Achromobacter xylosoxidans and application thereof in increasing content of rhoifolin in exocarpium citri grandis
By fermenting Citrus reticulata peel with Xylose-oxidizing Achromobacterium, the conversion of naringin to rosin was promoted, which solved the problem of low rosin content in Citrus reticulata peel and achieved a significant increase in rosin content and a reduction in production costs.
Patent Information
- Application Number
- CN202511513620.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-22
- Publication Date
- 2026-03-10
AI Technical Summary
The low content of rosin in Citrus reticulata peel makes extraction and purification difficult and costly, thus limiting its widespread application.
Achromobacter xylosoxidans YQ016 was used to ferment tangerine peel, promoting the conversion of naringin and other precursor substances into rosin, thereby increasing the rosin content.
It significantly increased the content of rosin in Huajuhong to over 6.70%, with an extraction rate of over 0.46%, reducing production costs and facilitating the high-value-added development of Huajuhong.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and specifically relates to a strain of xylose-oxidizing achromobacterium and its application in increasing the content of rosin in Citrus reticulata var. huali. Background Technology
[0002] Huazhou tangerine peel (also known as Huazhou pomelo peel) is the unripe or nearly ripe outer peel of the pomelo. It has effects such as regulating qi and resolving phlegm, strengthening the stomach and aiding digestion, and lowering blood pressure. It is one of the "Ten Rare Chinese Medicinal Herbs" of Guangdong Province. In 2024, Huazhou tangerine peel was included in the national list of substances with medicinal and edible homologous properties, making it an important raw material for medicinal cuisine, traditional Chinese medicine, and health foods, and a research hotspot in the pharmaceutical and health product fields. The active ingredients of Huazhou tangerine peel mainly consist of three categories: flavonoids, polysaccharides, and volatile substances. Among them, naringin and rhubarb glycoside are the two major flavonoids with significant medicinal effects in Huazhou tangerine peel.
[0003] Apigenin, also known as apigenin-7-O-β-neohesperidin, is a flavonoid glycoside compound with apigenin as its core. Studies have shown that apigenin possesses various biological activities, including antioxidant, hepatoprotective, anti-inflammatory, antihypertensive, bone-strengthening, neuroprotective, hypoglycemic, anticancer, and antiviral effects. It exhibits particularly strong inhibitory effects on various tumor cells, such as breast cancer cells, liver cancer cells, and lung cancer cells. However, the content of apigenin in Huajuhong (Citrus reticulata peel) is very low, typically ranging from 0.5% to 1.0%, less than 5% of the content of naringin. This low content makes extraction and purification difficult, resulting in high production costs. Comparing products of the same purity, apigenin is more than 10 times more expensive than naringin. Increasing the apigenin content in Huajuhong and reducing its extraction and production costs are crucial for promoting its widespread application. Summary of the Invention
[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a xylose-oxidizing colorless bacillus.
[0005] Another object of the present invention is to provide the application of the above-mentioned xylose-oxidizing achromobacterium in increasing the content of rosin in Citrus reticulatae.
[0006] Another object of the present invention is to provide a fermentation method for increasing the content of rosin in Citrus reticulata peel.
[0007] Another objective of this invention is to provide a method for preparing the extract of rosary glycosides from Citrus reticulata.
[0008] The objective of this invention is achieved through the following technical solution: a strain of xylose-oxidizing achromobacterium, named xylose-oxidizing achromobacterium (… Achromobacter xylosoxidans YQ016, with accession number GDMCC No: 67040, was deposited on September 26, 2025, at the Guangdong Provincial Microbial Culture Collection Center, Guangdong Academy of Sciences, located on the 5th floor of Building 59, Courtyard 100 Xianlie Middle Road, Guangzhou.
[0009] The application of the above-mentioned xylose-oxidizing achromobacterium in increasing the content of rosin in Citrus reticulata peel involves fermenting Citrus reticulata peel with xylose-oxidizing achromobacterium to promote the conversion of naringin and other precursor substances in Citrus reticulata peel into rosin, thereby increasing the content of rosin.
[0010] A fermentation method for increasing the content of rosin in Citrus reticulata peel includes the following steps: inoculating the above-mentioned xylose-oxidizing Achromobacterium seed liquid into a fermentation medium for fermentation culture to obtain fermented material with increased rosin content in Citrus reticulata peel.
[0011] The preferred composition of the fermentation medium is as follows: 28-32 g / L dried tangerine peel slices, 56-64 g / L fresh celery stalks, 130-150 g / L glucose, 65-75 g / L sodium citrate, 8-10 g / L peptone, 8-10 g / L yeast extract, 2.4-2.8 g / L potassium dihydrogen phosphate, and 0.22-0.26 g / L magnesium sulfate, with water as the solvent and a pH of 7.4-7.6.
[0012] The water mentioned includes purified water, deionized water, and distilled water.
[0013] The fermentation medium is preferably prepared by the following steps: mixing all the components of the fermentation medium, boiling and heating to homogenize, and then sterilizing to obtain the fermentation medium.
[0014] The preferred boiling and heating time is 3 to 5 minutes.
[0015] The preferred homogenization conditions are 5000-7000 r / min for 5-15 min; more preferably, 6000 r / min for 10 min.
[0016] The sterilization conditions are preferably 115-121℃ for 15-30 min; more preferably 121℃ for 20 min.
[0017] The Xylose-oxidizing Achromobacterium seed culture is preferably prepared by the following steps: inoculating the preserved strain of Xylose-oxidizing Achromobacterium into a seed culture medium, shaking and culturing to obtain the Xylose-oxidizing Achromobacterium seed culture.
[0018] The seed culture medium has the following composition: glucose 80-100 g / L, peptone 8-10 g / L, yeast extract 8-10 g / L, potassium dihydrogen phosphate 2.4-2.8 g / L, magnesium sulfate 0.22-0.26 g / L, pH 6.6-7.0, and water as the solvent.
