A kind of bifidobacterium fermentation method and application thereof

Abstract

The present invention relates to the field of microbial technology, and in particular to a fermentation method of bifidobacteria and its application. The method comprises adding 1 to 50 mg / L of edible plant-derived extracellular vesicles to the fermentation medium of bifidobacteria, and simultaneously adding 1 to 20 μg / ml of selenium. The present invention finds that adding edible plant-derived extracellular vesicles to the starter of bifidobacteria can significantly increase the yield of bifidobacterium extracellular vesicles, and adding selenium can effectively improve the antioxidant capacity of bifidobacterium extracellular vesicles, and finally makes bifidobacterium extracellular vesicles have a higher probiotic potential of increasing intestinal barrier function and delaying aging.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a fermentation method of bifidobacteria and application thereof. Background Art

[0002] Bifidobacterium is a common type of bacteria in the human intestine. It is a strictly anaerobic Gram-positive bacteria. It accounts for about 10% of the total bacteria in the intestine of adults. It is the most dominant type of bacteria in the intestine of infants, accounting for about 91% of the intestinal bacteria of breastfed infants and 75% of the intestinal bacteria of infants fed with milk powder. The difference between bifidobacterium and other lactic acid bacteria is that it can metabolize glucose using a unique 6-phosphate-fructose phosphoketolase pathway, and the GC content of its DNA is significantly higher than that of other lactic acid bacteria. Bifidobacterium (BFB), as a predominant probiotic colonizing in the intestine of healthy people, has a series of health functions such as immune regulation, anti-tumor, antibacterial and anti-inflammatory, anti-aging and lipid-lowering, and is closely related to many pathological and physiological phenomena.

[0003] Bifidobacterium is one of the most important physiological bacteria in the intestines of humans and animals and is the focus of current microecological research. Traditional methods for studying bifidobacteria are mainly based on isolation and culture. Studies by Moore and Holdman et al. found that bifidobacteria are present in high levels in human fecal samples. The bifidobacteria that can be isolated usually include: Bifidobacteriumadolescents, Bifidobacteriuminfantes and Bifidobacterium longum. Satokari et al. used group-specific primers combined with the DGGE method to analyze the composition of bifidobacteria in human fecal samples. The results showed that the composition of bifidobacteria is individual-specific and remains stable during a 4-month dynamic monitoring period. B. adolescentis is the most common type of bifidobacteria in human fecal samples. According to literature reports, the main bifidobacteria associated with humans are the following 12 species: Bifidobacterium adolescentis (B.adolescentis), Bifidobacterium infantis (B.infantis), Bifidobacterium longum (B.longum), Bifidobacterium bifidum (B.bifidum), Bifidobacterium breve (B.breve), Bifidobacterium catenulatum (B.catenulatum), Bifidobacterium pseudocatenulatum (B.pseudocatenulatum), Bifidobacterium angulatum (B.angulatum), Bifidobacterium gallicum (B.gallicum), Bifidobacterium inopinatum (B.inopinatum), Bifidobacterium dentium (B.denticolens) and Bifidobacterium denticolens (B.denticolens). The latter three are mainly found in the oral cavity.

[0004] At present, out of consideration for the safety of live probiotics, more and more studies have begun to focus on the beneficial effects of inactive probiotic components on the human body, such as probiotic wall components, heat-inactivated probiotic bodies, probiotic metabolites, etc. These beneficial substances are called "postbiotics". As studies have found that Gram-positive bacteria can also secrete extracellular vesicles under specific culture environments and conditions, more and more studies have begun to focus on the extracellular vesicles produced by probiotics such as Bifidobacterium, which are also a type of "postbiotics". Existing studies have shown that extracellular vesicles play an important role in the communication between bacteria and between bacteria and cells. Bifidobacterium extracellular vesicles are also widely used in food microbiology, food science, functional foods, health products and pharmaceuticals. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention provides a fermentation method of bifidobacterium and application thereof, and the yield of bifidobacterium extracellular vesicles can be effectively increased by adding edible plant-derived extracellular vesicles to the basic culture starter of bifidobacterium.

