CN115944085A

CN115944085A - Functional probiotic composition and quantitative adding device

Info

Publication number: CN115944085A
Application number: CN202211576071.7A
Authority: CN
Inventors: 晏卫东
Original assignee: Shandong Aobosen Biological Pharmaceutical Co ltd
Current assignee: Shandong Aobosen Biological Pharmaceutical Co ltd
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2023-04-11

Abstract

The invention provides a functional probiotic composition and a quantitative adding device, and relates to the technical field of functional foods. The functional probiotic composition comprises dihydroquercetin, a ginkgo biloba extract, lactobacillus reuteri, bifidobacterium adolescentis, lactobacillus rhamnosus, vitamin C, reduced glutathione, phosphatidylserine and gamma-aminobutyric acid. The active oxygen is eliminated and the oxidative stress is relieved through the composition of all the components, so that the active oxygen can well relieve atherosclerosis, diabetes and Alzheimer's disease.

Description

Functional probiotic composition and quantitative adding device

Technical Field

The invention relates to the technical field of functional foods, in particular to a functional probiotic composition and a quantitative adding device.

Background

It is generally accepted that the decline in cognitive function is a natural component of the aging process. However, given the increased overall health and life expectancy of people, coupled with the increasing demand in workplaces, these aging subjects are desirous of maintaining a high degree of cognitive function in addition to maintaining physical performance. Individuals with cognitive decline can be further divided into two groups, one group is age-related memory loss group, and the group is benign in this sense; another group of people, whose memory or other cognitive symptoms are the first clinical manifestations of dementia of the alzheimer's type (alzheimer's) or another type of dementia (i.e. mild cognitive impairment, MCI).

The prevalence and incidence of Alzheimer's Disease (AD) is expected to rise dramatically in the next 30 years. In 2010, over 500 million patients in the united states were affected by alzheimer's disease, and by 2050 this figure is expected to surge to 1600 million, with costs for this disease in the united states exceeding 1400 billion dollars per year. On a global scale, it is estimated that 3560 million people suffer from dementia, and this figure will reach 6570 million in 2030 and 1 hundred million 1540 million in 2050. Therefore, not only does the current status of Alzheimer's disease and dementia place a tremendous economic and social burden, but these burdens are expected to increase dramatically

The etiology of cognitive decline, the appearance of Mild Cognitive Impairment (MCI) and the progression to Alzheimer's Disease (AD) is complex, multifaceted and, to date, not fully understood. It is generally accepted that the accumulation of oxidative damage may begin at the pre-symptomatic stage, i.e. MCI, and that the progression to alzheimer's disease may be associated with a loss of antioxidant defense. Reactive oxygen species and reactive nitrogen species (ROS and RNS) are formed in normal metabolism and are present in increased levels in pathological situations, resulting in oxidation of amino acid residues in proteins to form protein carbonyls. Abnormalities in glucose or insulin metabolism may be reflected in the formation of glycosylation end products, which may have physiological effects on the brain. Insulin is a powerful hormone in the brain and may also exert potentially beneficial and protective effects on cognitive function. Insulin resistance refers to a decrease in the sensitivity of the target tissue to the beneficial effects of insulin production, and is associated with a variety of chronic diseases that may affect cognition and increase the risk of dementia. The incidence of alzheimer's disease and other cognitive disorders will continue to increase exponentially as insulin resistance-related diseases reach epidemic levels.

In addition, the powdery raw materials in the prior art are usually weighed and then stirred and mixed, so that the adding speed is not controllable, and in addition, part of ingredients are brought by wind along with the stirring effect, so that the effective ingredients enter the air, and ingredient loss is caused.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a functional probiotic composition and a quantitative adding device, and solves the problems that the prior art needs medicines for treating and/or preventing aging, the powdery raw materials are brought by wind to cause raw material loss and the adding speed is uncontrollable.

(II) technical scheme

In order to realize the purpose, the invention is realized by the following technical scheme: a functional probiotic composition comprises dihydroquercetin, semen Ginkgo extract, lactobacillus reuteri, bifidobacterium adolescentis, lactobacillus rhamnosus, vitamin C, reduced glutathione, phosphatidylserine and gamma-aminobutyric acid.

Preferably, the dihydroquercetin, the ginkgo biloba extract, lactobacillus reuteri, bifidobacterium adolescentis, lactobacillus rhamnosus, vitamin C, reduced glutathione, phosphatidylserine and gamma-aminobutyric acid are in a corresponding mass ratio of: 4:1.5:2:1:1:2:1:2:3.

Preferably, one or more pharmaceutically acceptable excipients or carriers are also included.