[0019] The preferred inoculation amount for the xylose-oxidizing achromobacterium strain is 2 loops of xylose-oxidizing achromobacterium bacterial motility per 100-200 mL of seed culture medium.
[0020] The preferred conditions for the shaking culture are 28-30℃ and 160-200 r / min for 18-20 h.
[0021] The inoculation amount of the xylose-oxidizing achromobacter seed liquid is preferably calculated as 0.5% to 1.0% of the volume of water used in the fermentation medium.
[0022] The preferred fermentation conditions are a temperature of 28-30℃, a stirring speed of 150-200 r / min, an aeration ratio of 0.10-0.2 vvm for 0-16 h of fermentation, an aeration ratio of 0.10 vvm for 17 h and thereafter, and a fermentation cycle of 40-44 h.
[0023] A method for preparing a glycoside extract from Citrus reticulata peel includes the following steps: (1) Reflux extraction and slag removal: The fermented material obtained by the above fermentation method is adjusted to alkalinity and placed into a reflux extraction device. After reflux extraction and solid-liquid separation, the extract is obtained. (2) Ultrafiltration to remove impurities: First, the extract is ultrafiltered with a 1000 Da ultrafiltration membrane to obtain the permeate; then, the permeate is ultrafiltered with a 360 Da ultrafiltration membrane to obtain the retentate. (3) Evaporation, concentration and drying: Adjust the pH of the retentate obtained in step (2) to neutral, then concentrate it further, and then dry the concentrate to obtain the extract of Citrus reticulata glycoside.
[0024] In step (1): The pH of the fermentation material is preferably adjusted to 7.5-8.0.
[0025] The preferred temperature for the reflux extraction is 90~100℃.
[0026] The preferred reflux extraction time is 40-60 min.
[0027] The preferred method for solid-liquid separation is centrifugation.
[0028] The preferred centrifugation conditions are: rotation speed 6000~7000 r / min, time 18~22 min.
[0029] In step (2): The preferred number of times the extract is ultrafiltered with a 1000 Da ultrafiltration membrane is 2, specifically as follows: the degree of ultrafiltration in the first ultrafiltration is 80% to 84% of the original volume of the permeate; in the second ultrafiltration, water with a volume of 2.5 to 3.5 times that of the retentate obtained in the first ultrafiltration is added to the ultrafiltration retentate, and the degree of ultrafiltration is 73% to 77% of the original volume of the permeate.
[0030] The preferred number of times the permeate is ultrafiltered with the 360 Da ultrafiltration membrane is 2, specifically as follows: the degree of ultrafiltration in the first ultrafiltration is 16% to 20% of the original volume of the retentate; in the second ultrafiltration, water with a volume of 2.5 to 3.5 times that of the retentate is added to the ultrafiltration retentate obtained in the first ultrafiltration, and the degree of ultrafiltration is 23% to 27% of the original volume of the permeate.
[0031] In step (3): The preferred method of concentration is vacuum evaporation concentration.
[0032] The preferred conditions for vacuum evaporation concentration are: vacuum degree -81~-76 kPa and temperature 60~65℃.
[0033] The degree of vacuum evaporation concentration is such that the concentrate is 20% to 30% of the original volume.
[0034] The preferred drying method is spray drying.
[0035] The preferred conditions for spray drying are: inlet air temperature 160~170℃ and outlet air temperature 40~50℃.
[0036] The extract of Citrus reticulata extract contains more than 6.70% rosin, and the extraction yield of rosin from Citrus reticulata is more than 0.46%.
[0037] The water used in this invention is preferably purified water, deionized water, or distilled water.
[0038] The present invention has the following advantages and effects compared with the prior art: (1) The inventors of this invention have tried fermenting Citrus reticulata peel using various lactate dehydrogenase-producing bacteria or gluconate dehydrogenase-producing bacteria, such as Lactobacillus plantarum, Lactobacillus casei, Leuconostoc mesenteroides, and Bacillus subtilis. However, these methods not only failed to increase the content of rosin but actually decreased it. This may be because the glycosidases produced by these bacteria have a decomposing effect on rosin. This invention has screened out a strain of Xylose-oxidizing Achromobacterium YQ016 that produces dehydrogenase, which can be used to increase the rosin content of Citrus reticulata peel by fermentation.
[0039] (2) This invention uses xylose-oxidizing achromobacterium to ferment Citrus reticulata peel, promoting the conversion of Citrus reticulata peel naringin and other precursor substances into rhubarb glycosides, resulting in a significant increase in the rhubarb glycoside content in the extract. Compared with the unfermented control, the rhubarb glycoside content is more than 1.3 times that of the control. The obtained extract can be used as a raw material in the pharmaceutical field, which is conducive to promoting the high-value-added development and utilization of Citrus reticulata peel. Detailed Implementation
[0040] The present invention will now be described in further detail, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.
[0041] Example 1 1. Enrichment culture Enrichment medium: glucose 100 g / L, peptone 10 g / L, yeast extract 10 g / L, KH2PO4 2.5 g / L, MgSO4•7H2O 0.5 g / L, pH adjusted to 7.0, solvent: deionized water. Prepare 100 mL of enrichment medium, place it in a 500 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, cool, and then inoculate with 1 g of petroleum-contaminated soil (sampled from Maoming City, Guangdong Province). Incubate at 30℃ and 100 r / min for 12 h.
[0042] 2. Plate separation Plate isolation medium: glucose 20 g / L, peptone 10 g / L, yeast extract 10 g / L, KH2PO4 2 g / L, agar 20 g / L, adjusted to pH 7.0, solvent is deionized water. After preparation, the medium is sterilized at 121℃ for 20 min, poured into petri dishes, and plated.
[0043] The enriched culture medium was serially diluted and spread onto agar plates, then incubated at 30°C for 36 h. Eighty colonies with larger diameters were picked and inoculated onto slant agar (the composition of the slant agar was the same as that of the agar plate). The slant agar was incubated at 30°C for 36 h and stored at 4°C for later use.
