: 35 ℃; Detection wavelength: 230 nm; Injection volume: 10 μL. Method validation (precision, repeatability, stability, and recovery) all met the requirements.

[0090] In vitro antioxidant activity assay

[0091] The in vitro antioxidant activity of the biotransformation fermentation broth of the optimal combination (ER6-L) was compared with that of the basic fermentation broth. Total reducing power was determined using the potassium ferricyanide method; DPPH scavenging rate was determined using the method of Brand-Williams et al.; ABTS⁺ scavenging rate was determined using the method of Miller et al. All measurements were performed in triplicate.

[0092] Statistical analysis

[0093] All experiments were performed in at least three independent replicates. Data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 29.0 software. One-way ANOVA was used for comparisons among multiple groups, followed by Duncan's multiple range test. P < 0.05 was considered statistically significant.

[0094] Isolation, identification and preliminary screening of endophytic fungi in peony roots

[0095] Seven endophytic fungi with distinct morphologies were isolated from peony roots using the tissue block method and named ER1 to ER7. (See [link to relevant documentation]). Figure 1 .Depend on Figure 1 It was observed that each fungal species exhibited a uniform morphology and good growth. Among them, ER-3, ER-5, and ER-6 were anaerobic bacteria, while the remaining endophytic fungi of peony roots were facultative anaerobic bacteria. Subsequent molecular biological identification of these seven isolated endophytic fungi was performed to further clarify their phylogenetic positioning. Using fungal genomic DNA as a template, amplification was performed using ITS-1 and ITS-4 primers, and the PCR products were detected by agarose gel electrophoresis.

[0096] Based on morphological observation and molecular biological identification, the strains belonged to the genera Byssochlamys, Paecilomyces, Rhizoctonia, Cephalosporium, and Clavispora, respectively, indicating that the endophytic fungal community of peony roots has rich diversity.

[0097] The content of monoterpene glycosides in the basal fermentation broths of seven fungal strains was further determined by alkaline hydrolysis-benzoic acid method combined with HPLC. The results showed that strains ER1, ER2, and ER4 possessed the ability to produce monoterpene glycosides under basal conditions, with yields of 8.26, 2.86, and 8.44 mg·L⁻¹, respectively. -1 (Table 1), among which ER4 has the highest production.

[0098] Table 1. Yield of monoterpenoid glycosides by endophytic fungi ( ±S, n=3)

[0099]

[0100] The yield of monoterpene glycosides obtained was significantly higher than the highest yield reported in the literature by endophytic fungi of *Paeonia lactiflora* (0.44 mg·L⁻¹). -1 This indicates that the endophytic fungi isolated from Paeonia lactiflora produced in Heilongjiang Province have greater potential in the synthesis of monoterpene glycosides.

[0101] Biotransformation strategies significantly improve monoterpene glycoside yield

[0102] To activate and enhance the monoterpene glycoside synthesis capacity of endophytic fungi, the biotransformation effects of five different substrates—peony root, cork bark, aboveground parts of peony (stem and leaf mixture), pyruvate, and naphthaleneacetic acid—were systematically evaluated. Figure 4 As shown in Table 2, the induction effects of different substrates on different strains varied significantly. Among them, the "ER6-paeonia aerial parts" (ER6-L) combination exhibited superior induction efficiency, increasing the monoterpene glycoside yield from 0 to 297.03 mg·L⁻¹. -1 In addition, the "ER1-peony root" combination also increased the yield from 8.26 mg·L⁻¹. -1 Significantly increased to 282.83 mg·L -1 .

[0103] Table 2. Yield of monoterpenoid glycosides by endophytic fungi after biotransformation (n=3)

[0104]

[0105] Subsequently, process optimization was performed on the optimal combination ER6-L. For example... Figure 5 As shown, when 1.25 g of peony aerial parts (0.0083 g·mL⁻¹) were added to each 150 mL fermentation system... -1 At that time, the monoterpene glycoside yield reached its peak (304.67 mg·L⁻¹). -1 Excessively high substrate concentrations (>1.0%) may lead to decreased yield due to substrate inhibition effects. Figure 5 (Table 3). The results of fermentation time optimization show that ( Figure 6(Table 4) The yield of monoterpene glycosides reached its highest level (306.61 mg·L⁻¹) after 7 days of cultivation. -1 Extending the fermentation time actually led to a decrease in content, suggesting that the product may have been degraded by subsequent fungal metabolism. Therefore, the optimal process was determined to be: adding 0.0083 g·mL. -1 The above-ground parts of peony were fermented for 7 days, repeated 3 times, yielding a monoterpene glycoside yield of 297.0 ± 13.8 mg·L⁻¹. -1 .

