Portulaca oleracea fermentation process

Abstract

The invention provides a portulaca oleracea fermentation process, which utilizes lactobacillus plantarum BJ-M8 with carbohydrate amylase, glucosidase and cellulase production capacity to ferment portulaca oleracea, and promotes the conversion of macromolecular glycoside substances to small molecular aglycones. Metabonomics analysis shows that through fermentation treatment of the strain BJ-M8, the composition of alkaloids, phenolic acids, flavonoids and the like of purslane is effectively improved. Compared with the prior art, the target product calycosin is increased by 10 times, acacetin is increased by 7 times, kaempferol is increased by 4 times, apigenin is increased by 4 times, naringenin is increased by 3 times, dihydroferulic acid is increased by 177 times, dihydrocaffeic acid is increased by 27 times, and spinogenin is increased by 6 times. According to an in-vitro evaluation test, the inhibition effect of the fermented purslane on salmonella and escherichia coli is enhanced, the anti-inflammatory effect on LPS treated macrophage RAW264.7 and the oxidation resistance on Caco-2 cells are improved, and the application prospect in the aspect of functional product preparation is wide.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation, in particular to a purslane fermentation process. Background Art

[0002] Purslane is a plant with both medicinal and edible properties, has strong environmental adaptability, and is widely distributed in my country. Purslane extracts are rich in active ingredients such as alkaloids, flavonoids, polysaccharides, and organic acids, and have promising development and application prospects, including thirst-quenching, diuretic, heat-clearing, detoxifying, anti-inflammatory, and swelling-reducing properties. However, the dense structure of the purslane plant cell wall hinders the release of its active ingredients. Furthermore, the complex and low content of purslane's active ingredients significantly weaken its effectiveness.

[0003] Although the addition of enzymes can promote the degradation of plant leaf cell walls, cost constraints limit their large-scale use. Lactobacillus plantarum is a widely used edible microorganism in fermentation and is a microbial feed additive approved for use in feed by the Ministry of Agriculture and Rural Affairs. However, the functionalities of strains vary significantly. Finding strains with extensive enzyme production capacity could potentially achieve high-value conversion of substrates. Furthermore, the mechanism of Lactobacillus plantarum biotransformation of purslane remains unclear and requires further investigation.

[0004] CN117838598A discloses a purslane extract. It uses purslane residue as the raw material, extracts it with water, then inoculates it with Lactobacillus plantarum. The fermented purslane liquor is then freeze-dried and mixed with purslane distillate. However, the specific components of the purslane extract are not analyzed. Furthermore, there is significant variation between strains, and there is currently a lack of research on fermentation processes based on Lactobacillus plantarum to promote the conversion of macromolecular glycosides in purslane into micromolecular aglycones. Summary of the Invention

[0005] The present invention aims to provide a purslane fermentation process, specifically relates to fermentation of purslane by Lactobacillus plantarum BJ-M8, and especially relates to fermentation of purslane and in vitro biological activity evaluation.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a purslane fermentation process, using the plant lactobacillus (CGMCC No. 31307) Lactobacillus plantarum )BJ-M8 is used for purslane fermentation.

[0007] Lactobacillus plantarum BJ-M8 was isolated and purified from sauerkraut and classified as Lactobacillus plantarumThe strain has been deposited in the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, with the deposit number CGMCC No.31307 and the deposit date July 15, 2024.

[0008] Furthermore, the fermentation process comprises: crushing dry purslane, mixing purslane and water at a material-liquid ratio of 1 g: (1-2) mL as a fermentation substrate, inoculating Lactobacillus plantarum bacterial liquid at a volume ratio of 5% into the fermentation substrate for sealed fermentation; wherein the content of Lactobacillus plantarum in the bacterial liquid is 1×10 8- 1×10 10 CFU / mL.

[0009] Preferably, the material-liquid ratio of purslane and water is 1 g:1 mL or 1 g:2 mL.

[0010] Preferably, the fermentation conditions are: fermentation at 37° C. for 14-16 days.

[0011] In a second aspect, the present invention provides a fermented purslane product prepared according to the process.

[0012] In a third aspect, the present invention provides any of the following applications of the purslane fermented product: (1) Used in the preparation of feed additives; (2) Used for livestock and poultry breeding; (3) Used in the preparation of antioxidants; (4) Used in the preparation of antibacterial agents; (5) Used in food processing; (6) Used in the cosmetics industry.

[0013] Furthermore, the antibacterial agent described in (4) can inhibit the growth of Salmonella ( Salmonella ) and Escherichia coli ( Escherichia coli ).