[0044] 3. Screening of dehydrogenase-producing bacteria by fermentation Dehydrogenase fermentation medium: 30 g / L dried tangerine peel, 60 g / L celery stalks, 120 g / L glucose, 60 g / L sodium citrate, 10 g / L peptone, 8 g / L yeast extract, 2.5 g / L KH₂PO₄, 0.4 g / L MgSO₄•7H₂O, adjusted to pH 7.0, solvent: deionized water. After preparation, the medium was boiled, homogenized at 6000 r / min for 10 min, and transferred to 1000 mL Erlenmeyer flasks, with 200 mL of liquid in each flask. The flasks were sterilized at 121℃ for 20 min. After cooling, 80 selected bacterial strains were inoculated, with 2 loops of bacterial growth per flask. Fermentation was carried out at 30℃ and 25 r / min for 32 h. The dehydrogenase activity in the fermentation broth of each strain was measured, and the 8 strains with higher activity were selected for further screening experiments.
[0045] Following the instructions of the lactate dehydrogenase release assay kit (WST-8 method; Shanghai Beyotime Biotechnology Co., Ltd.), as shown in Table 1, LDH standard stock solution (50 mU / mL) and LDH detection working solution were added to the wells of the microplate reader. The wells were wrapped with aluminum foil (to protect from light) and incubated on a shaker at 37 ℃ and 50 rpm for 20 min. Then, 20 μL of stop solution was added to each well to terminate the reaction. The absorbance was measured at 450 nm, and a curve showing the relationship between absorbance and enzyme activity was plotted to establish a regression equation for the curve.
[0046] Table 1. Reaction system for dehydrogenase activity assay
[0047] Before measuring the fermentation broth, centrifuge the broth at 4 ℃ and 6000 rpm for 20 min, and collect the supernatant, which is the crude enzyme solution. For the measurement, appropriately dilute the crude enzyme solution, take 10 μL of the diluent and 90 μL of the working solution, place them in a well plate, and incubate on a shaker at 37 ℃ and 50 rpm for 20 min. Stop the reaction by adding 20 μL of stop solution, and measure the absorbance at 450 nm. Calculate the dehydrogenase activity in the fermentation broth according to the regression equation.
[0048] After screening 80 strains by enzyme-producing fermentation, 36 strains were found to have the ability to produce dehydrogenases. Among them, 8 strains with strong enzyme-producing ability are shown in Table 2.
[0049] Table 2. Eight strains with strong enzyme production and their dehydrogenase activities.
[0050] 4. Screening of strains that produce rhubarb glycosides The fermentation medium was the same as the dehydrogenase fermentation medium described above, consisting of 200 mL of liquid in a 1000 mL Erlenmeyer flask. The eight selected bacterial strains were inoculated into the fermentation medium, with two loops of bacterial growth in each flask. Fermentation was carried out at 30 ℃ and 25 r / min for 32 h. The content of rhododendron glycosides in the fermentation broth of each strain was measured, and the strain with the highest rhododendron glycoside content was selected.
[0051] The improved liquid chromatography method for determining naringin (Reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.) was described. The chromatographic conditions were as follows: column: Shim-pack GIST C18 (4.6×250 mm, 5μm); column temperature: 30℃; mobile phase: methanol (A)-0.1% phosphoric acid (B), gradient elution (0~5 min, 75%B; 5~10 min, 75%→70%B; 10~25 min, 70%→60%B; 25~45 min, 60%→45%B; 45~50 min, 45%→40%B; 50~60 min, 40%→75%B; 60~70 min, 75%B); detection wavelength: 333 nm. nm; Injection volume: 5 μL; Flow rate: 1.0 mL / min.
[0052] The standard of rutin was dissolved in methanol to prepare standard solutions of 0.94, 4.70, 9.40, 18.80, 47.00, and 94.00 μg / mL. The standard solutions were determined by liquid chromatography (LC). A standard curve of rutin was plotted with rutin concentration on the x-axis and peak area on the y-axis. Before the rutin sample analysis, the pH of the fermentation broth was adjusted to 7.5, and the sample was refluxed at 100 °C for 60 min. Then, the sample was centrifuged at 7000 r / min for 20 min, and the supernatant was collected. The concentration of rutin in the supernatant was then determined by LC.
[0053] Eight strains with strong dehydrogenase production underwent fermentation transformation experiments, and the concentrations of rosin in the fermentation broth are shown in Table 3. Among them, strain YQ016 had the highest concentration of rosin in its fermentation broth, therefore, this strain is the preferred strain.
[0054] Table 3. Concentration of rhubarb glycosides in the fermentation broth of 8 bacterial strains
[0055] 5. Strain identification Gram staining and optical microscopy examination revealed that strain YQ016 is a Gram-negative bacillus. Strain YQ016 was sent to a gene sequencing company for 16S rDNA analysis. The detected sequence was retrieved and compared using the NCBI data platform, and the strain was identified as *Achromobacter xylose oxidizing* (…). Achromobacter xylosoxidans The strain was named *Xylose-oxidizing achromobacter*. Achromobacter xylosoxidans YQ016 was deposited on September 26, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67040.
[0056] Xylose-oxidizing achromobacterium ( Achromobacter xylosoxidans The 16S rDNA sequence of YQ016 is as follows:
[0057] Example 2 1. Culture of Xylose-oxidizing Achromobacterium Prepare 150 mL of liquid culture medium (culture medium formula: glucose 90 g / L, peptone 9 g / L, yeast extract 9 g / L, potassium dihydrogen phosphate 2.6 g / L, magnesium sulfate 0.24 g / L, pH adjusted to 6.8, solvent: deionized water), put it into a 1000 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, cool, inoculate with two loops of xylose-oxidizing achromobacter YQ016 slant culture, and incubate at 29℃ and 180 r / min for 19 h.