Preferably, the probiotic composition is applied to a medicament for treating and/or preventing aging.

Preferably, the dihydroquercetin is replaced by cherry fruit powder, and the reduced glutathione is replaced by any one of glutamic acid, cysteine and glycine.

A quantitative adding device for a functional probiotic composition is used for adding the probiotic composition and comprises a machine head, wherein a plurality of charging barrels with electromagnetic valves are arranged on the periphery of the upper side face of the machine head, a quantitative cavity and a blanking cavity which are vertically distributed and communicated are arranged in the machine head, a motor and a lifting capacity adjusting structure are arranged on the upper portion of the machine head, a connecting shaft is fixedly connected to the output end of the motor, and a push plate is fixedly connected to the lower end of the connecting shaft in the machine head;

an inner cavity is formed in the machine head, the blanking cavity is communicated with the inner cavity, an inclined guide plate inclined towards the outer side of the machine head is fixed at the bottom of the blanking cavity, and an inner cavity shell is fixedly connected to the upper inner wall of the inner cavity and the edge of the lower end of the inclined guide plate;

the lifting capacity adjusting structure comprises an electric push rod, a pressing frame, an inner connecting shell, an inner tube, an inner shell, a filling sleeve and a top head, wherein the output end of the electric push rod is fixedly connected with the pressing frame, the pressing frame vertically penetrates through a machine head to downwards extend into the inner side of the inner cavity shell and is fixedly connected with the inner connecting shell, the outer side of the inner connecting shell is connected with a liquid nitrogen tank through a conveying pipe, one end of the inner tube is fixed with the inner connecting shell, the other end of the inner tube vertically upwards penetrates through an inclined guide plate and extends into the inner shell, an inner limiting ring is fixedly connected to the end part of the inner connecting shell in a sliding mode, a spherical elastic head is fixedly connected to the upper end of the inner shell, the upper end of the top head is fixedly connected with the inner wall of the elastic head, the filling sleeve is arranged on the outer side of the lower portion of the inner shell, and the lower end of the inner shell is limited to the inner side of a blanking cavity;

the quantitative cavity is upwards connected with the charging barrel, the upper part of the quantitative cavity is fixedly connected with a silica gel pad and a thin steel sheet which are distributed up and down, through holes are densely distributed at the outer edge of the silica gel pad, a guide pipe inserted into the through holes is fixedly connected onto the thin steel sheet, the elastic head is upwards fixedly connected with the thin steel sheet, and the middle part of the thin steel sheet is of a convex structure;

the utility model discloses a vibrating reed, including the aircraft nose, the lateral surface of aircraft nose just is located transition department fixedly connected with in ration chamber and unloading chamber and goes up the backup pad, go up the downside fixedly connected with graphite alkene pad of backup pad, the inboard of graphite alkene pad is provided with the trembler, the vibrator of trembler is pushed down to the downside side fixedly connected with of graphite alkene pad, the upper portion medial surface department that the trembler level passed the unloading chamber in the aircraft nose, the middle part of trembler is in unsettled state in the aircraft nose.

Preferably, the lower part of the machine head is conical, the two sides of one end, away from the connecting shaft, of the push plate are fixedly connected with conical strips, and the conical strips and the push plate are in contact with the conical inner wall of the machine head.

Preferably, the inner chamber shell is the toper for upper and lower part, and the bellied setting that makes progress in bottom, the connecting axle rotates with the bellying that makes progress of inner chamber shell to be connected, and the bellying union coupling that makes progress of inner chamber shell has the air exhauster, the push pedal is located the below of inner chamber shell.

Preferably, the conveyer pipe is made by wire hose and nonrust steel pipe, wire hose and nonrust steel pipe are connected, wire hose's the other end fixed connection link shell in, the liquid nitrogen container is connected to the nonrust steel pipe other end.

Preferably, the connecting shaft penetrates through the middle part of the inner connecting shell, and the inner connecting shell is positioned on the inner side of the inner cavity shell.

Preferably, the outside fixedly connected with bottom suspension fagging of aircraft nose toper part, fixedly connected with bracing piece between bottom suspension fagging and the last backup pad, the lower extreme department of aircraft nose toper part is the stub bar.

(III) advantageous effects

The invention provides a functional probiotic composition and a quantitative adding device. The method has the following beneficial effects:

1. according to the invention, the active oxygen scavenging and oxidative stress relieving are realized through the components, so that the active oxygen scavenging and oxidative stress relieving effects on atherosclerosis, diabetes and Alzheimer's disease are well relieved.

2. According to the invention, the purpose of controllable adding amount is realized through the quantitative adding device, compared with the method of weighing firstly and then mixing, the method can control the mixing speed, and the loss of effective components caused by the fact that the components enter the air under the action of wind brought by the mixing equipment during mixing is avoided.

