Fructus aurantii processing method based on functional strain fermentation and fermented fructus aurantii

Abstract

The invention belongs to the technical field of traditional Chinese medicine processing and microbial fermentation. The invention relates to a fructus aurantii processing method, in particular to a fructus aurantii processing method based on functional strain fermentation and fermented fructus aurantii. The invention provides a strain composition. The strain composition contains Aspergillus urumi CQM3. Preferably, the composition can be further compounded with rhizopus arrhizus SGGM1 and / or aspergillus niger HQM2. When the strain composition is used for fermenting the fructus aurantii, the quality of a fructus aurantii processed product can be remarkably improved, white hyphae are uniformly and densely distributed on the appearance, the total content of flavonoid components is increased, the content proportion of flavonoid aglycone components with higher bioavailability is increased, and the antioxidant activity is improved. The invention also develops an adaptive fructus aurantii fermentation process aiming at the strain composition, and the whole fermentation process is controllable in process parameter and strong in stability, has good large-scale production adaptability, and has good application prospect and application value.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine processing and microbial fermentation technology. More specifically, it relates to a method for processing Citrus aurantium based on functional bacterial strain fermentation and the fermented Citrus aurantium. Background Technology

[0002] Citrus aurantium is a commonly used traditional Chinese medicine, derived from the Rutaceae plant Citrus aurantium (Citrus aurantium var. aurantium). Citrus aurantium The dried, immature fruit of *Citrus aurantium* (L.) and its cultivated varieties. Its main medicinal components are flavonoids, such as naringin, hesperidin, and neohesperidin. However, these components mostly exist in glycoside form, generally suffering from poor water solubility and low bioavailability, thus limiting their full efficacy to some extent. Existing research has shown that microbial fermentation can promote the deglycosylation of flavonoids in *Citrus aurantium*, converting them into aglycone forms with higher biological activity and easier absorption by the body, thereby enhancing their pharmacological effects.

[0003] Traditional processing of Citrus aurantium relies heavily on natural fermentation, a method dependent on environmental microorganisms and exhibiting significant drawbacks: ① long fermentation cycle (typically 5-7 days), making it difficult to determine the endpoint; ② uncontrollable microbial community structure, susceptible to contamination by other microorganisms, leading to large batch-to-batch quality fluctuations; ③ low flavonoid conversion efficiency and unclear direction, resulting in insufficient accumulation of highly active aglycone components. This makes it difficult to meet the requirements of batch consistency and quality stability for industrial production.

[0004] Therefore, there is an urgent need to develop a novel controlled fermentation technology for Citrus aurantium. By screening efficient and stable fermentation strains, optimizing process parameters, and establishing a standardized fermentation process control system, it is expected to significantly improve the quality uniformity and efficacy stability of fermented Citrus aurantium products, laying a solid foundation for their in-depth development in innovative drug research and development and functional health foods. Summary of the Invention

[0005] This invention aims to overcome the defects and shortcomings of existing natural fermentation processing methods for Citrus aurantium, such as uncontrollable fermentation microorganisms, long cycle, susceptibility to contamination by other microorganisms, low flavonoid conversion efficiency, and large batch quality fluctuations. It provides a microbial composition and a method for processing Citrus aurantium using this microbial composition for fermentation. This method can produce Citrus aurantium processed products with excellent appearance, efficient and targeted conversion of flavonoid glycosides into highly bioavailable aglycones, controllable process, and stable quality.

[0006] The first objective of this invention is to provide a strain of Aspergillus luchuensis CQM3.

[0007] A second objective of this invention is to provide a microbial composition.

[0008] A third object of the present invention is to provide the application of the above-described strains or bacterial compositions.

[0009] The fourth objective of this invention is to provide a method for processing fermented bitter orange peel.

[0010] The fifth objective of this invention is to provide a fermented bitter orange peel.

[0011] The sixth object of this invention is to provide a product.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a strain of Aspergillus ryukyu ( Aspergillus luchuensis CQM3, this bacterium was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with the accession number GDMCC No: 63972.

[0013] The present invention provides a microbial composition, characterized in that it contains the above-mentioned Aspergillus ryuki CQM3.

[0014] Preferably, the above-mentioned microbial composition further contains Rhizopus oligosporus (Rhizopus spp.) Rhizopus arrhizus SGGM1 or Aspergillus niger ( Aspergillus niger HQM2; The Rhizopus SGGM1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63974, and the Aspergillus niger HQM2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63973.

[0015] Preferably, in the above-mentioned microbial composition, the mass ratio of Aspergillus ryuki CQM3 to Rhizopus SGGM1 or Aspergillus niger HQM2 is (1~3):(1~2).

[0016] More preferably, the above-mentioned microbial composition further contains Rhizopus oligosporus (Rhizopus spp.) Rhizopus arrhizus SGGM1 and Aspergillus niger ( Aspergillus niger HQM2; The Rhizopus SGGM1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63974, and the Aspergillus niger HQM2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63973.

[0017] More preferably, in the above-mentioned microbial composition, the mass ratio of Aspergillus ryukiensis, Rhizopus septemlobus, and Aspergillus niger is (1~3):(1~2):(1~2).

[0018] More preferably, the mass ratio of Aspergillus ryukiensis, Rhizopus septemlobus, and Aspergillus niger is (1~2):1:1.

[0019] Optionally, the mass ratio of Aspergillus ryuki, Rhizopus spp. and Aspergillus niger is 2:1:1.

[0020] This invention provides an application of any one of a1 to a2 in any one of b1 to b2; a1. The above-mentioned Aspergillus ryuki CQM3 or its fermentation broth a2. The above-mentioned microbial composition or its fermentation broth; b1. Fermented bitter orange peel, b2. Preparation of products for fermenting bitter orange peel. b3. Increase the content of flavonoids in bitter orange peel. b4. Enhance the antioxidant activity of Citrus aurantium.