[0058] 2. Fermentation of Citrus reticulata peel Weigh out 0.6 kg of dried Citrus reticulata peel slices, 1.2 kg of fresh celery stalks, 2.8 kg of glucose, 1.5 kg of sodium citrate, 180 g of peptone, 180 g of yeast extract, 52 g of potassium dihydrogen phosphate, and 4.8 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.5 with sodium carbonate, boil for 4 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, inoculate with 150 mL of Xylose-oxidizing Achromobacterium culture medium, start stirring, and control the stirring speed at 175 r / min. Control the fermentation temperature at 29℃. During fermentation, the aeration ratio is controlled at 0.10~0.20 vvm from 0 to 16 h, and at 0.10 vvm from 17 h onwards. The fermentation cycle is 42 h.
[0059] 3. Reflux extraction and slag removal The pH of the fermentation material was adjusted to 7.7 with sodium carbonate, and then refluxed at 95℃ for 50 min. The extract was then centrifuged at 6500 r / min for 20 min, and the supernatant was collected, yielding 17.1 L. The concentration of naringin was determined using a modified liquid chromatography method (reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.), and was found to be 175.2 mg / L.
[0060] 4. Ultrafiltration for impurity removal The extract supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane to obtain 14 L of permeate. 9.3 L of purified water was added to the concentrate, and a second ultrafiltration using a 1000 Da ultrafiltration membrane was performed to obtain another 9.3 L of permeate. The permeates from both ultrafiltrations were combined, and a first ultrafiltration using a 360 Da ultrafiltration membrane was performed to obtain 4.2 L of retentate. 12.6 L of purified water was added to the concentrate (i.e., the aforementioned retentate), and a second ultrafiltration using a 360 Da ultrafiltration membrane was performed to obtain another 4.2 L of retentate.
[0061] 5. Evaporation, Concentration, and Drying The pH of the 4.2 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The concentrate was then placed in a vacuum evaporator and evaporated and concentrated under a vacuum of -81 kPa and a temperature of 60 °C to obtain 1.05 L of concentrate. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170 °C and the outlet air temperature at 40–50 °C, yielding 40.2 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of arugula glycosides in the powder was 6.98%. Therefore, the extraction rate of arugula glycosides from the dried Citrus reticulata peel raw material in the powder was 0.47%.
[0062] Example 3 1. Culture of Xylose-oxidizing Achromobacterium Prepare 200 mL of liquid culture medium (culture medium formula: glucose 100 g / L, peptone 10 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate 2.8 g / L, magnesium sulfate 0.28 g / L, adjust pH to 7.0, solvent is deionized water), put it into a 1000 mL Erlenmeyer flask, sterilize at 121 ℃ for 20 min, cool, inoculate with two loops of xylose-oxidizing achromobacter YQ016 slant culture, and incubate at 30 ℃ and 200 r / min for 20 h.
[0063] 2. Fermentation of Citrus reticulata peel Weigh out 0.64 kg of dried Citrus reticulata peel slices, 1.28 kg of fresh celery stalks, 3.0 kg of glucose, 1.5 kg of sodium citrate, 200 g of peptone, 200 g of yeast extract, 56 g of potassium dihydrogen phosphate, and 5.2 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.6 with sodium carbonate, boil for 5 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, inoculate with 200 mL of Xylose-oxidizing Achromobacterium culture medium, start stirring, and control the stirring speed at 200 r / min. Control the fermentation temperature at 30℃. During fermentation, the aeration ratio is controlled at 0.10~0.20 vvm from 0 to 16 h, and at 0.10 vvm from 17 h onwards. The fermentation cycle is 40 h.
[0064] 3. Reflux extraction and slag removal The pH of the fermentation material was adjusted to 8.0 with sodium carbonate, and then placed in a reflux extraction device for extraction at 100 °C for 40 min. The extract was then centrifuged at 7000 r / min for 18 min, and the supernatant was collected, yielding 16.7 L of supernatant. The concentration of naringin was determined using a modified liquid chromatography method from the reference (Reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.), and was found to be 187.5 mg / L.
[0065] 4. Ultrafiltration for impurity removal The extract supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 13.4 L of permeate. 11.6 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 11.5 L of permeate. The permeates from both ultrafiltrations were combined, and subjected to a first ultrafiltration using a 360 Da ultrafiltration membrane, yielding 5 L of retentate. 17.5 L of purified water was added to the concentrate (i.e., the retentate obtained from the first ultrafiltration using the 360 Da ultrafiltration membrane), followed by a second ultrafiltration using a 360 Da ultrafiltration membrane, yielding 6.1 L of retentate.
[0066] 5. Evaporation, Concentration, and Drying The pH of the 6.1 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The concentrate was then placed in a vacuum evaporator and evaporated and concentrated under a vacuum of -76 kPa and a temperature of 65 °C to obtain 1.2 L of concentrate. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170 °C and the outlet air temperature at 40–50 °C, yielding 42.9 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of arugula glycosides in the powder was 6.79%. Therefore, the extraction rate of arugula glycosides from the dried Citrus reticulata peel raw material in the powder was 0.46%.
[0067] Example 4 1. Culture of Xylose-oxidizing Achromobacterium Prepare 100 mL of liquid culture medium (culture medium formula: glucose 80 g / L, peptone 8 g / L, yeast extract 8 g / L, potassium dihydrogen phosphate 2.4 g / L, magnesium sulfate 0.22 g / L, adjust pH to 6.6, solvent is deionized water), put it into a 500 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, cool, inoculate with two loops of xylose-oxidizing achromobacter YQ016 slant culture, and incubate at 28℃ and 200 r / min for 18 h.
[0068] 2. Fermentation of Citrus reticulata peel Weigh out 0.56 kg of dried Citrus reticulata peel slices, 1.12 kg of fresh celery stalks, 2.6 kg of glucose, 1.3 kg of sodium citrate, 160 g of peptone, 160 g of yeast extract, 48 g of potassium dihydrogen phosphate, and 4.4 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.4 with sodium carbonate, boil for 3 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, inoculate with 100 mL of Xylose-oxidizing Achromobacterium culture medium, start stirring, and control the stirring speed at 150 r / min. Control the fermentation temperature at 28 ℃. During fermentation, the aeration ratio is controlled at 0.10~0.20 vvm from 0 to 16 h, and at 0.10 vvm from 17 h onwards. The fermentation cycle is 44 h.