[0006] In a first aspect, the present invention provides a fermentation method for increasing the production of extracellular vesicles of bifidobacteria, comprising: adding 1-50 mg / L of edible plant-derived extracellular vesicles into a fermentation medium of bifidobacteria.

[0007] Furthermore, the edible plant-derived extracellular vesicles include: carrot-derived extracellular vesicles and / or tomato-derived extracellular vesicles.

[0008] Furthermore, it also includes a selenium ion salt; the selenium ion salt is preferably sodium selenite.

[0009] Furthermore, the added amount of the selenium ion salt is 1 to 20 μg / ml.

[0010] Furthermore, the content of the edible carrot-derived extracellular vesicles is 5-20 mg / L; and / or, the tomato-derived extracellular vesicles are 10-30 mg / ml. Furthermore, these two types of vesicles can be used at the same time, for example, 20 mg / ml carrot-derived extracellular vesicles + 30 mg / ml tomato-derived extracellular vesicles.

[0011] Furthermore, the basic culture starter comprises, by weight: 10 to 30 parts of glucose, 5 to 20 parts of peptone, 5 to 20 parts of beef extract, 1 to 10 parts of yeast extract, 1 to 10 parts of sodium acetate, 1 to 5 parts of disodium hydrogen phosphate, 1 to 5 parts of diammonium hydrogen citrate, 0.1 to 2 parts of Tween-80, 0.1 to 1 part of anhydrous magnesium sulfate, 0.1 to 1 part of manganese sulfate monohydrate and 0.1 to 1 part of L-cysteine ​​hydrochloride.

[0012] In a second aspect, the present invention provides a starter additive, comprising: edible plant-derived extracellular vesicles and selenium ion salts.

[0013] Furthermore, the edible plant-derived extracellular vesicles include: carrot-derived extracellular vesicles and / or tomato-derived extracellular vesicles; the content of the edible plant-derived extracellular vesicles is 1-50 mg / L, and the content of the selenium ion salt is 1-20 μg / ml.

[0014] Furthermore, the content of the edible carrot-derived extracellular vesicles is 5-20 mg / L; and / or, the content of the tomato-derived extracellular vesicles is 10-30 mg / ml; and / or, the content of the selenium ion salt is 5-20 μg / ml.

[0015] In a third aspect, the present invention provides a method for improving the antioxidant capacity of extracellular vesicles of bifidobacteria, comprising: adding the starter additive to the fermentation medium of bifidobacteria.

[0016] The present invention further provides the fermentation method, or the use of the fermentation agent additive in increasing the yield of bifidobacterium extracellular vesicles.

[0017] The present invention further provides the fermentation method, or the use of the fermentation agent additive in improving the antioxidant activity of bifidobacterium extracellular vesicles.

[0018] The present invention further provides the fermentation method, or the use of the fermentation agent additive in improving the anti-aging ability.

[0019] The present invention has the following beneficial effects:

[0020] The present invention specifically adds growth-promoting factors, edible plants-carrot and / or tomato-derived extracellular vesicles and selenium ion salt sodium selenite to the improved culture fermentation agent. Among them, the edible plants-carrot and / or tomato-derived extracellular vesicles can significantly increase the yield of bifidobacterium extracellular vesicles, while sodium selenite and edible plant-derived extracellular vesicles (carrot and / or tomato-derived extracellular vesicles) can enrich more selenoproteins in the bifidobacterium extracellular vesicles and improve the antioxidant capacity of the bifidobacterium extracellular vesicles.

[0021] The method provided by the present invention can be used for the mass production of bifidobacterium extracellular vesicles in factories, and has important value for the application of bifidobacterium extracellular vesicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the effects of sodium selenite and edible plant-derived exogenous vesicles on the production of extracellular vesicles of Bifidobacterium provided in Example 2 of the present invention.

[0023] Figure 2This is the detection result of the bifidobacterium provided in Example 3 of the present invention binding to the extracellular vesicles derived from carrot or tomato.

[0024] Figure 3 This is a schematic diagram showing the effect of the addition amount of 0, 5, 10, 20, 30 and 50 mg / L of carrot or tomato-derived extracellular vesicles provided in Example 4 of the present invention on the yield of bifidobacterium extracellular vesicles.