[0021] This invention provides a method for processing fermented immature bitter orange peel, using any one of a1 to a2 fermented immature bitter orange peel. a1. The above-mentioned Aspergillus ryuki CQM3 or its fermentation broth a2. The above-mentioned microbial composition or its fermentation broth.

[0022] Preferably, in the above-mentioned processing method, the fermentation time is 12-60 h.

[0023] More preferably, in the above-mentioned preparation method, the fermentation time is 48 hours.

[0024] Optionally, the fermentation temperature is 35~39 ℃ and the relative humidity is 85~95%.

[0025] Optionally, the fermentation temperature is 37 ℃ and the relative humidity is 90%.

[0026] Preferably, in the above processing method, the fermentation liquid in a1~a2 is used to inoculate the bitter orange peel for fermentation, and the inoculation amount is 10~20%.

[0027] More preferably, in the above processing method, the fermentation liquid in a1~a2 is used to inoculate and ferment the bitter orange peel, and the inoculation amount is 15%.

[0028] Specifically, the inoculation amount refers to the percentage (w / w) of the fermentation broth mass to the total mass of the bitter orange peel.

[0029] As an alternative implementation method, the fermented bitter orange peel is dried.

[0030] Optionally, the drying process is carried out at 40-60°C for 16-24 hours.

[0031] The fermented bitter orange peel product prepared by the fermentation and processing method described in this invention has significantly better quality than the naturally fermented bitter orange peel product: it has a uniform appearance with dense white mycelium, an increased total content of flavonoid components, an increased proportion of flavonoid aglycones with higher bioavailability, and improved antioxidant activity of bitter orange peel.

[0032] Therefore, the present invention provides a fermented bitter orange peel, which is prepared by the above method.

[0033] The present invention provides a product containing the above-mentioned fermented bitter orange peel.

[0034] The present invention has the following beneficial effects: This invention isolated three functional strains: *Aspergillus ryukyu* CQM3, *Rhizopus oligosporus* SGGM1, and *Aspergillus niger* HQM2. This invention provides a microbial composition containing *Aspergillus ryukyu* CQM3; preferably, it can be further combined with *Rhizopus oligosporus* SGGM1 and / or *Aspergillus niger* HQM2. Compared with natural fermentation, fermentation of *Citrus aurantium* using this microbial composition can significantly improve the quality of processed *Citrus aurantium* products: the surface of the processed *Citrus aurantium* products is uniformly covered with white mycelium, without discoloration or spoilage, and the appearance score reaches full marks; at the same time, the total content of flavonoids increases, the proportion of flavonoid aglycones with higher bioavailability increases, and the antioxidant activity is improved.

[0035] This invention also establishes a mixed-culture fermentation process with well-defined parameters and controllable process (inoculation ratio Z1:Z2:Z3 = 2:1:1, inoculation amount 15%, fermentation time 48 h, temperature 37 ℃, relative humidity 90%). The process is robust and has good repeatability. The resulting fermented bitter orange peel has significantly enhanced antioxidant activity, and possesses good appearance, high content of active ingredients, and functional value. It provides a reliable technical path for the standardization, large-scale production, and modernization of bitter orange peel processing, and has excellent application prospects and value. Attached Figure Description

[0036] Figure 1 The results of microbial community determination during the natural fermentation of Citrus aurantium (Figure A shows the species distribution of fungi at the phylum level during Citrus aurantium fermentation; Figure B shows the species distribution of fungi at the genus level during Citrus aurantium fermentation).

[0037] Figure 2 Figure A shows the results of flavonoid composition determination during the fermentation of Citrus aurantium; Figure B shows the content determination of 10 flavonoid compounds in Citrus aurantium; Figure B shows the heatmap of flavonoid composition and fungal species abundance.

[0038] Figure 3 Results of flavonoid content determination in fermented samples of Citrus aurantium prepared for different treatment groups.

[0039] Figure 4The results of free radical scavenging of fermented Citrus aurantium samples prepared for different treatment groups are shown in Figure A. The results of DPPH free radical scavenging are shown in Figure B. The results of ABTS free radical cation scavenging are shown in Figure B.

[0040] Figure 5 The liquid chromatograms are of 11 flavonoid mixed reference standards.

[0041] Figure 6 Composite score chart for single-factor samples with different inoculation ratios.

[0042] Figure 7 Composite score chart for single-factor samples with different inoculation amounts.

[0043] Figure 8 Composite score graph for single-factor samples with different fermentation times.

[0044] Figure 9 The response surface plots are shown for the effects of inoculation ratio, inoculation amount, and fermentation time on the composite score of the mixed fermented product of Citrus aurantium (Figure A is the three-dimensional response surface plot of the effect of inoculation amount and inoculation ratio on the composite score; Figure B is the three-dimensional response surface plot of the effect of fermentation time and inoculation ratio on the composite score; Figure C is the three-dimensional response surface plot of the effect of inoculation amount and fermentation time on the composite score).

[0045] Figure 10 The liquid chromatogram shows the flavonoid components of the mixed bacterial fermentation sample of Citrus aurantium prepared under optimal process conditions.

[0046] Figure 11 This is a picture of a mixed bacterial fermentation sample of Citrus aurantium prepared under optimal process conditions. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] The Citrus aurantium was purchased from Guangzhou Zhixin Pharmaceutical Co., Ltd. (batch number 190701).