[0069] 3. Reflux extraction and slag removal The pH of the fermentation material was adjusted to 7.5 with sodium carbonate, and then refluxed at 90 °C for 60 min. The extract was then centrifuged at 6000 r / min for 22 min, and the supernatant was collected, yielding 18.4 L of supernatant. The concentration of naringin was determined to be 160.3 mg / L using a modified liquid chromatography method (Reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.).
[0070] 4. Ultrafiltration for impurity removal The extract supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 15.5 L of permeate. 7.3 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 7.4 L of permeate. The permeates from both ultrafiltrations were combined and subjected to a first ultrafiltration using a 360 Da ultrafiltration membrane, yielding 3.7 L of retentate. 9.3 L of purified water was added to the concentrate (i.e., the retentate obtained from the first ultrafiltration using the 360 Da ultrafiltration membrane), followed by a second ultrafiltration using a 360 Da ultrafiltration membrane, yielding 3 L of retentate.
[0071] 5. Evaporation, Concentration, and Drying The pH of the above 3 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The solution was then placed in a vacuum evaporator and concentrated under a vacuum of -79.5 kPa and a temperature of 62 °C to obtain 0.9 L of concentrate. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170 °C and the outlet air temperature at 40–50 °C, yielding 38.8 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of argutoin in the powder was 7.09%. Therefore, the extraction rate of argutoin from the powder for the dried Citrus reticulata peel raw material was 0.49%.
[0072] Comparative Example 1 1. Reflux extraction and slag removal Weigh out 0.6 kg of dried tangerine peel slices, 1.2 kg of fresh celery stalks, 2.8 kg of glucose, 1.5 kg of sodium citrate, 180 g of peptone, 180 g of yeast extract, 52 g of potassium dihydrogen phosphate, and 4.8 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.5 with sodium carbonate, boil for 4 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, add 150 mL of purified water, start stirring, and control the stirring speed at 175 r / min. Control the fermentation temperature at 29℃. During fermentation, the aeration ratio is controlled at 0.10~0.20 vvm from 0 to 16 h, and at 0.10 vvm from 17 h onwards. Stir for 42 h.
[0073] Then, the material was placed in a reflux extraction device and refluxed at 95 °C for 50 min. The extracted material was then centrifuged at 6500 r / min for 20 min, and the supernatant was collected to obtain 17.2 L of supernatant. The concentration of naringin was determined by the improved liquid chromatography method described in the reference (Reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.), and the concentration of naringin was found to be 122.6 mg / L.
[0074] 2. Ultrafiltration for impurity removal The supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 14.1 L of permeate. 9.3 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 1000 Da ultrafiltration membrane, yielding another 9.3 L of permeate. The permeates from both ultrafiltrations were combined and subjected to a first ultrafiltration using a 360 Da ultrafiltration membrane, yielding 4.2 L of retentate. 12.6 L of purified water was added to the concentrate (i.e., the aforementioned retentate), followed by a second ultrafiltration using a 360 Da ultrafiltration membrane, yielding another 4.2 L of retentate.
[0075] 5. Evaporation, Concentration, and Drying The pH of the 4.2 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The concentrate was then placed in a vacuum evaporator and evaporated and concentrated under a vacuum of -81 kPa and a temperature of 60 °C to obtain 1.05 L of concentrate. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170 °C and the outlet air temperature at 40–50 °C, yielding 40.9 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of arugula glycosides in the powder was 4.86%. Therefore, the extraction rate of arugula glycosides from the dried Citrus reticulata peel raw material in the powder was 0.33%.
[0076] Comparative Example 2 1. Culture of Bacillus subtilis Prepare 150 mL of liquid culture medium (culture medium formula: glucose 90 g / L, peptone 9 g / L, yeast extract 9 g / L, potassium dihydrogen phosphate 2.6 g / L, magnesium sulfate 0.24 g / L, pH adjusted to 6.8, solvent: deionized water), put it into a 1000 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, cool, and inoculate with two loops of Bacillus subtilis CICC 21368 slant culture, and incubate at 30℃ and 180 r / min for 18 h.
[0077] 2. Fermentation of Citrus reticulata peel Weigh out 0.6 kg of dried Citrus reticulata peel slices, 1.2 kg of fresh celery stalks, 2.8 kg of glucose, 1.5 kg of sodium citrate, 180 g of peptone, 180 g of yeast extract, 52 g of potassium dihydrogen phosphate, and 4.8 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.5 with sodium carbonate, boil for 4 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, inoculate with 150 mL of Bacillus subtilis culture medium, start stirring, and control the stirring speed at 175 r / min and the fermentation temperature at 30℃. During fermentation, the aeration ratio was controlled at 0.20-0.32 vvm for 0-16 h, 0.32-0.36 vvm for 17-24 h, 0.32-0.30 vvm for 25-36 h, and 0.28 vvm for 37 h and beyond. The fermentation cycle was 42 h.
[0078] 3. Reflux extraction and slag removal The pH of the fermentation material was adjusted to 7.7 with sodium carbonate, and then refluxed at 95℃ for 50 min. The extract was then centrifuged at 6500 r / min for 20 min, and the supernatant was collected, yielding 16.8 L of supernatant. The modified liquid chromatography method described in the reference (Reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringenin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.) was used to determine naringenin. No naringenin was detected (i.e., the concentration of naringenin was 0 mg / L).
[0079] 4. Ultrafiltration for impurity removal The extract supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 13.8 L of permeate. 9 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 1000 Da ultrafiltration membrane, yielding another 9 L of permeate. The permeates from both ultrafiltrations were combined, and the mixture was subjected to a first ultrafiltration using a 360 Da ultrafiltration membrane, yielding 4.1 L of retentate. 12.3 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 360 Da ultrafiltration membrane, yielding another 4.1 L of retentate.