[0025] Figure 4 Schematic diagram of the effect of sodium selenite and carrot or tomato derived extracellular vesicles on the anti-lipid peroxidation rate of bifidobacterium extracellular vesicles provided in Example 5 of the present invention.

[0026] Figure 5 Schematic diagram of the effect of sodium selenite and carrot or tomato derived extracellular vesicles on the DPPH radical scavenging rate of bifidobacterium extracellular vesicles provided in Example 5 of the present invention.

[0027] Figure 6 This is a schematic diagram of the scavenging DPPH free radical effect of sodium selenite at concentrations of 5, 10, 20, and 30 ug / ml on the extracellular vesicles of bifidobacteria provided in Example 5 of the present invention.

[0028] Figure 7 This is a schematic diagram of the effects of sodium selenite and plant-derived extracellular vesicles on the extracellular vesicles of Bifidobacterium longum BL in the intestinal barrier function provided by Example 6 of the present invention.

[0029] Figure 8 Schematic diagram of the effects of sodium selenite and plant-derived extracellular vesicles on the anti-aging ability of bifidobacterium extracellular vesicles provided in Example 7 of the present invention.

[0030] Fig. 9 This is a schematic diagram of the effects of sodium selenite, plant-derived carrot or tomato extracellular vesicles on the production and antioxidant effect of Lactobacillus extracellular vesicles provided in Example 8 of the present invention.

[0031] Fig.10 Schematic diagram of the effects of sodium selenite, plant-derived carrot or tomato extracellular vesicles on the production of bifidobacterium extracellular vesicles in conventional TPY medium and MRS medium provided in Example 9 of the present invention.

[0032] Fig.11 Schematic diagram of the effects of sodium selenite, plant-derived carrot or tomato extracellular vesicles on the antioxidant effect of bifidobacterium extracellular vesicles in conventional TPY medium and MRS medium provided in Example 9 of the present invention. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0034] Example 1 Preliminary screening of vesicle-producing Bifidobacterium

[0035] The present invention screens out bifidobacteria that can produce vesicles from 33 bifidobacteria existing in the laboratory, and obtains 3 bifidobacteria that can produce vesicles, wherein Bifidobacterium longum BL is taken as the research object.

[0036] Example 2 Production of Bifidobacterium Extracellular Vesicles

[0037] 1. The ratio of the improved culture starter is: 20g glucose, 10g peptone, 10g beef extract, 5g yeast extract, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 1mL Tween-80, 0.58g anhydrous magnesium sulfate, 0.25g manganese sulfate monohydrate, 0.4g L-cysteine ​​hydrochloride, add distilled water to 1L, adjust the pH to 6.5-7.0, fill the aliquots with nitrogen, and sterilize at 121℃ for 15min. In addition, add 1-50mg / L of sterile growth-promoting carrot and / or tomato extracellular vesicles and 1-20ug / ml of selenium ion salt sodium selenite.

[0038] 2. The preparation method of the above-mentioned plant-derived carrot extracellular vesicles is as follows:

[0039] (1) Homogenization: Peel and wash fresh carrots or tomatoes, put them into a high-speed blender, and homogenize for 1 minute.

[0040] (2) Filtration: Remove the carrot or tomato solids by filtering through a vacuum filter to obtain carrot or tomato juice.

[0041] (3) Centrifugation: At 4°C, the carrot or tomato juice was centrifuged at 2000 g for 10 min, 5000 g for 20 min, and 10000 g for 60 min, and then at 150000 g for 90 min. Sterile saline was added to resuspend the vesicle precipitate and stored at -80°C for later use.

[0042] 3. Preparation of Bifidobacterium Extracellular Vesicles:

[0043] (1) Fermentation: 2×10 7 The modified culture starter was inoculated with an inoculum of CFU / ml, and the modified culture starter without the sterile growth-promoting factor carrot-derived or tomato-derived extracellular vesicles and without the selenium ion salt sodium selenite was used as a control. The culture was incubated at 37°C for 48 hours until the OD 600 =1.5.

[0044] (2) Centrifugation: At 4°C, the fermentation broth was centrifuged at 5000 g for 10 min and 10000 g for 20 min. The supernatant was filtered using a 0.45 μm filter and then centrifuged at 150000 g for 90 min. Finally, sterile saline was added to resuspend the vesicle precipitate.