[0050] Example 1: Analysis of Microbial Community Diversity During Natural Fermentation of Citrus aurantium Based on High-Throughput Sequencing Technology I. Experimental Methods 1. Preparation of naturally fermented bitter orange peel Take the raw material of Citrus aurantium, weigh it, soak it in water for 4 hours, and then place the soaked Citrus aurantium under natural conditions for fermentation. Take samples on days 0, 1, 2, 3, 4 and 5 of fermentation, and number them N0d to N5d. Put them into sterile sealed bags and store them in a -80℃ refrigerator for later use. Take 3 biological replicates for each sample to obtain the corresponding fermented Citrus aurantium product.

[0051] 2. Collection of total DNA from samples Take 10 g of fermented bitter orange peel (N0d~N5d) and place them in sterile centrifuge tubes. Add 50~100 mL of sterile water and shake at 200 r / min for 60 min. Sonicate for 5 min and shake again at 200 r / min for 60 min. After shaking, transfer the supernatant to a new sterile centrifuge tube and centrifuge at 8000 r / min. Discard the supernatant, collect the precipitate, freeze it quickly in liquid nitrogen, and store it in a freezer at -80℃ for later use.

[0052] 3. Library construction and sequencing After extracting total DNA from the sample, primers were designed based on conserved regions. Sequencing adapters were added to the primer ends, and PCR amplification was performed. The products were then purified, quantified, and homogenized to form sequencing libraries. The constructed libraries underwent quality control, and those that passed quality control were sequenced using Illumina NovaSeq 6000. The raw image data files obtained from high-throughput sequencing were converted into raw sequencing reads through base calling analysis. The results were stored in FASTQ (fq) file format, which included the sequence information of the sequencing reads and their corresponding sequencing quality information.

[0053] 4. High-throughput sequencing data processing and analysis The high-throughput sequencing work was submitted to Qingke Technology Co., Ltd. (Guangzhou, China) for completion. First, Trimmomatic v0.33 software was used to filter the raw reads obtained from sequencing. Then, cutadapt 1.9.1 software was used to identify and remove primer sequences, obtaining clean reads that do not contain primer sequences. Noise was removed using the dada2 method in QIIME2 2020.6, and paired-end sequences were spliced ​​and chimeric sequences were removed to obtain the final valid data (non-chimeric reads).

[0054] II. Experimental Results Experimental results are as follows Figure 1As shown, the results indicate that the microbial community structure and metabolic characteristics have a decisive influence on fermentation efficiency and product composition during the fermentation of Citrus aurantium. Through systematic analysis of the dynamic changes in the fungal community throughout the fermentation process, combined with the determination of metabolite content and enzyme activity, the genus *Aspergillus* was identified as the dominant microorganism. Aspergillus The fact that this genus of fungi occupies an absolutely dominant position in the fermentation system indicates that it has stable ecological functions and plays a core metabolic role during fermentation. Other fungal genera, such as Botryotrichum , Naganishia , Cladosporium The proportions of each were relatively low and fluctuated significantly with the fermentation process, suggesting that they were easily inhibited by bacterial competition and changes in the metabolic environment, thus contributing little to the overall fermentation effect.

[0055] Example 2: Study on the dynamic changes of flavonoid components during the fermentation of Citrus aurantium I. Experimental Methods 1. Preparation of reference solution Accurately weigh appropriate amounts of the following reference standards: rutin, neo-northern rutin, naringin, hesperidin, neo-hesperidin, hesperidin-7-O-glucoside, citric acid, naringin, and hesperidin. Dissolve them in methanol to prepare single reference stock solutions with concentrations of 0.318, 0.258, 0.822, 1.500, 0.873, 1.500, 0.804, 0.834, 0.705, and 0.762 mg / mL, respectively. Accurately pipette appropriate amounts of each of the above 10 reference stock solutions into 10 mL volumetric flasks, dilute to the mark with methanol, and shake well to prepare mixed reference solutions with concentrations of 5.30, 4.30, 13.70, 75.00, 13.40, 75.00, 13.40, 13.90, 11.75, and 12.68 μg / mL, respectively.

[0056] 2. Preparation of the test solution Take 0.5 g of the crude powder of fermented Citrus aurantium samples N0d~N5d prepared in Example 1, weigh accurately, place in a round-bottom flask, add 45 mL of methanol, weigh, heat under reflux for 1.5 h, cool, weigh, make up with methanol, shake well, filter, accurately measure 0.1 mL of the filtrate, place in a 2 mL volumetric flask, dilute with methanol to the mark, shake well, and filter through a 0.22 μm microporous membrane to obtain the corresponding test solution.

[0057] 3. Chromatographic conditions Waters UPLC® BEH C18 column (100 mm × 2.1 mm, 1.7 μm); mobile phase: 0.1% phosphoric acid water-acetonitrile, gradient elution; 0–2 min, 5% acetonitrile; 2–26 min, 5%–55% acetonitrile; 26–28 min, 55%–20% acetonitrile; 28–30 min, 20%–5% acetonitrile; flow rate: 0.3 mL / min; column temperature: 35 °C; detector wavelength: 283 nm; injection volume: 2 μL; PDA detector.

[0058] 4. Content determination Inject the sample according to the chromatographic conditions of this embodiment, and calculate the content of each component based on the peak area and the reference standard curve.

[0059] II. Experimental Results Experimental results are as follows Figure 2 As shown, the results indicate that during fermentation, the levels of flavonoid glycosides (including naringin, neohesperidin, and hesperidin) gradually decreased with fermentation time, while their corresponding aglycones or monosaccharide derivatives (such as naringenin, hesperidin, and hesperidin-7-O-glucoside) showed a trend of first increasing and then decreasing. This metabolic dynamics suggests that a biotransformation process of flavonoid glycosides exists in the fermentation system, catalyzed by specific enzymes secreted by Aspergillus (such as naringinase and hesperidinase). Figure 2 Figure A). Further correlation analysis revealed that *Aspergillus* was significantly positively correlated with hesperidin, naringenin, and hesperidin-7-O-glucoside (p<0.05), while *Rhizopus* (… Rhizopus It showed a significant negative correlation with various flavonoid glycosides (p<0.01), while other genera such as Botryotrichum , Naganishia , Mortierella , Cladosporium , Rhodotorula Although unclassified fungi showed significant correlations with some components, their functional impact was relatively limited due to their low proportion and large fluctuations in the fungal community. Figure 2 (Figure B).