[0080] 5. Evaporation, Concentration, and Drying The pH of the 4.1 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The concentrate was then placed in a vacuum evaporator and concentrated under a vacuum of -81 kPa and a temperature of 60 °C to obtain 1.03 L of concentrate. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170 °C and the outlet air temperature at 40–50 °C, yielding 39.8 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of argutoin in the powder was 0. Therefore, the extraction rate of argutoin from the dried Citrus reticulata peel raw material in the powder was 0.
[0081] Comparative Example 3 1. Culture of Lactobacillus plantarum Prepare 150 mL of liquid culture medium (culture medium formula: glucose 90 g / L, peptone 9 g / L, yeast extract 9 g / L, potassium dihydrogen phosphate 2.6 g / L, magnesium sulfate 0.24 g / L, pH adjusted to 6.8, solvent: deionized water), put it into a 1000 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, cool, inoculate with two loops of Lactobacillus plantarum CICC 25125 slant culture, and incubate at 37℃ for 18 h.
[0082] 2. Fermentation of Citrus reticulata peel Weigh out 0.6 kg of dried Citrus reticulata peel slices, 1.2 kg of fresh celery stalks, 2.8 kg of glucose, 1.5 kg of sodium citrate, 180 g of peptone, 180 g of yeast extract, 52 g of potassium dihydrogen phosphate, and 4.8 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.5 with sodium carbonate, boil for 4 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, inoculate with 150 mL of Lactobacillus plantarum culture, start stirring, and control the stirring speed at 175 r / min. Control the fermentation temperature at 37℃. Do not introduce sterile air during fermentation, and the fermentation period is 42 h.
[0083] 3. Reflux extraction and slag removal The pH of the fermentation material was adjusted to 7.7 with sodium carbonate, and then placed in a reflux extraction device for extraction at 95℃ for 50 min. The extracted material was then centrifuged at 6500 r / min for 20 min, and the supernatant was collected, yielding 17.4 L of supernatant. The modified liquid chromatography method described in the reference (Reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringenin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.) was used to determine naringenin. No naringenin was detected (i.e., the concentration of naringenin was 0 mg / L).
[0084] 4. Ultrafiltration for impurity removal The extract supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 14.3 L of permeate. 9.3 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 1000 Da ultrafiltration membrane, yielding another 9.3 L of permeate. The permeates from both ultrafiltrations were combined, and a first ultrafiltration using a 360 Da ultrafiltration membrane yielded 4.2 L of retentate. 12.6 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 360 Da ultrafiltration membrane, yielding another 4.2 L of retentate.
[0085] 5. Evaporation, Concentration, and Drying The pH of the 4.2 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The concentrate was then placed in a vacuum evaporator and concentrated under a vacuum of -81 kPa and a temperature of 60°C to obtain 1.05 L of concentrated solution. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170°C and the outlet air temperature at 40–50°C, yielding 40.6 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of arugula glycosides in the powder was 0. Therefore, the extraction rate of arugula glycosides from the dried Citrus reticulata peel raw material in the powder was 0.
[0086] Comparative Example 4 1. Culture of Leuconostoc mesenteroides Prepare 150 mL of liquid culture medium (culture medium formula: glucose 90 g / L, peptone 9 g / L, yeast extract 9 g / L, potassium dihydrogen phosphate 2.6 g / L, magnesium sulfate 0.24 g / L, pH adjusted to 6.8, solvent: deionized water), put it into a 1000 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, cool, and inoculate with two loops of Leuconostoc mesenteroides CICC 21860 slant culture, and incubate at 37℃ and 100 r / min for 24 h.
[0087] 2. Fermentation of Citrus reticulata peel Weigh out 0.6 kg of dried Citrus reticulata peel slices, 1.2 kg of fresh celery stalks, 2.8 kg of glucose, 1.5 kg of sodium citrate, 180 g of peptone, 180 g of yeast extract, 52 g of potassium dihydrogen phosphate, and 4.8 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.5 with sodium carbonate, boil for 4 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, inoculate with 150 mL of Leuconostoc mesenteroides culture, start stirring, and control the stirring speed at 175 r / min and the fermentation temperature at 37℃. During fermentation, the aeration ratio was controlled at 0.10~0.24 vvm for 0~16 h, 0.24~0.32 vvm for 17~24 h, 0.28~0.24 vvm for 25~36 h, and 0.24 vvm for 37 h and thereafter. The fermentation cycle was 42 h.
[0088] 3. Reflux extraction and slag removal The pH of the fermentation material was adjusted to 7.7 with sodium carbonate, and then refluxed at 95℃ for 50 min. The extract was then centrifuged at 6500 r / min for 20 min, and the supernatant was collected to obtain 17 L of supernatant. The concentration of naringin was determined by a modified liquid chromatography method from the reference (Reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.), and was found to be 104.2 mg / L.
[0089] 4. Ultrafiltration for impurity removal The extract supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 13.9 L of permeate. 9.3 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 1000 Da ultrafiltration membrane, yielding another 9.3 L of permeate. The permeates from both ultrafiltrations were combined, and a first ultrafiltration using a 360 Da ultrafiltration membrane yielded 4.2 L of retentate. 12.6 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 360 Da ultrafiltration membrane, yielding another 4.2 L of retentate.
[0090] 5. Evaporation, Concentration, and Drying The pH of the 4.2 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The concentrate was then placed in a vacuum evaporator and concentrated under a vacuum of -81 kPa and a temperature of 60°C to obtain 1.05 L of concentrated solution. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170°C and the outlet air temperature at 40–50°C, yielding 39.6 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of arugula glycosides in the powder was 4.15%. Therefore, the extraction rate of arugula glycosides from the dried Citrus reticulata peel raw material in the powder was 0.27%.