[0014] In a fourth aspect, the present invention provides an antioxidant or antibacterial agent, the active ingredient of which is the water extract or alcohol extract of the purslane fermented product, preferably the water extract.

[0015] In a specific embodiment of the present invention, 30 grams of fermented purslane product (i.e., fermented purslane) was added to 300 mL of purified water and soaked in cold water for 30 minutes. The mixture was boiled over high heat and simmered over low heat for 3 times. The decoctions were combined to obtain the water extract.

[0016] In a fifth aspect, the present invention provides any of the following applications of the process: (1) To improve the composition of alkaloids, phenolic acids and flavonoids in fermented purslane; (2) Used to increase the content of calycosin, acacetin, kaempferol, apigenin, naringenin, dihydroferulic acid, dihydrocaffeic acid, and jujube saponin in fermented purslane.

[0017] Furthermore, the flavonoid compound includes flavonoid aglycones.

[0018] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention utilizes a strain of Lactobacillus plantarum BJ-M8 with good carbohydrate amylase, glucosidase, and cellulase production to ferment and treat purslane, effectively improving the composition of alkaloids, phenolic acids, and flavonoids in purslane. The carbohydrate amylase, glucosidase, and cellulase produced by Lactobacillus plantarum promote the degradation of plant leaf cell walls and catalyze the hydrolysis of glycosidic bonds, thereby promoting the conversion of purslane macromolecular glycosides into small molecule aglycones. The present invention also demonstrates that fermented purslane increases the inhibitory effect on Salmonella and Escherichia coli through in vitro evaluation tests, improves the anti-inflammatory effect on LPS-treated macrophages RAW264.7, and the antioxidant capacity on Caco-2 cells. The present invention provides a theoretical basis for the rational application of fermented purslane in terms of anti-inflammatory and antioxidant effects by conducting a functional evaluation of fermented purslane, and has good prospects in the development of functional products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The flavonoid content and pH changes of fermented purslane in a preferred embodiment of the present invention.

[0020] Figure 2 This is a diagram showing the categories and composition of metabolites of purslane and fermented purslane in a preferred embodiment of the present invention.

[0021] Figure 3 This is a clustering heat map of differential metabolites in a preferred embodiment of the present invention.

[0022] Figure 4 This is the KEGG enrichment map of the metabolic pathway of differential metabolites in a preferred embodiment of the present invention.

[0023] Figure 5 Figure 2 is a diagram of the core metabolic pathway for flavonoid biosynthesis in a preferred embodiment of the present invention.

[0024] Figure 6 This is the effect of purslane and fermented purslane on the NO content in macrophages in a preferred embodiment of the present invention.

[0025] Figure 7 This is the effect of purslane and fermented purslane on Salmonella and Escherichia coli in the preferred embodiment of the present invention.

[0026] Figure 8This is the effect of purslane and fermented purslane on antioxidant enzymes in Caco-2 cells in preferred embodiments of the present invention. DETAILED DESCRIPTION

[0027] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0028] The purslane used in the following examples was purchased from Anguo Changda Chinese Medicinal Materials Pieces Co., Ltd. (Baoding, Hebei Province), and was crushed and passed through a 40-mesh sieve for later use.

[0029] Lactobacillus plantarum (BJ-M8) was isolated and identified by the laboratory of the Feed Resources and Bioconversion Team of the Institute of Feed Research, Chinese Academy of Agricultural Sciences. It has excellent acid production performance. Lactobacillus plantarum BJ-M8 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 15, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31307.

[0030] MRS liquid medium was purchased from Shanghai Solebao Biotechnology Co., Ltd. Caco-2 cells, RAW264.7 cells, 30% H₂O₂, DMEM / F12 medium, DMEM medium, fetal bovine serum, double-streptomycin antibodies (penicillin and streptomycin), PBS, trypsin, hydrocortisone, and CCK-8 reagent were all purchased from the Feed Resources and Bioconversion Team Laboratory, Institute of Feed Research, Chinese Academy of Agricultural Sciences. Superoxide dismutase (SOD) assay kit, glutathione peroxidase (GSH-Px) assay kit, total antioxidant capacity (T-AOC) assay kit, and nitric oxide assay kit were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0031] The experimental data were statistically analyzed using SPSS 26.0 (IBM) software, using a one-way analysis of variance (ANOVA) procedure. Data are presented as mean and standard error of the mean (SEM). P < 0.05 indicated a significant difference. GraphPad Prism was used to create bar and line graphs.