[0060] Example 3: Isolation, purification, and identification of microorganisms during the fermentation of Citrus aurantium. I. Strain Isolation 100 μL of the diluted fermentation solution of the Citrus aurantium sample after 5 days of fermentation (N5d) was evenly spread onto MRS plates, LB plates, PDA plates, and Gao's No. 1 culture plates. The MRS and LB culture plates were incubated at 37℃ for 1-2 days; the PDA and Gao's No. 1 culture plates were incubated under the same conditions for 2-3 days.

[0061] II. Strain Purification After incubation on each plate for a period of time, single colonies with different morphologies on each culture medium were picked using the tip hyphae picking method, transferred to new plates for further incubation, and purified using the streak plate method to obtain pure and uncontaminated single colonies. The strains isolated from different culture media were then classified and numbered.

[0062] III. Preservation of Microbial Strains The strain was preserved by slant culture. The strain was inoculated into slant culture medium, the test tube was sealed with sealing film or kraft paper, and then placed in the dark at 26°C until the hyphae covered the entire slant. Then it was stored in a refrigerator at 4°C.

[0063] IV. Morphological Identification of Microorganisms The colony morphology and microscopic characteristics of the isolated fungal strains were initially classified and identified. Molecular biological identification of the fungi was performed through DNA extraction, PCR amplification, agarose gel electrophoresis, and DNA sequencing.

[0064] DNA extraction was performed on the isolated fungi using a DNA extraction kit (D2300 Fungal Genome Kit, Beijing Solarbio Science & Technology Co., Ltd.) following the kit's instructions. For PCR amplification, universal primers ITS1 and ITS4 were used. After 1% agarose gel electrophoresis to check the amplification results, the samples were sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The ITS sequences were then compared with the NCBI database using BLAST to perform molecular biological identification of the strains.

[0065] V. Results of Microbial Isolation, Purification and Identification Eight fungi were isolated in this embodiment, numbered Z1 to Z8. Based on morphological observation and molecular biological methods (ITS sequence alignment), the classification of each strain is as follows: Z1 and Z6: Aspergillus ryukyu ( Aspergillus luchuensis ); Z2: Rhizopus oligosporus ( Rhizopus arrhizus ); Z3 and Z8: Aspergillus niger ( Aspergillus niger ); Z4: Aspergillus fasciculata ( Aspergillus caespitosus ); Z5: Neoniger Aspergillus ( Aspergillus neoniger ); Z7: Aspergillus fumigatus ( Aspergillus fumigatus ). Some of these strains have been preserved; details are as follows: Z1 is designated as Aspergillus ryuko ( Aspergillus luchuensis The CQM3 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63972.

[0066] Z2 is denoted as Rhizopus septum ( Rhizopus arrhizus SGGM1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63974.

[0067] Z3 is designated as Aspergillus niger ( Aspergillus niger HQM2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63973.

[0068] Example 4 Enzyme activity assay of isolated strains I. Plate Screening Experiment: Naringinase solid screening medium: beef extract 0.1 g, K2HPO4 0.1 g, MgSO4·7H2O 0.05 g, FeSO4·7H2O 0.001 g, distilled water 100 mL, naringin 0.1 g, agar 1.5 g, pH natural. (Enzyme production capacity depends on the presence and size of the clear zone).

[0069] Hesperidinase solid screening medium: beef extract 0.1 g, K2HPO4 0.1 g, MgSO4·7H2O 0.05 g, FeSO4·7H2O 0.001 g, distilled water 100 mL, hesperidin 0.1 g, agar 1.5 g, pH natural. (Enzyme production capacity depends on the presence and size of the clear zone).

[0070] Separate screening media for naringinase and hesperidinase were prepared, sterilized, and poured into plates. After cooling, four small wells were punched in each plate using a sterilized punch. The same amount of bacterial suspension was then transferred to each well, with each group repeated three times. After incubation, the clear zone was observed. The presence of a clear zone indicates enzyme production (naringinase or hesperidinase), and a larger clear zone indicates stronger enzyme production. The absence of a clear zone indicates that the bacterial strain does not produce the corresponding enzyme and cannot decompose the substrate.

[0071] II. Determination of enzyme activity by ultraviolet spectrophotometry Extraction of crude enzyme solution: The bacterial strain stored at 4℃ was taken out and inoculated onto PDA medium for activation. It was cultured at 28℃ for 3 days to obtain mature spores of each bacterium. The spores were washed with sterile physiological saline or water and placed in an Erlenmeyer flask containing sterile glass beads and 50 mL of sterile physiological saline. The flask was shaken thoroughly for 30 min. After the spores were completely dispersed and homogenized, the OD value of the bacterial suspension was measured. 600 The OD value was measured, and then adjusted to 0.2 with sterile physiological saline to obtain the corresponding single-spore fungal suspension. The suspension was then centrifuged at 12000 r / min for 15 min, and the bacterial cells were filtered off. The resulting supernatant was the crude enzyme solution.

[0072] Naringinase activity assay: Mix 0.2 mL of crude enzyme solution with 0.8 mL of naringin solution and hydrolyze at 50 °C for 30 min. Then add 0.1 mL of the reaction solution to 5 mL of 90% (v=v) diethylene glycol and 0.1 mL of 1 mol / L NaOH. Let the mixture develop color at room temperature for 10 min. After correcting the blank solvent, measure the absorbance value at 420 nm. The naringinase activity can be obtained by using a standard curve.