[0045] (3) Extracellular vesicle quantification: The vesicles were quantitatively analyzed using the NanoSight NS300 nanoparticle analyzer.

[0046] The results show that Figure 1 ): Compared with the blank control group, the addition of sodium selenite and edible plant-carrot extracellular vesicles (20 mg / L) (Se-CNPs) group, tomato-derived extracellular vesicles (30 mg / L) (Se-TNPs) group and carrot + tomato-derived extracellular vesicles (20+30 mg / L) (Se-C+TNPs) group can significantly increase the extracellular vesicle production of Bifidobacterium longum BL, which increased by 192%, 180% and 203% respectively. However, the addition of sodium selenite 20ug / ml alone had no effect on the extracellular vesicle production of Bifidobacterium longum BL compared with the blank group without sodium selenite.

[0047] Example 3: Identification of Bifidobacteria Binding to Extracellular Vesicles from Carrot or Tomato

[0048] 1. PKH26 fluorescent dye kit for fluorescent labeling of carrot or tomato derived extracellular vesicles

[0049] Add 4ul of Sigma's PKH26 fluorescent dye to 500ul of carrot or tomato extracellular vesicle solution resuspended in sterile saline, stain at room temperature in the dark for 30 minutes, then add 500ul of diluent C solution to react at room temperature in the dark for 10 minutes. Centrifuge at 13000g for 5 minutes, and add 500ul of sterile saline to resuspend the precipitate.

[0050] 2. Identification of the binding of Bifidobacterium longum BL to carrot-derived extracellular vesicles

[0051] Using 1×10 7 Each Bifidobacterium longum BL was cultured with 1 mg PKH26-labeled carrot-derived extracellular vesicles at room temperature for 30 minutes, washed twice with sterile saline, and then fluorescent images were taken under a confocal microscope.

[0052] The results show that Figure 2 ): Compared with the blank control group, the fluorescence on Bifidobacterium longum BL in the group with added PKH26-labeled carrot-derived extracellular vesicles was significantly increased, proving that carrot-derived extracellular vesicles can be taken up and combined with Bifidobacterium longum BL.

[0053] Example 4: Determination of the amount of ingredients added and the yield-increasing effect in the improved culture starter

[0054] Experiments were conducted with the addition of extracellular vesicles from carrots and / or tomatoes at a concentration of 0, 5, 10, 20, 30 and 50 mg / L, respectively, to compare the production of extracellular vesicles of Bifidobacterium longum BL.

[0055] The results show that Figure 3 ): The minimum effective addition amount of carrot-derived extracellular vesicles is 5 mg / L, and the maximum effective addition amount is 20 mg / L; the minimum effective addition amount of tomato-derived extracellular vesicles is 10 mg / L, and the maximum effective addition amount is 30 mg / L.

[0056] Example 5: Determination of the effect of plant-derived extracellular vesicles and selenium ion salt sodium selenite on the antioxidant activity of extracellular vesicles of Bifidobacterium longum BL in improved culture starter

[0057] 1. Anti-lipid peroxidation test

[0058] (1) The reaction system consists of: 1 ml of linoleic acid emulsion, 0.2 ml of FeSO4 (0.01%), 0.2 ml of H2O2 (0.02%), and 1 ml of PBS (0.02 mol / L, pH 7.4), with a total volume of 2.4 ml, wherein the linoleic acid emulsion is composed of 0.1 ml of linoleic acid, 0.2 ml of Tween 20, and 19.7 ml of deionized water.

[0059] (2) Samples were added according to the above reaction system, and the extracellular vesicles of Bifidobacterium longum BL cultured in ordinary culture starter were used as the blank control (NPs) group, and the extracellular vesicles of Bifidobacterium longum BL cultured in the improved culture starter with sodium selenite (20ug / ml) (Se-NPs) group, sodium selenite and carrot-derived extracellular vesicles (20mg / L) (Se-CNPs group), sodium selenite and tomato-derived extracellular vesicles (30mg / L) (Se-TNPs group), and sodium selenite and plant-derived extracellular vesicles (carrot-derived extracellular vesicles 20mg / L, tomato-derived extracellular vesicles 30mg / L) (Se-C+TNPs) group were used as experimental groups, and samples were added at a dose of 20ug / ml of the above-mentioned Bifidobacterium longum BL extracellular vesicles.