[0091] Comparative Example 5 1. Culture of Lactobacillus casei Prepare 150 mL of liquid culture medium (culture medium formula: glucose 90 g / L, peptone 9 g / L, yeast extract 9 g / L, potassium dihydrogen phosphate 2.6 g / L, magnesium sulfate 0.24 g / L, pH adjusted to 6.8, solvent: deionized water), put it into a 1000 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, cool, and inoculate with two loops of Lactobacillus casei CICC 20335 slant culture, and incubate at 37℃ and 100 r / min for 24 h.
[0092] 2. Fermentation of Citrus reticulata peel Weigh out 0.6 kg of dried Citrus reticulata peel slices, 1.2 kg of fresh celery stalks, 2.8 kg of glucose, 1.5 kg of sodium citrate, 180 g of peptone, 180 g of yeast extract, 52 g of potassium dihydrogen phosphate, and 4.8 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.5 with sodium carbonate, boil for 4 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, inoculate with 150 mL of Lactobacillus casei culture medium, start stirring, and control the stirring speed at 175 r / min and the fermentation temperature at 37℃. During fermentation, the aeration ratio was controlled at 0.10~0.24 vvm for 0~16 h, 0.24~0.32 vvm for 17~24 h, 0.28~0.24 vvm for 25~36 h, and 0.24 vvm for 37 h and thereafter. The fermentation cycle was 42 h.
[0093] 3. Reflux extraction and slag removal The pH of the fermentation material was adjusted to 7.7 with sodium carbonate, and then refluxed at 95℃ for 50 min. The extract was then centrifuged at 6500 r / min for 20 min, and the supernatant was collected, yielding 16.9 L of supernatant. The concentration of naringin was determined using a modified liquid chromatography method from the reference (Reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.), and was found to be 45.6 mg / L.
[0094] 4. Ultrafiltration for impurity removal The extract supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 13.9 L of permeate. 9 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 1000 Da ultrafiltration membrane, yielding another 9 L of permeate. The permeates from both ultrafiltrations were combined, and the mixture was subjected to a first ultrafiltration using a 360 Da ultrafiltration membrane, yielding 4.1 L of retentate. 12.3 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 360 Da ultrafiltration membrane, yielding another 4.1 L of retentate.
[0095] 5. Evaporation, Concentration, and Drying The pH of the 4.1 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The concentrate was then placed in a vacuum evaporator and concentrated under a vacuum of -81 kPa and a temperature of 60°C to obtain 1.03 L of concentrated solution. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170°C and the outlet air temperature at 40–50°C, yielding 39.2 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of arugula glycosides in the powder was 1.84%. Therefore, the extraction rate of arugula glycosides from the dried Citrus reticulata peel raw material in the powder was 0.12%.
[0096] Comparative Example 6 1. Culture of Xylose-oxidizing Achromobacterium Prepare 150 mL of liquid culture medium (culture medium formula: glucose 90 g / L, peptone 9 g / L, yeast extract 9 g / L, potassium dihydrogen phosphate 2.6 g / L, magnesium sulfate 0.24 g / L, pH adjusted to 6.8, solvent: deionized water), put it into a 1000 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, cool, inoculate with two loops of xylose-oxidizing achromobacter YQ016 slant culture, and incubate at 29℃ and 180 r / min for 19 h.
[0097] 2. Fermentation of Citrus reticulata peel Weigh out 0.6 kg of dried Citrus reticulata peel slices, 2.8 kg of glucose, 1.5 kg of sodium citrate, 180 g of peptone, 180 g of yeast extract, 52 g of potassium dihydrogen phosphate, and 4.8 g of magnesium sulfate. Mix with 20 L of water, adjust the pH to 7.5 with sodium carbonate, boil for 4 min, and then homogenize in a high-speed homogenizer at 6000 r / min for 10 min. Transfer to a 30 L fermenter and sterilize at 121℃ for 20 min. After cooling, inoculate with 150 mL of Xylose-oxidizing Achromobacterium culture medium, start stirring, and control the stirring speed at 175 r / min. Control the fermentation temperature at 29℃. During fermentation, the aeration ratio is controlled at 0.10~0.20 vvm from 0 to 16 h, and at 0.10 vvm from 17 h onwards. The fermentation cycle is 42 h.
[0098] 3. Reflux extraction and slag removal The pH of the fermentation material was adjusted to 7.7 with sodium carbonate, and then refluxed at 95℃ for 50 min. The extract was then centrifuged at 6500 r / min for 20 min, and the supernatant was collected, yielding 17.6 L. The concentration of naringin was determined using a modified liquid chromatography method (reference: Zhou Guifen, Chen Suhong, Lü Guiyuan, et al. Determination of naringin content in Dendrobium officinale by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(4):520-523.), and was found to be 150.3 mg / L.
[0099] 4. Ultrafiltration for impurity removal The extract supernatant was subjected to a first ultrafiltration using a 1000 Da ultrafiltration membrane, yielding 14.4 L of permeate. 9.6 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 1000 Da ultrafiltration membrane, yielding another 9.6 L of permeate. The permeates from both ultrafiltrations were combined, and a first ultrafiltration using a 360 Da ultrafiltration membrane yielded 4.3 L of retentate. 12.9 L of purified water was added to the concentrate, followed by a second ultrafiltration using a 360 Da ultrafiltration membrane, yielding another 4.3 L of retentate.
[0100] 5. Evaporation, Concentration, and Drying The pH of the 4.3 L retentate (the retentate from the second ultrafiltration of the 360 Da ultrafiltration membrane) was adjusted to 7.0 using a 10% v / v phosphoric acid solution. The concentrate was then placed in a vacuum evaporator and evaporated and concentrated under a vacuum of -81 kPa and a temperature of 60 °C to obtain 1.08 L of concentrate. This concentrate was then spray-dried, with the inlet air temperature controlled at 160–170 °C and the outlet air temperature at 40–50 °C, yielding 39.5 g of powder. High-performance liquid chromatography (HPLC) analysis showed that the content of arugula glycosides in the powder was 6.25%. Therefore, the extraction rate of arugula glycosides from the dried Citrus reticulata peel raw material in the powder was 0.41%.