[0073] Hesperidinase activity assay: Take 2 mL of hesperidin solution, add 1.7 mL of pH 4.0 acetate buffer, mix well, preheat at 100℃ for 5 min, and after cooling to room temperature, add 0.3 mL of crude enzyme solution, and accurately hold at 40℃ for 30 min. Quickly pipette 0.3 mL of the reaction solution into a test tube containing 5 mL of diethylene glycol, 0.5 mL of 1 mol / L NaOH, and 0.2 mL of distilled water, mix thoroughly, and develop color at 30℃ for 30 min. After correcting the blank solvent, measure the absorbance value at 420 nm. The hesperidinase activity can be obtained through a standard curve.

[0074] III. Experimental Results Enzyme activity assays were performed on eight strains extracted from the surface of Citrus aurantium. The results are shown in Table 1. The results indicate that strains Z1, Z2, and Z3 exhibited the highest enzyme activities in naringinase and hesperidinase. Based on enzyme activity, Z1, Z2, and Z3 were preliminarily screened as candidate strains with high-efficiency flavonoid conversion capabilities.

[0075] Table 1. Enzyme activity results of microorganisms extracted from the surface of Citrus aurantium.

[0076] Example 5: Pure and mixed culture fermentation I. Fermentation of pure and mixed strains After soaking the raw Citrus aurantium in sterile water for 4 hours, it was transferred to a sterile culture bottle for fermentation.

[0077] Experimental group: At an inoculation rate of 15% (v / w), the bitter orange peel was evenly inoculated with bacterial solutions of different single strains (Z1, Z2 or Z3), or with mixed bacterial solutions prepared according to a preset ratio. Blank control group (N): Each bottle was filled with an equal volume (15%) of sterile water as a control.

[0078] All culture flasks were placed in a constant temperature and humidity incubator and fermented at 37℃ and 90% relative humidity for 48 hours. The resulting samples were the corresponding pure culture fermented products or mixed culture fermented products.

[0079] The experimental group design is as follows: 1. Raw Citrus aurantium control group (R): Unfermented Citrus aurantium; 2. Natural fermentation group (N): Only sterile water is added to simulate non-inoculation fermentation under natural conditions; 3. Pure culture fermentation group (3 groups): The selected strains Z1, Z2 and Z3 were inoculated respectively and recorded as group Z1, group Z2 and group Z3; 4. Mixed-strain fermentation group (4 groups): Group H1 was a mixed bacterial solution obtained by mixing strain Z2 and strain Z3 at a volume ratio of 1:1; Group H2 was a mixed bacterial solution obtained by mixing strain Z1 and strain Z2 at a volume ratio of 1:1; Group H3 was a mixed bacterial solution obtained by mixing strain Z1 and strain Z3 at a volume ratio of 1:1. Group H4 was a mixed bacterial solution obtained by mixing strains Z1, Z2 and Z3 in a volume ratio of 1:1:1.

[0080] The contents of flavonoid components (senna-senna, neo-senna-senna, rutin, naringin, hesperidin, neo-hesperidin, hesperidin-7-O-glucoside, limonene, naringin, and hesperidin) in the fermented samples of Citrus aurantium prepared from different treatment groups were determined using the same method as in Example 2.

[0081] II. AHP-Entropy Weight Method for Comprehensive Quality Evaluation of Pure Culture and Mixed Culture Fermentation Samples The content of major flavonoid components and appearance characteristics were selected as quality evaluation indicators for the fermented samples of Citrus aurantium. The composite weight of each evaluation indicator was established by using the AHP-entropy weight method, which combines subjective and objective methods, and the composite score of each group of samples was calculated to comprehensively evaluate their quality and fermentation effect.

[0082] Appearance evaluation of fermented bitter orange peel samples: Based on traditional experience standards, the appearance of mold growth on the surface was observed for judgment, with the highest score being when the sample was completely covered with white mycelium. Specific scoring criteria are shown in Table 2.

[0083] Table 2 Appearance Scoring of Fermented Citrus Aurantium

[0084] III. Evaluation of Antioxidant Activity 1. DPPH free radical scavenging rate determination: Accurately weigh approximately 0.5 g of the coarse sample powder and place it in a round-bottom flask. Add 45 mL of methanol, weigh the mixture, heat under reflux for 1.5 h, cool, and weigh again. Make up the weight loss with methanol, shake well, filter, and accurately measure 0.1 mL of the filtrate. Dilute to the mark with methanol in a 2 mL volumetric flask and shake well to obtain the corresponding sample solution. Mix an equal volume of 0.1 mM DPPH methanol solution with the sample solution, vortex, and react at room temperature in the dark for 30 minutes. Measure the absorbance at 517 nm. Use an equal volume of methanol instead of the sample solution as a control group, and an equal volume of methanol instead of the DPPH solution as a sample blank. The clearance rate is calculated using the following formula: DPPH clearance rate (%) = [1 - (sample-blank) / control] × 100% 2. Determination of ABTS free radical cation scavenging rate: Mix an equal volume of 7 mM ABTS solution with 2.45 mM potassium persulfate solution and react at room temperature in the dark for 12–16 hours to prepare an ABTS stock solution. Before use, dilute with anhydrous ethanol to an absorbance of 0.70 ± 0.02 at 734 nm. Mix an appropriate amount of the diluted ABTS working solution with an equal volume of the sample solution, react for 30 minutes, and then measure the absorbance at 734 nm. Calculate using the same method as for DPPH scavenging.

[0085] IV. Experimental Results 1. Analysis results of flavonoid content The results of flavonoid content determination of fermented Citrus aurantium samples prepared from different treatment groups are as follows: Figure 3 As shown, the total flavonoid content of groups Z1, H1, H2, H3, and H4 was significantly higher than that of the naturally fermented group, with group Z1 having the highest total flavonoid content.