[0060] After reacting at 37°C for 12 hours, add 0.2 ml of trichloroacetic acid (4%), 2 ml of thiobarbituric acid (0.8%), and 0.2 ml of 2,6-di-tert-butyl-p-cresol (0.4%), mix thoroughly, react at 100°C for 30 minutes, and then cool. Finally, centrifuge, and measure the absorbance of the supernatant at 532 nm in a colorimetric dish using a spectrophotometer.

[0061] Anti-lipid peroxidation rate (%) = [1-A 532(Sample) / A 532 (blank)]×100

[0062] The results show that Figure 4 ): Adding sodium selenite can significantly increase the anti-lipid peroxidation rate of extracellular vesicles of Bifidobacterium longum BL. Adding plant-derived carrot extracellular vesicles under selenium-rich conditions can significantly enhance the anti-lipid peroxidation rate of extracellular vesicles of Bifidobacterium longum BL, but adding tomato-derived extracellular vesicles has no significant effect. Compared with the Se-NPs group, adding 20 mg / L carrot-derived extracellular vesicles and 30 mg / L tomato-derived extracellular vesicles under selenium-rich conditions can significantly increase the anti-lipid peroxidation rate.

[0063] 2. DPPH free radical scavenging ability test

[0064] The extracellular vesicles of Bifidobacterium longum BL cultured in ordinary blank culture starter were used as the blank control (NPs) group, and the extracellular vesicles of Bifidobacterium longum BL cultured in the improved culture starter with sodium selenite (20ug / ml) (Se-NPs), sodium selenite and carrot-derived extracellular vesicles (20mg / L) (Se-CNPs), sodium selenite and tomato-derived extracellular vesicles (30mg / L) (Se-TNPs), and sodium selenite and plant-derived extracellular vesicles (carrot-derived extracellular vesicles 20mg / L, tomato-derived extracellular vesicles 30mg / L) (Se-C+TNPs) were used as experimental groups.

[0065] The sample was added at a concentration of 20ug / ml of the extracellular vesicles of Bifidobacterium longum BL, and PBS was used to make up to 2.0ml, and then 3.0ml of 0.1mmol / L DPPH solution (95% ethanol) was added and mixed, and the mixture was vigorously shaken and reacted at room temperature in the dark for 30min, centrifuged, and the absorbance of the supernatant was measured at 517nm. 200μg / L of the antioxidant 2,6-di-tert-butyl-p-cresol (BHT) was used as a positive control.

[0066] DPPH free radical scavenging rate (%) = [1-A 517 (Sample) / A 517 (blank)]×100

[0067] In the formula, A 517 (Sample) is the absorbance value of the sample group after reaction; A 517 (Blank) is the absorbance value of the blank control group after reaction. The results show that ( Figure 5): Adding sodium selenite can significantly increase the DPPH radical scavenging rate of extracellular vesicles of Bifidobacterium longum BL. Adding plant-derived carrot-derived extracellular vesicles under selenium-rich conditions can enhance the DPPH radical scavenging rate of extracellular vesicles of Bifidobacterium longum BL. Compared with the Se-NPs group, the DPPH radical scavenging rate of extracellular vesicles of Bifidobacterium longum BL in the Se-C+TNPs group was significantly increased.

[0068] 3. Identification of the antioxidant capacity of extracellular vesicles of Bifidobacterium longum BL by different concentrations of sodium selenite

[0069] The extracellular vesicles of Bifidobacterium longum BL cultured in ordinary blank culture starter were used as the blank control (NPs) group, and the extracellular vesicles of Bifidobacterium longum BL cultured in improved culture starter with sodium selenite added at concentrations of 5, 10, 20, and 30 ug / ml were used as experimental groups.

[0070] The DPPH free radical scavenging experiment was measured according to the above method.

[0071] The results show that Figure 6 ): 5ug / ml sodium selenite concentration is the minimum effective addition concentration to increase the scavenging DPPH free radical effect of the extracellular vesicles of Bifidobacterium longum BL, and 20ug / ml concentration is the maximum effective addition concentration to increase the scavenging DPPH free radical effect of the extracellular vesicles of Bifidobacterium longum BL.