[0101] 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 Achromobacter xylosoxidans, characterized in that: Achromobacter xylosoxidans (ATCC 19367) Achromobacter xylosoxidans YQ016, deposited on September 26, 2025, at the Guangdong Microbial Culture Collection Center, located at the 5th Floor, Building 59, Guangzhou Academy of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou, with the accession number GDMCC 67040, GDMCC No: 67040.
2. The use of the Achromobacter xylosoxidans in claim 1 in improving the content of wild maslinic glycoside in Citrus grandis.
3. A fermentation method for increasing the content of wild orange red lacquertree glycoside, characterized in that The method comprises the following steps: inoculating the seed liquid of the Achromobacter xylosoxidans in claim 1 into a fermentation medium for fermentation culture, so as to obtain a fermentation material with improved content of wild maslinic glycoside in Citrus grandis. The fermentation medium comprises the following components: 28-32 g / L of dried Citrus grandis slices, 56-64 g / L of fresh celery stems, 130-150 g / L of glucose, 65-75 g / L of sodium citrate, 8-10 g / L of proteose peptone, 8-10 g / L of yeast extract, 2.4-2.8 g / L of potassium dihydrogen phosphate, and 0.22-0.26 g / L of magnesium sulfate, and water is used as the solvent, and the pH value is 7.4-7.
6.
4. The fermentation method for improving the content of wild maslinic glycoside in Citrus grandis according to claim 3, characterized in that: The fermentation medium is prepared by the following steps: mixing all the components in the fermentation medium, homogenizing after boiling and heating, and then sterilizing, so as to obtain the fermentation medium. The seed liquid of the Achromobacter xylosoxidans is prepared by the following steps: inoculating the preserved seed of the Achromobacter xylosoxidans into a seed culture medium, and oscillating and culturing, so as to obtain the seed liquid of the Achromobacter xylosoxidans.
5. The fermentation method for improving the content of wild maslinic glycoside in Citrus grandis according to claim 4, characterized in that: The boiling and heating time is 3-5 min; The homogenizing condition is 5000-7000 r / min for 5-15 min; The sterilizing condition is 115-121 ℃ for 15-30 min; The seed culture medium comprises the following components: 80-100 g / L of glucose, 8-10 g / L of proteose peptone, 8-10 g / L of yeast extract, 2.4-2.8 g / L of potassium dihydrogen phosphate, and 0.22-0.26 g / L of magnesium sulfate, and the pH value is 6.6-7.0, and water is used as the solvent; The inoculation amount of the preserved seed of the Achromobacter xylosoxidans is 2 loops of Achromobacter xylosoxidans bacterial film per 100-200 mL of the seed culture medium; The oscillating and culturing condition is 28-30 ℃, 160-200 r / min for 18-20 h.
6. The fermentation method for improving the content of wild maslinic glycoside in Citrus grandis according to claim 3, characterized in that: The inoculation amount of the seed liquid of the Achromobacter xylosoxidans is calculated according to 0.5%-1.0% of the water volume of the fermentation medium; The fermentation culture condition is that the temperature is 28-30 ℃, the stirring speed is 150-200 r / min, the aeration ratio is controlled to be 0.10-0.2 vvm for 0-16 h of fermentation, the aeration ratio is controlled to be 0.10 vvm after 17 h of fermentation, and the fermentation cycle is 40-44 h.
7. A method for preparing an extract of wildgumi, characterized in that The method comprises the following steps: (1) reflux extraction and deslagging: the fermented material obtained by the fermentation method of any one of claims 3-6 is adjusted to alkaline, put into a reflux extraction device, and subjected to reflux extraction and solid-liquid separation to obtain an extract; (2) ultrafiltration impurity removal: the extract is first subjected to ultrafiltration using a 1000 Da ultrafiltration membrane to obtain a permeate; the permeate is then subjected to ultrafiltration using a 360 Da ultrafiltration membrane to obtain a retentate; (3) evaporation concentration and drying: the retentate finally obtained in step (2) is adjusted to neutral pH, further concentrated, and then the obtained concentrated solution is dried to obtain the extract of mallotus japonicus fructus.
8. The preparation method of the extract of mallotus japonicus fructus according to claim 7, characterized in that: in step (2), the number of times of ultrafiltration of the extract using a 1000 Da ultrafiltration membrane is 2, and the specific operation is as follows: the degree of the first ultrafiltration is that the permeate is 80%-84% of the original volume; when the second ultrafiltration is performed, 2.5-3.5 times the volume of water of the retentate of the first ultrafiltration is added to the retentate, and the degree of the ultrafiltration is that the permeate is 73%-77% of the original volume; the number of times of ultrafiltration of the permeate using a 360 Da ultrafiltration membrane is 2, and the specific operation is as follows: the degree of the first ultrafiltration is that the retentate is 16%-20% of the original volume; when the second ultrafiltration is performed, 2.5-3.5 times the volume of water of the retentate of the first ultrafiltration is added to the retentate, and the degree of the ultrafiltration is that the permeate is 23%-27% of the original volume.
9. The preparation method of the extract of mallotus japonicus fructus according to claim 7, characterized in that: in step (1), the pH of the fermented material is adjusted to 7.5-8.0; the temperature of the reflux extraction is 90-100℃; the time of the reflux extraction is 40-60 min; the mode of the solid-liquid separation is centrifugation; in step (3), the mode of the concentration is vacuum evaporation concentration; the mode of the drying is spray drying.
10. The preparation method of the extract of mallotus japonicus fructus according to claim 9, characterized in that: the centrifugation is performed at a speed of 6000-7000 r / min for 18-22 min; the vacuum evaporation concentration is performed at a vacuum degree of-81--76 kPa and a temperature of 60-65℃; the degree of the vacuum evaporation concentration is that the concentrated solution is 20%-30% of the original volume; the spray drying is performed at an inlet air temperature of 160-170℃ and an outlet air temperature of 40-50℃.