[0086] Further analysis of various flavonoid components and the appearance quality of fermentation samples was conducted using the AHP-entropy weight method. The composite weight Z of the AHP-entropy weight method was calculated. W j and R j The coefficients are shown in Table 3. The comprehensive score results of different treatment groups evaluated by the AHP entropy weight method are shown in Table 4. The results show that: (1) Among the pure culture fermentation groups, only the Z1 group scored significantly better than the natural fermentation group; (2) Among the mixed culture fermentation groups, the scores of H4, H2, and H3 were significantly better than those of the pure culture fermentation group and the natural fermentation group; (3) Overall, the H4 group scored the highest, followed by the H2 and H3 groups, then the Z1 group, and the natural fermentation group and the Z3 group scored the lowest (45 points).

[0087] The above results indicate that inoculation with specific strains and combinations of strains can significantly improve the overall quality of fermented bitter orange peel.

[0088] 2. Free radical scavenging rate results The results of free radical scavenging rate determination of fermented Citrus aurantium samples prepared in different treatment groups are as follows: Figure 4 As shown, the results indicate that in the three-strain mixed fermentation group (H4), the DPPH free radical scavenging ability and ABTS free radical scavenging ability were both higher than those in the raw bitter orange peel control group (R) and the natural fermentation group (N), further demonstrating that the strain combination used in this invention can not only improve the conversion efficiency of flavonoids, but also enhance the functional value of the product.

[0089] In summary, this invention, by selecting and combining strains with high enzyme activity, effectively promotes the conversion of flavonoid glycosides in Citrus aurantium into aglycone forms with higher bioavailability, thereby improving the fermentation efficiency and product quality of Citrus aurantium.

[0090] Table 3. Composite weight Z of AHP-entropy weight method. W j and R j coefficient

[0091] Table 4. Comprehensive scores of different fermentation groups evaluated using the AHP entropy weight method.

[0092] Example 6: Exploration of mixed-culture fermentation process for Citrus aurantium Using the appearance and content of 11 flavonoid components (senna-senna, neo-senna-senna, rutin, naringin, hesperidin, neo-hesperidin, naringin-7-O-glucoside, hesperidin-7-O-glucoside, limonin, naringin, and hesperidin) of the fermented product, a composite score was obtained by combining subjective and objective methods using the AHP-entropy weight method. This comprehensive evaluation of the fermented product's quality provides a stable and feasible quality evaluation method, laying the foundation for the subsequent optimization of process parameters for the fermentation of trifoliate orange peel.

[0093] The scoring criteria for the appearance characteristics of the mixed-culture fermented products of Citrus aurantium are the same as those in Table 2.

[0094] The importance of 12 indicators—appearance traits, sennain, neo-sennain, rutin, naringin, hesperidin, neo-hesperidin, naringin-7-O-glucoside, hesperidin-7-O-glucoside, limonin, naringin, and hesperidin content—was quantified using the analytic hierarchy process (AHP-entropy method). The selection criteria are as follows: 1. Appearance: It is very important to judge whether processed products are qualified. Only if they meet the traditional experience indicators can it be meaningful to judge other indicators. 2. The index components of Citrus aurantium in the 2020 edition of the Chinese Pharmacopoeia are naringin and neohesperidin. However, after processing, both of these components will change and generate corresponding monosaccharides or aglycones, resulting in a decrease in content.

[0095] In the selection of chemical components, those chemical components that have undergone significant changes before and after processing should not be ignored. Therefore, in addition to selecting the two main components in the pharmacopoeia, a number of components that have undergone significant changes before and after fermentation should also be selected as indicator components for the mixed fermentation product of Citrus aurantium. 3. Determine the composite weights of each indicator using the analytic hierarchy process (AHP) and entropy weight method. The Analytic Hierarchy Process (AHP) constructs a hierarchical relationship between indicators according to their importance on a certain scale, and then calculates the weight values ​​of the indicators accordingly. It belongs to the subjective weighting method. The AHP determines the priority order of each indicator: naringin = neohesperidin > naringenin = hesperidin = hesperidin-7-O-glucoside = naringin-7-O-glucoside > rutin = neo-northern rutin = rutin = hesperidin = appearance traits. The specific judgment matrix is ​​shown in Table 5. W j and R j These are the weighting coefficients obtained by the entropy weight method and the AHP method, respectively, and the composite weights of each index in the samples of single-factor experiments and orthogonal experiments. F j * See Tables 6 and 7.

[0096] The data was analyzed using Excel using a sum-product method, yielding the weights of rutin, neo-northern rutin, naringin, hesperidin, neo-hesperidin, hesperidin-7-O glucoside, citric acid, naringenin, hesperidin, and appearance traits as 0.050, 0.050, 0.050, 0.180, 0.050, 0.090, 0.180, 0.090, 0.050, 0.090, 0.090, and 0.050, respectively. The consistency ratio (CR) calculated using Excel was 0 (<0.1), indicating that the matrix is ​​reasonable and effective, and the weight coefficients are reliable. A higher consistency ratio indicates poorer matrix consistency, while a consistency ratio of 0 indicates complete matrix consistency.

[0097] Table 5. Priority Matrix of Analytic Hierarchy Process for Each Indicator

[0098] Table 6. Weights of each evaluation index in single-factor experiments

[0099] Table 7. Weights of each evaluation index in response surface methodology.