[0072] Example 6: Identification of the effects of adding sodium selenite and plant-derived extracellular vesicles on the extracellular vesicles of Bifidobacterium longum BL in the intestinal barrier function

[0073] 1. Determination of the transmembrane electrical resistance (TEER) of intestinal epithelial cells

[0074] (1) The extracellular vesicles of Bifidobacterium longum BL cultured in ordinary blank culture medium were used as the control (NPs) group, and the extracellular vesicles of Bifidobacterium longum BL cultured in the modified culture medium with the addition of sodium selenite (20ug / ml) (Se-NPs), sodium selenite and carrot-derived extracellular vesicles (20mg / L) (Se-CNPs), sodium selenite and tomato-derived extracellular vesicles (30mg / L) (Se-TNPs), and sodium selenite and plant-derived extracellular vesicles (carrot-derived extracellular vesicles 20mg / L, tomato-derived extracellular vesicles 30mg / L) (Se-C+TNPs) were used as experimental groups.

[0075] (2) Intestinal epithelial Caco-2 cells were selected and cultured at 2x10 5 The cells were plated at a density of 10 / ml and cultured for 2 weeks to form a monolayer. 8The above-mentioned Bifidobacterium longum BL extracellular vesicles were added to Caco-2 cells at a concentration of 100 / ml, and then the TEER resistance was measured.

[0076] The results show that Figure 7 ): Compared with the blank control (NPs) group, the addition of sodium selenite can significantly increase the TEER resistance value. Under selenium-enriched conditions, the addition of plant-derived carrot extracellular vesicles group can significantly increase the TEER resistance value of Caco-2 cells, but the addition of tomato-derived extracellular vesicles has no significant effect on the TEER resistance value of Caco-2 cells.

[0077] Example 7: Identification of the anti-aging ability of extracellular vesicles of Bifidobacterium longum BL cultured in an improved culture starter supplemented with sodium selenite and plant-derived extracellular vesicles

[0078] Experiments were conducted with Caenorhabditis elegans (C.elegans). Newly born (day 0) nematodes were cultured in OP50 dishes until maturity and then transferred to OP50 control group, OP50+extracellular vesicles of Bifidobacterium longum BL (OP50+NPs) group, OP50+extracellular vesicles of Bifidobacterium longum BL cultured in sodium selenite culture starter (OP50+Se-NPs) group, OP50+extracellular vesicles of Bifidobacterium longum BL cultured in sodium selenite+extracellular vesicles of carrot-derived culture starter (OP50+Se-CNPs) group, and OP50+extracellular vesicles of Bifidobacterium longum BL cultured in sodium selenite+extracellular vesicles of carrot and tomato-derived culture starter (OP50+Se-C+TNPs) group. Each group contained 20 nematodes and was incubated at 20°C. The worms were transferred to new dishes every other day. The number of live or dead nematodes was counted at the time of transfer each day.

[0079] Survival was calculated as the percentage of surviving nematodes / total number of nematodes. Nematodes that died due to sticking to the plate wall were excluded from the analysis. Lifespan assessment was performed at least three times.

[0080] The results show that Figure 8 ): Compared with the blank control OP50 group, the addition of Bifidobacterium longum BL extracellular vesicles (NPs) can significantly increase the maximum average lifespan of nematodes. The beneficial effect of Bifidobacterium longum BL extracellular vesicles (NPs) in increasing the maximum average lifespan of nematodes can be effectively amplified under biological selenium-enriched conditions. Compared with the (Se-NPs) group, the addition of plant-derived carrot extracellular vesicles (Se-CNPs) group and carrot + tomato extracellular vesicles (Se-C+TNPs) group can significantly prolong the maximum average lifespan of nematodes. This experiment shows that the addition of sodium selenite and plant-derived extracellular vesicles in the improved culture medium can significantly increase the effect of Bifidobacterium longum BL extracellular vesicles (NPs) on anti-aging ability.