[0032] Example 1: Fermentation of Purslane by Lactobacillus plantarum BJ-M8 The Lactobacillus plantarum stored at -20℃ was quickly thawed and inoculated into autoclaved MRS liquid culture medium. The culture was incubated at 37℃ for 24 hours to observe the activation of the bacteria. Then, 5% of the inoculum was inoculated into MRS liquid culture medium and incubated at 37℃ for 24 hours. After microscopic examination and counting, 1×10 9The optimal inoculum size was 100 CFU / mL, and its content was adjusted to prepare the solid fermentation strain. Dried purslane was ground through a 40-mesh sieve and divided into two groups, each with three replicates: Group A (purslane feedstock to water ratio 1:2 (g:mL)) and Group B (purslane feedstock to water ratio 1:1 (g:mL). Each group was packaged in 35 mm × 45 mm plastic film fermentation bags. Lactobacillus plantarum culture was inoculated into the fermentation substrate at a volume ratio of 5%, and sealed and fermented at 37°C for 30 days.

[0033] Example 2 Fermentation of Purslane Flavonoids Content and pH Changes 1. pH determination Samples were collected from multiple points in the four experimental groups on days 0, 2, 4, 6, 8, 10, 12, 16, and 20. A total of 1 g of pH was collected from the upper, middle, lower, left, and right points of each group. 10 mL of deionized water was added, and the samples were vortexed for 30 minutes before measurement using a pH meter.

[0034] 2. Determination of flavonoid content (1) Prepare 60% ethanol extract; (2) Add 10 mg of rutin to 1 mL of standard diluent to prepare a 10 mg / mL standard solution; (3) Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 470 nm; (4) Dilute the 10 mg / mL rutin standard solution with the standard diluent in a 1.5 ml centrifuge tube to 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, and 0.039 mg / mL; (5) Dry the purslane at 37°C to constant weight, grind it, pass it through a 40-mesh sieve, weigh about 0.1 g, add 1 mL of extract, and extract it using ultrasonic extraction at an ultrasonic power of 300 W and a temperature of 60°C for 30 minutes. Centrifuge at 12000rpm, 25℃ for 10min, take the supernatant, and dilute to 1mL with 60% ethanol; (6) Add the extract to a 1.5ml centrifuge tube in sequence according to the instructions of the flavonoid reagent, vortex and mix, place in a 37℃ water bath for accurate reaction for 45min, then centrifuge at 10000g for 10min at room temperature, take 200µL of the supernatant in a 96-well plate, and use an enzyme reader to measure the OD 470 .

[0035] The results are as follows Figure 1As shown, when the material-water ratio was 1:2 and 1:1: the pH of purslane showed a significant decline on the second day of fermentation, and then maintained a stable pH. On the 12th day, the pH began to decline again, and after 20 days, the purslane reached the lowest pH of 3.73; the initial flavonoid content of purslane was 9.23 mg / g; the flavonoid content of purslane with a material-water ratio of 1:2 and 1:1 continued to rise during the fermentation process, reaching the highest content on the 16th day. The flavonoid content of purslane with a material-water ratio of 1:1 on the 16th day was 12.05 mg / g, an increase of 30.55%. Therefore, the final number of days selected for fermentation was 16 days.

[0036] Example 3 Targeted metabolomics of purslane and fermented purslane The sample was placed in a freeze dryer and vacuum freeze-dried for 63 h. After grinding into powder, 50 mg of sample powder was weighed and added with 1200 μL of 70% methanol water internal standard extract pre-cooled at -20 °C. The sample was vortexed 6 times for 30 seconds and centrifuged (12000 rpm, 3 minutes). The supernatant was aspirated, filtered with a 0.22 μm microporous filter membrane, and stored in an injection vial for UPLC-MS / MS analysis.

[0037] The UPLC liquid phase used an AgilentSB-C18 1.8 µm, 2.1 mm × 100 mm column; the mobile phase was ultrapure water (with 0.1% formic acid) in phase A and acetonitrile (with 0.1% formic acid) in phase B; the elution gradient was 5% phase B at 0.00 min, linearly increasing to 95% within 9.00 min and maintaining at 95% for 1 min; from 10.00 to 11.10 min, the phase B proportion was reduced to 5% and equilibrated at 5% for 14 min; the flow rate was 0.35 mL / min; the column temperature was 40°C; and the injection volume was 2 μL.

[0038] MS / MS spectra were acquired using an electrospray ionization (ESI) source at 500°C and an ion spray voltage (IS) of 5500 V (positive ion mode) / -4500 V (negative ion mode). Ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and collision-induced ionization parameters were set to high. QQQ scanning was performed in MRM mode with a collision gas (nitrogen) set to medium. Declustering potential (DP) and collision energy (CE) optimization were performed for each MRM transition. A specific set of MRM transitions was monitored in each period based on the metabolites eluting during that period.