[0106] Table 3. Effect of substrate addition amount on monoterpene glycoside production by endophytic fungi (n=3)

[0107]

[0108] Table 4. Effect of fermentation time on monoterpene glycoside production by endophytic fungi (n=3)

[0109]

[0110] Biotransformation enhances the in vitro antioxidant capacity of fermentation broth

[0111] The in vitro antioxidant activity of the fermentation broth before and after ER6-L biotransformation was evaluated. For example... Figure 7-9 As shown in Tables 5-7, compared with the ER6 basic fermentation broth without substrate, the ER6-L fermentation broth showed significant enhancements in total reducing power, DPPH· scavenging rate, and ABTS⁺· scavenging rate (P < 0.05). This result indicates that the addition of the aboveground parts of peony not only specifically induced the synthesis of monoterpenoid glycosides but also globally enhanced the in vitro antioxidant potential of the fermentation system. This may stem from the release of exogenous antioxidant components from plant tissues and the synergistic synthesis of endogenous antioxidant metabolites from fungi.

[0112] Table 5. Measurement of total reducing power (n=3)

[0113]

[0114] Table 6. Results of DPPH· Scavenging Rate Measurement (n=3)

[0115]

[0116] Table 7 ABTS + • Scavenging rate measurement results (n=3)

[0117]

[0118] Conclusion: Phylogenetic analysis based on ITS sequence showed that this strain had a sequence similarity of more than 95% with Hymenula cereal strain (GenBank accession number MF574268.1), and was therefore a new strain, named Hymenulacereal ER6.

[0119] Endophytic fungus ER6 does not produce monoterpene glycosides under basic conditions, reflecting the "silent" nature of the gene clusters involved in the synthesis of microbial secondary metabolites. In this embodiment, the addition of the aboveground parts of peony—a "waste biomass"—instead of a single chemical precursor successfully unlocked the monoterpene glycoside synthesis capacity of ER6. This was achieved by adding 0.0083 g·mL⁻¹. -1 After the substrate was applied, its yield jumped from 0 to 308.7 ± 11.2 g·mL. -1 This phenomenon goes far beyond the scope of nutritional supplementation; its essence lies in the comprehensive biological stress initiated by plant tissues as a complex "biological signal reservoir" against fungi. Specific polysaccharides, phenolic acids, or cell wall degradation fragments contained in the aboveground parts, once recognized by fungi, may trigger their defense and stress response programs by activating specific transcriptional regulatory factors. The synthesis of monoterpenoid glycosides is a key step in this strongly activated process. This demonstrates the strategic advantage of using plant-microbe interactions for "directed induction" of target products and provides an excellent example of the high-value utilization of agricultural by-products.

[0120] Most importantly, this metabolic reprogramming, while increasing yield, directly endowed the fermentation broth with stronger in vitro antioxidant capacity. Results showed that ER6-L's DPPH· and ABTS... + The significantly improved clearance rate and total reducing power indicate that the addition of the substrate resulted in a holistic alteration of the metabolites, constructing a synergistic antioxidant network. This suggests that our "cellular factory" produces a complex, functionally enhanced system whose value transcends that of a single product.

[0121] Through strain isolation and substrate screening, the ER6-L fermentation system constructed in this embodiment successfully solved the problem of increasing the yield of monoterpene glycosides from zero to 308.7 ± 11.2 g·mL. -1 This breakthrough overcomes the bottleneck of low production of secondary metabolites in endophytic fungi. Crucially, this functional enhancement occurs simultaneously with the synthesis of the target product, demonstrating that the biotransformation yields a complex product rich in natural antioxidants.