[0100] Example 7: Method for determining the content of 11 flavonoids in fermented products of Citrus aurantium mixed with bacteria I. Chromatographic conditions Agilent 1260 high performance liquid chromatograph (Agilent Technologies, Inc., USA); Phenomenex Luna® C18(2) column (150 mm × 4.6 mm, 5 μm); mobile phase: 0.1% phosphoric acid aqueous solution-acetonitrile, flow rate: 1 mL / min; column temperature: 35℃; detection wavelength: 283 nm; injection volume: 10 μL; PDA detector, detailed elution program is shown in Table 8.

[0101] Table 8. Elution Procedure for Content Determination of Mixed Bacterial Inoculum Products of Citrus aurantium

[0102] II. Preparation of reference solution The following reference standards were prepared: 6.360, 5.160, 5.480, 40.030, 5.820, 4.160, 38.170, 13.400, 5.560, 7.050, and 7.605 mg / L, respectively. Accurately weigh and dilute to volume 1 mg to prepare single reference stock solutions with concentrations of 0.636, 0.516, 0.548, 4.003, 0.582, 0.416, 3.817, 1.340, 0.556, 0.705, and 0.761 mg / mL. Accurately pipette appropriate amounts of each of the 11 reference stock solutions, mix them, and add methanol to prepare 11 flavonoid components (in order of...). A mixed reference solution containing rutin, neohesperidin, naringin, naringin, hesperidin, naringin-7-O-glucoside, neohesperidin, hesperidin-7-O-glucoside, limonin, naringin, and hesperidin at concentrations of 63.60, 25.80, 54.80, 400.30, 58.20, 41.60, 381.75, 134.00, 55.60, 70.50, and 76.05 μg / mL. III. Preparation of the test solution Take approximately 0.5 g of the sample coarse powder, weigh it accurately, place it in a round-bottom flask, add 45 mL of methanol, weigh it, heat under reflux for 1.5 h, cool it, weigh it again, add methanol to make up the weight, mix well, filter it, dilute it with methanol by a certain factor, and then filter it through a 0.22 μm microporous membrane to obtain the corresponding test solution. IV. Preparation of Standard Curve Accurately measure 0.3, 0.4, 0.5, 1.0, 2.5, and 5.0 mL of the mixed reference solution into 5 mL volumetric flasks, dilute to volume with methanol, and mix thoroughly. Perform linear regression with the mass concentration of the 11 flavonoid components as the x-axis (X) and peak area as the y-axis to obtain the regression equation. The chromatogram of the mixed reference solution is shown below. Figure 1 The linear regression equations for each reference standard are shown in Table 9. The liquid chromatograms of the 11 flavonoid mixed reference standards are shown below. Figure 5 As shown.

[0103] Table 9. Results of linear relationship investigation of 11 components in the mixed-strain fermented product of Citrus aurantium.

[0104] Example 8: Single-factor experiments and response surface methodology were used to determine the optimal fermentation conditions for mixed cultures of Citrus aurantium. Based on the methods of Examples 6 and 7, this example uses the appearance and content of 11 flavonoid components (senna-derived glycoside, neo-northern senna-derived glycoside, rutin, naringin, hesperidin, neo-hesperidin, naringenin-7-O-glucoside, hesperidin-7-O-glucoside, limonene, naringenin, and hesperidin) of the fermented Citrus aurantium sample as evaluation indicators. A composite scoring model is constructed using the AHP-entropy weight method, and single-factor experiments and response surface experiments are systematically carried out to determine the optimal range of fermentation process parameters.

[0105] I. Single-factor experimental design After soaking the raw Citrus aurantium in sterile water for 4 hours, it was transferred to a sterile culture bottle and mixed bacterial solution (Z1, Z2 and Z3) was evenly inoculated according to different treatment methods. Fermentation was carried out at 37℃ and 90% relative humidity to obtain fermented Citrus aurantium samples.

[0106] The effects of the following three key process parameters on fermented samples of Citrus aurantium were examined sequentially: 1. Vaccination ratio (Z1:Z2:Z3, v / v / v): 3:1:1, 2:1:1, 1:1:1, 1:1:2, 1:1:3; 2. Inoculation amount (percentage of wet bitter orange peel by weight): 5%, 10%, 15%, 20%, 25%; 3. Fermentation time: 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h.

[0107] When examining single-factor experiments, other factors were kept constant. Specifically, when examining the inoculation ratio, the inoculation amount was 15% and the fermentation time was 48 hours; when examining the inoculation amount, the inoculation ratio was 1:1:1 and the fermentation time was 48 hours; and when examining the fermentation time, the inoculation ratio was 1:1:1 and the inoculation amount was 15%.

[0108] Each group of experiments was repeated three times. After fermentation, the fermented Citrus aurantium samples were prepared into test solutions according to the method in Example 7. The content of each flavonoid component was determined, and the appearance characteristics were scored according to Table 2. The composite weights of each index in Table 6 were combined ( F j * The comprehensive composite score (Z) for each sample is calculated using the following formula:

[0109] The results of the single-factor experiment are as follows Figures 6-8 As shown, the results indicate that the overall score is highest when the inoculation ratio is 2:1:1 (Z1:Z2:Z3); the optimal inoculation amount is 15%; the fermentation time reaches its peak at around 48 h, and the score tends to stabilize or slightly decrease after being extended to 60 h.

[0110] Based on this, the initial optimization interval was determined as follows: the inoculation ratio of Z1:Z2:Z3 was (1.5~2.5):1:1, the inoculation amount was 12%~18%, and the fermentation time was 36~60 h, which will be used for subsequent response surface optimization.

[0111] II. Optimization of Box-Behnken Response Surface Experiment A three-factor, three-level Box-Behnken design (BBD) was adopted, with inoculation ratio (A), inoculation amount (B), and fermentation time (C) as independent variables, and the composite score (Z) as the response value, to establish a quadratic multinomial regression model. The response value was calculated based on the composite weights (Fj*) of each indicator in the response surface experiment in Table 7, and the regression equation was fitted. Analysis of variance and significance testing were then performed (p<0.05 was considered significant).