[0039] The results are shown in Table 1 and Figure 2As shown, a total of 1,522 metabolites were identified in purslane and fermented purslane, with 744 showing significant changes in content, including 236 up-regulated and 508 down-regulated. Among the metabolites, there were 218 flavonoids (29.3%), 136 phenolic acids (18.28%), 131 alkaloids (17.61%), 117 terpenes (15.73%), 52 lignans and coumarins (6.99%), 11 quinones (1.48%), 6 tannins (0.81%), and 73 other compounds, including lactones (9.81%).

[0040] Table 1 Some significantly upregulated metabolites in fermented Purslane

[0041] At the same time, the metabolites of fermented purslane that changed significantly in Table 1 and Figure 3 Cluster heat maps of differential metabolites and target metabolites show that fermentation of purslane with Lactobacillus plantarum effectively improves the alkaloid, phenolic acid, and flavonoid composition of purslane. Lactobacillus plantarum was also found to promote the conversion of macromolecular glycosides to small-molecule aglycones. This is likely due to the carbohydrate amylase, glucosidase, and cellulase produced by Lactobacillus plantarum promoting the degradation of plant leaf cell walls and catalyzing the hydrolysis of glycosidic bonds, thereby promoting the conversion of glycosides to aglycones.

[0042] according to Figure 4 KEGG enrichment map of differential metabolites metabolic pathways and Figure 5 Analysis of the core metabolic pathways for flavonoid biosynthesis revealed that most compounds were enriched in the secondary metabolite synthesis and phenylpropanoid biosynthesis pathways, with flavonoid and flavonol biosynthesis and flavonoid biosynthesis being more prominent. Ultimately, flavonoid biosynthesis was identified as the most dynamic metabolic pathway during fermentation. Naringenin chalcone isomerizes to naringenin, which is then oxidized to citrinin. Citrinin is further oxidized to tamarind, which is then oxidized and dehydroxylated to kaempferol and quercetin. Finally, quercetin reacts with glucose to form rutin. Similarly, naringenin can be oxidized to apigenin and eriodictyol, which are then oxidized to luteolin and eriodictyol. eriodictyol is then dehydrogenated to diosgenin, and luteolin reacts with glucose to form luteolin. During the fermentation process, the content of key precursor substances for the synthesis of flavonoids such as naringenin, kaempferol, quercetin, apigenin, and luteolin increased significantly, while the content of rutin decreased significantly. These reflected the significant upregulation of flavonoid aglycones in fermented purslane, and its antioxidant and anti-inflammatory active substances increased significantly. Moreover, flavonoid aglycones are more easily absorbed by the body than flavonoid glycosides. This also shows that Lactobacillus plantarum greatly improved the antioxidant and anti-inflammatory effects of purslane.

[0043] Example 4 Evaluation of the in vitro effects of purslane and fermented purslane Take 30 grams of purslane and fermented purslane, add 300 mL of pure water, soak in cold water for 30 minutes, boil over high heat, and simmer over low heat three times. Combine the decoctions, filter and concentrate them to a concentration of 1 g of raw medicinal material per 1 mL of oral solution, and obtain 1 g / mL of drug stock solution.

[0044] 1. Anti-inflammatory test of macrophage RAW264.7 RAW264.7 cells in the logarithmic growth phase were cultured at a rate of 2.0×10 6 Cells were seeded at a density of 100 μL cells / mL in a 96-well plate. 100 μL of cell suspension was added to each well and incubated at 37°C in a 5% CO2 incubator for 24 hours. The cells were then incubated with the various extracts and reagents for 24 hours. The experiments were divided into a normal group, an LPS model group (4.0 μg / mL LPS), a positive control group (final concentration of 10 μmol / L hydrocortisone and 2.0 μg / mL LPS), a purslane group (1 mg / mL purslane extract + final concentration of 2.0 μg / mL LPS), and a fermented purslane group (1 mg / mL fermented purslane extract + final concentration of 2.0 μg / mL LPS). After the experiment, 50 μL of the supernatant was collected and assayed using a nitric oxide detection kit. After vortexing, the OD value was measured at 540 nm. Remove Griess Reagent I and II and return to room temperature; dilute the standard (1-100 μM) with the 1 mmol / L NaNO2 solution used for the sample to be tested; add the standard and sample to a 96-well plate at a rate of 50 μl / well; add room-temperature Griess Reagent I to each well at a rate of 50 μl / well; add room-temperature Griess Reagent II to each well at a rate of 50 μl / well; measure the absorbance through a 540 nm filter; calculate the nitric oxide concentration in the sample based on the standard curve.