Claims

1. A method of increasing the yield of monoterpene glycosides from a fermentation product of an endophytic fungus isolated from Paeonia, characterized in that: Specifically comprising the following steps: S1, material processing: fresh paeony roots are washed with running water and sterilized; S2, strain isolation: the root tissues after surface sterilization are cut into 1 cm small pieces, placed on PDA plates, and cultured in an incubator at 28 ℃, and when the colonies grow around the tissues, they are immediately transferred to new PDA plates for purification until pure endophytic fungal cultures are obtained; S3, strain identification: observe the colony morphology and microscopic structure of the culture on PDA plate for 7-15 d, extract the genomic DNA of the strain using the kit, perform PCR amplification with ITS1 and ITS4 as primers, sequence the PCR product, perform BLAST comparison of the sequence in NCBI database, and construct a phylogenetic tree for identification, one of the strains is identified as an endophytic fungus by the above method Hymenula cereal ER6; S4, strain and inducer preparation: the endophytic fungus ER6 is isolated and identified in step S3, the aboveground parts of paeony are dried, crushed, sieved through an 80-mesh sieve, made into powder, and sterilized to serve as a key inducer; S5, fermentation culture: using PDB liquid medium as the basic fermentation medium, the activated ER6 strain is inoculated into a fermentation container containing sterilized PDB medium, and the dry powder of the aboveground parts of paeony is added as a biological inducer; shake culture; S6, fermentation broth harvesting: after fermentation, the fermentation broth is harvested.

2. A method of increasing the yield of monoterpene glycosides from a fermentation product of an endophytic fungus isolated from Paeonia lactiflora according to claim 1, characterized in that: The mass percentage concentration of the dry powder of the aboveground parts of paeony added in step S5 is 0.5-1.5%.

3. A method of increasing the yield of monoterpene glycosides from a fermentation product of endophytic fungi isolated from Paeonia lactiflora according to claim 1 or 2, characterized in that: The fermentation conditions in step S5 are a temperature of 28 ℃, a rotation speed of 120 rpm, and a fermentation time of 7-28 d.

4. The method of increasing the yield of monoterpene glycosides from a fermentation product of an endophytic fungus isolated from Paeonia lactiflora of claim 2, wherein: The mass percentage concentration of the dry powder of the aboveground parts of paeony added in step S5 is 1%.

5. The method of increasing the yield of monoterpene glycosides from a fermentation product of an endophytic fungus isolated from Paeonia lactiflora of claim 2, wherein: The mass percentage concentration of the dry powder of the aboveground parts of paeony added in step S5 is 0.83%.

6. The method of increasing the yield of monoterpene glycosides from a fermentation product of an endophytic fungus isolated from Paeonia lactiflora of claim 3, wherein: The fermentation time in step S5 is 7 d.

7. The method of increasing the yield of monoterpene glycosides from a fermentation product of an endophytic fungus isolated from Paeonia lactiflora of claim 1, wherein: After the fermentation broth obtained in step S6 is dried at 50 ℃, a solid extract is obtained.

8. The Paeonia endophytic fungus of claim 1 Hymenula cereal Method for identifying ER6: The DNA of each strain is extracted using a fungal genomic DNA extraction kit; PCR amplification is performed using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCG CTTATTGATATGC-3'); The total volume of the PCR reaction solution is 50 μL, 1.0 μL of genomic DNA, 5.0 μL of 10×Buffer (containing Mg²⁺), 1.0 μL of Taq polymerase, 1.0 μL of dNTPs, 1.5 μL of each of the forward and reverse primers, and ddH2O to 50 μL; The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min, followed by the following steps: denaturation at 95 ℃ for 30 s, annealing at 58 ℃ for 30 s, extension at 72 ℃ for 1 min, a total of 35 cycles, and after the cycles, extension at 72 ℃ for 7 min; After the PCR product is detected by agarose gel electrophoresis, sequencing is performed, the obtained sequences are subjected to BLAST comparison in the NCBI database, and a phylogenetic tree is constructed using MEGA 11.0 software.

9. The fermentation broth obtained by the preparation method according to any one of claims 1-7.

10. The use of the fermentation broth according to claim 9 in the preparation of a natural growth-promoting feed additive.

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