[0112] Response surface analysis results are as follows Figure 9 As shown, the results indicate that there is a significant interaction among the three factors; the model predicts the optimal process conditions as follows: inoculation ratio (Z1:Z2:Z3) = 2:1:1; inoculation = 15%; fermentation time = 48 h.

[0113] Example 9: A method for processing Citrus aurantium based on mixed-culture fermentation Fermented samples of Citrus aurantium were prepared according to the optimal process conditions in Example 8.

[0114] I. Raw material pretreatment Take the cleaned Citrus aurantium, weigh it, irradiate it under a UV lamp (254 nm) for 25 min, then soak it in sterile water for 4 h, and then take it out for later use.

[0115] II. Preparation of Mixed Bacterial Solution: Z1: Aspergillus ryukyu ( Aspergillus luchuensis CQM3; Z2: Rhizopus oligosporus ( Rhizopus arrhizus SGGM1; Z3: Aspergillus niger ( Aspergillus niger HQM2.

[0116] Strains Z1, Z3, and Z2 were inoculated onto potato dextrose agar (PDA) medium for activation and cultured at 28°C for 36 h. The activated strains were then inoculated into 50 mL of sterilized liquid potato dextrose agar and cultured at 28°C and 150 rpm for 36 h to obtain three seed cultures: Z1, Z2, and Z3. The three seed cultures were mixed thoroughly at a volume ratio of Z1:Z2:Z3 = 2:1:1 to obtain a mixed bacterial culture.

[0117] III. Inoculation and Fermentation The pretreated wet bitter orange peel was placed into a sterile, breathable, and leak-proof container, and the mixed bacterial solution was inoculated onto the surface of the bitter orange peel at an inoculation rate of 15% (w / w) (based on the wet weight of the bitter orange peel). Then it was placed in a constant temperature and humidity incubator and statically fermented for 48 h at 37 ℃ and 90% relative humidity.

[0118] 4. Product collection: After fermentation, clean the product, slice it into thin slices (2-3 mm), and dry it (dry in a 50 ℃ oven for 20 h) to obtain a deep yellow fermented product of Citrus aurantium mixed culture (PFA) with a uniform surface covered with white mycelium.

[0119] 5. Quality evaluation and effect verification The contents of 11 flavonoid components (including naringin, neohesperidin, naringenin, hesperidin-7-O-glucoside, etc.) in the fermented product were determined by high performance liquid chromatography (HPLC), and the results were combined with appearance score.

[0120] IV. Experimental Results The liquid chromatogram of flavonoid components in the mixed-culture fermented product of Citrus aurantium prepared under optimal conditions is shown below. Figure 10 As shown in the image, the appearance of the fermented product of mixed bacteria and trifoliate orange peel is as follows. Figure 11 As shown, its appearance is deep yellow with a uniform and dense layer of white mycelium on the surface, without any discoloration or spoilage, and its appearance score reaches the maximum (10 points). Liquid chromatography analysis revealed that the optimal conditions for preparing the mixed-culture fermented product of Citrus aurantium resulted in a high comprehensive content of various flavonoid components, with increased content of highly bioavailable components such as flavonoid monosaccharides and flavonoid aglycones, making the finished product easier to absorb and utilize, thereby improving its efficacy.

[0121] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Aspergillus Ryukyu ( Aspergillus luchuensis CQM3, characterized in that, The strain has been preserved in Guangdong Microbial Culture Collection Center on November 1, 2023, and the preservation number is GDMCC No: 63972.

2. A starter culture composition, characterized in that, containing the Aspergillus luchuensis CQM3 of claim 1.

3. The bacterial composition of claim 2, wherein It also contains Rhizopus spp. ( Rhizopus arrhizus SGGM1 or Aspergillus niger ( Aspergillus niger HQM2; The Rhizopus SGGM1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63974, and the Aspergillus niger HQM2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63973.

4. The bacterial composition according to claim 3, characterized in that, The mass ratio of the Aspergillus luchuensis CQM3 to the Rhizopus arrhizus SGGM1 or the Aspergillus niger HQM2 is (1~3):(1~2).

5. The microbial composition according to claim 2, characterized in that, It also contains Rhizopus spp. ( Rhizopus arrhizus SGGM1 and Aspergillus niger ( Aspergillus niger HQM2; The Rhizopus SGGM1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63974, and the Aspergillus niger HQM2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 1, 2023, with accession number GDMCC No: 63973.

6. The bacterial composition of claim 5, wherein the bacterial composition is a probiotic composition. The mass ratio of the Aspergillus luchuensis, the Rhizopus arrhizus and the Aspergillus niger is (1~3):(1~2):(1~2); preferably, the mass ratio of the Aspergillus luchuensis, the Rhizopus arrhizus and the Aspergillus niger is (1~2):1:

1.

7. Use of any of a1-a2 in any of b1-b4. a1. the Aspergillus luchuensis CQM3 or the fermentation liquor thereof of claim 1, a2. the strain composition of any of claims 2-6 or the fermentation liquor thereof; b1. fermenting Aurantii Fructus, b2. preparing a product for fermenting Aurantii Fructus, b3. increasing the content of flavonoid components in Aurantii Fructus, b4. increasing the antioxidant activity of Aurantii Fructus.

8. A method for processing of Citrus aurantium L. by fermentation, characterized in that, fermenting Aurantii Fructus using any of a1-a2, a1. the Aspergillus luchuensis CQM3 or the fermentation liquor thereof of claim 1, a2. the strain composition of any of claims 2-6 or the fermentation liquor thereof.

9. A fermented fructus aurantii, characterized in that, obtained by the method of claim 8.

10. A product characterized by, containing the fermented Aurantii Fructus of claim 9.

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