[0045] The results are as follows Figure 6 As shown in the data, under the stimulation of LPS, macrophage RAW264.7 cells underwent inflammatory response. The addition of purslane, fermented purslane and anti-inflammatory drug hydrocortisone all alleviated the inflammatory response, and the inhibitory effect of fermented purslane was better than that of purslane.

[0046] 2. Antibacterial test Prepare LB medium and solid medium (agar content 1.5%); activate Salmonella ATCC14028 and Escherichia coli K88: inoculate 2% of the culture medium into a 10 ml LB tube and culture at 37°C, 200 rpm for 4 hours until the culture is about 10 8CFU / ml; Pour 10ml of sterilized nutrient agar medium (agar content 1.5%) into the culture dish, place it horizontally, and after solidification, use sterile tweezers to gently place a sterilized stainless steel tube with a diameter of 6mm and smooth ends on the bottom of the culture medium; Pour 20ml of LB solid medium (agar content 1.5%) into the culture dish, and after solidification, use sterile tweezers to pick out the stainless steel tube to form a 6mm diameter circular hole on the culture medium (double-layer agar can prevent bacterial liquid leakage); Add 100ul 10 8 Spread the Salmonella and Escherichia coli bacterial liquid at CFU / ml evenly; add about 150 μl of liquid to each well, making sure it is full but not overflowing.

[0047] The results are as follows Figure 7 As shown, fermented purslane enhanced the inhibitory effect on Salmonella and Escherichia coli compared with purslane.

[0048] 3. Antioxidant test of Caco-2 cells Caco-2 cells in logarithmic growth phase were cultured at a rate of 8.0×10 5 Cells were seeded at a density of 10 cells / mL into 6-well plates. 2 ml of cell suspension was added to each well and incubated in a 37°C, 5% CO2 incubator for 12 hours. Then, 2 ml of a 1.2 mmol / L H₂O₂ solution was added. Three parallel wells were set up for each group, along with a blank and control group. After 4 hours of co-incubation, 2 ml of 100 μg / mL purslane and fermented purslane were added to each well and incubated for another 24 hours. The enzymes were then assayed according to the respective antioxidant enzyme assay kits.

[0049] The results are as follows Figure 8 As shown in the data, under the stimulation of H2O2, the total antioxidant capacity, glutathione peroxidase and superoxide dismutase activities of Caco-2 cells were significantly reduced, while after adding purslane and fermented purslane, the total antioxidant capacity, glutathione peroxidase and superoxide dismutase activities were significantly increased compared with the enzyme activity of the damaged group, but the enzyme activity did not return to normal, and the fermented purslane had a better antioxidant effect than the normal group.

[0050] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Purslane fermentation process, characterized in that, Using the Lactobacillus plantarum (CGMCC No. 31307) Lactobacillus plantarum )BJ-M8 is used for purslane fermentation.

2. The process according to claim 1, characterized in that The fermentation process comprises: crushing dry purslane, mixing purslane and water in a ratio of 1 g: (1-2) mL as a fermentation substrate, inoculating Lactobacillus plantarum bacterial liquid in a volume ratio of 5% into the fermentation substrate, and performing sealed fermentation; wherein the content of Lactobacillus plantarum in the bacterial liquid is 1×10 8 -1×10 10 CFU / mL.

3. The process according to claim 2, characterized in that The material-liquid ratio of purslane and water is 1g:1mL or 1g:2mL.

4. The process according to claim 2 or 3, characterized in that Fermentation conditions: ferment at 37℃ for 14-16 days.

5. A fermented purslane product prepared according to the process of any one of claims 1 to 4.

6. Any of the following uses of the fermented purslane product according to claim 5: (1) Used in the preparation of feed additives; (2) Used for livestock and poultry breeding; (3) Used in the preparation of antioxidants; (4) Used in the preparation of antibacterial agents; (5) Used in food processing; (6) Used in the cosmetics industry.

7. The use according to claim 6, characterized in that 4) The antibacterial agents mentioned above can inhibit the growth of Salmonella ( Salmonella ) and Escherichia coli ( Escherichia coli ).

8. An antioxidant or antibacterial agent, characterized in that The active ingredient is the water extract or alcohol extract of the purslane fermented product according to claim 5, preferably the water extract.

9. Any of the following applications of the process according to any one of claims 1 to 4: (1) To improve the composition of alkaloids, phenolic acids and flavonoids in fermented purslane; (2) Used to increase the content of calycosin, acacetin, kaempferol, apigenin, naringenin, dihydroferulic acid, dihydrocaffeic acid, and jujube saponin in fermented purslane.

10. The use according to claim 9, characterized in that The flavonoid compounds include flavonoid aglycones.

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