[0081] Example 8

[0082] This example verifies the effect of adding sodium selenite, plant-derived carrot and / or tomato extracellular vesicles on the extracellular vesicle production and antioxidant effect of other bacteria

[0083] Ordinary blank culture starter was used as the blank control (LNPs) group, and Lactobacillus plantarum was cultured in the following groups: sodium selenite (20ug / ml) (Se-LNPs) group, sodium selenite and carrot-derived extracellular vesicles (20mg / L) (Se-CLNPs) group, tomato-derived extracellular vesicles (30mg / L) (Se-TLNPs) group, and carrot-derived extracellular vesicles 20mg / L + tomato-derived extracellular vesicles 30mg / L (C+TLNPs) group.

[0084] The results show that Fig. 9 ): Compared with the blank control (LNPs) group, the addition of sodium selenite, plant-derived carrot or tomato extracellular vesicles could not increase the extracellular vesicle production and antioxidant effect of Lactobacillus plantarum.

[0085] Example 9

[0086] In this example, other conventional culture media were used to add key nutrients such as sodium selenite and extracellular vesicles from plant-derived carrots and / or tomatoes to evaluate the effects of the extracellular vesicle production and antioxidant effects of Bifidobacterium longum BL

[0087] Conventional TPY medium and MRS medium were used for the experiment, which were divided into a blank control group (Control) and a group added with sodium selenite (20ug / ml) + carrot-derived extracellular vesicles (20mg / L) + tomato-derived extracellular vesicles (30mg / L) (Se+C+T). Bifidobacterium longum BL was cultured according to the above grouping, and extracellular vesicles of Bifidobacterium longum BL were obtained for identification of extracellular vesicle production and antioxidant effect.

[0088] The results show that Fig.10 and Fig.11 ): In conventional TPY medium and MRS medium, compared with the blank control (Control) group, the Se+C+T group can significantly increase the extracellular vesicle production and antioxidant effect of Bifidobacterium longum BL.

[0089] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A fermentation method for increasing the production of extracellular vesicles of bifidobacteria, characterized in that: include: Adding edible plant-derived extracellular vesicles to the fermentation medium of bifidobacteria; The bifidobacterium is Bifidobacterium longum BL; The edible plant-derived extracellular vesicles are: 5-20 mg / L of carrot-derived extracellular vesicles and / or 10-30 mg / L of tomato-derived extracellular vesicles.

2. The fermentation method according to claim 1, characterized in that The fermentation medium also includes a selenium ion salt.

3. The fermentation method according to claim 2, characterized in that The selenium ion salt is sodium selenite.

4. The fermentation method according to claim 3, characterized in that The added amount of the selenium ion salt is 1-20 μg / mL.

5. The fermentation method according to any one of claims 1 to 4, characterized in that: The fermentation medium comprises, by weight, 10 to 30 parts of glucose, 5 to 20 parts of peptone, 5 to 20 parts of beef extract, 1 to 10 parts of yeast extract, 1 to 10 parts of sodium acetate, 1 to 5 parts of disodium hydrogen phosphate, 1 to 5 parts of diammonium hydrogen citrate, 0.1 to 2 parts of Tween-80, 0.1 to 1 part of anhydrous magnesium sulfate, 0.1 to 1 part of manganese sulfate monohydrate and 0.1 to 1 part of L-cysteine ​​hydrochloride.

6. Use of the fermentation method or starter additive according to any one of claims 1 to 5 in increasing the production of extracellular vesicles of Bifidobacterium longum BL; the starter additive comprises: edible plant-derived extracellular vesicles and selenium ion salts; the amount of edible plant-derived extracellular vesicles added is: 5-20 mg / L of carrot-derived extracellular vesicles and / or 10-30 mg / L of tomato-derived extracellular vesicles.

7. Use of the fermentation method or starter additive according to any one of claims 1 to 5 in improving the antioxidant activity of extracellular vesicles of Bifidobacterium longum BL; the starter additive comprises: edible plant-derived extracellular vesicles and selenium ion salts; the added amount of the edible plant-derived extracellular vesicles is: 5-20 mg / L of carrot-derived extracellular vesicles and / or 10-30 mg / L of tomato-derived extracellular vesicles.

8. Use of the extracellular vesicles of Bifidobacterium longum BL prepared by the fermentation method according to any one of claims 1 to 5 in the preparation of a drug for improving anti-aging ability.

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