Drawings

FIG. 1 is a chart showing that composition 1 detected by the L-012 probe in the invention relieves oxidative stress injury of nerve cells of an aged mouse;

FIG. 2 is a graph showing the comparison of the composition 1 for alleviating oxidative stress injury in aged mice according to the animal in vivo imaging test;

FIG. 3 is a chart showing that the L-012 probe of the invention detects the remission of the composition 2 on the oxidative stress injury of the nerve cells of the aged mice;

FIG. 4 is a graph showing the composition 2 for alleviating oxidative stress injury in aged mice according to the animal in vivo imaging test;

FIG. 5 is a chart showing that the L-012 probe of the invention detects that composition 3 relieves oxidative stress injury of nerve cells of an aged mouse;

FIG. 6 is a graph showing the comparison of the composition 3 for alleviating oxidative stress injury in aged mice according to the present invention;

FIG. 7 is a graph showing the comparison of the

composition

1, 2, 3 (from left to right) in the animal living body imaging experiment according to the present invention for alleviating oxidative stress injury in aged mice;

FIG. 8 is a first schematic view of a quantitative adding device according to the present invention;

FIG. 9 is a schematic structural view of a quantitative adding device according to the present invention;

FIG. 10 is a third schematic view of the quantitative adding device of the present invention;

FIG. 11 is a cross-sectional view of the dosing device of the present invention;

FIG. 12 is an enlarged view taken at A in FIG. 11;

FIG. 13 is a cross-sectional view of the dosing device of the present invention at the inner housing;

FIG. 14 is an enlarged view at B of FIG. 11;

FIG. 15 is a schematic view of the structure of the pushing plate of the quantitative adding device of the present invention.

Wherein, 1, a machine head; 2. a lower support plate; 3. a controller; 4. an exhaust fan; 5. an upper support plate; 6. a charging barrel; 7. an electric push rod; 8. a motor; 9. a vibrator; 10. a support bar; 11. a delivery pipe; 12. a liquid nitrogen tank; 13. discharging a stub bar; 14. pressing a frame; 15. a graphene pad; 16. a vibrating piece; 17. a dosing chamber; 18. a blanking cavity; 19. a tapered sleeve; 20. an inclined guide plate; 21. an inner cavity shell; 22. pushing a plate; 23. a connecting shaft; 24. an inner connecting shell; 25. a silica gel pad; 26. a conduit; 27. a thin steel sheet; 28. an elastic head; 29. ejecting the head; 30. an inner limiting ring; 31. an inner tube; 32. an inner shell; 33. filling the sleeve; 34. a support frame.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows:

as shown in fig. 1 to 6, the embodiment of the present invention provides a functional probiotic composition, including dihydroquercetin, a ginkgo biloba extract, lactobacillus reuteri, bifidobacterium adolescentis, lactobacillus rhamnosus, vitamin C, reduced glutathione, phosphatidylserine, and γ -aminobutyric acid, in a mass ratio: 4:1.5:2:1:1:2:1:2:3.

Also comprises one or more pharmaceutically acceptable excipients or carriers.

The probiotic composition is applied to the medicines for treating and/or preventing aging.

Detecting contents of Glutathione (GSH), malondialdehyde (MDA) and superoxide dismutase (SOD) in hippocampus, serum and liver of mice supplemented with different compositions and normal saline control to obtain antioxidant effect of the composition; meanwhile, the oxidative stress condition in nerve cells and living mice is detected by an active oxygen detection probe L-012.

Composition 1

The formula is as follows: lactobacillus reuteri powder: bifidobacterium adolescentis powder: lactobacillus rhamnosus powder: vitamin C = 2.

The active bacteria cell number of each gram of lactobacillus reuteri powder is 5.5 multiplied by 10 ⁷ ；

The Bifidobacterium adolescentis powder contains active bacteria cells of 4.8 × 10/g ⁷ ；

The number of active bacteria cells contained in each gram of lactobacillus rhamnosus powder is 2.7 multiplied by 10 ⁷ ；

The purity of the vitamin C is 93 percent;

TABLE 1 Effect of composition 1 on GSH in hippocampus, serum and liver of aged mice (n = 20)

Note: * Denotes p <0.01.

TABLE 2 Effect of composition 1 on MDA in hippocampus, serum and liver of aged mice (n = 20)

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Note: * Representing p <0.05.

TABLE 3 Effect of composition 1 on SOD in hippocampus, serum and liver of aging mice (n = 20)

Note: * Denotes p <0.01.

The L-012 probe in figure 1 detects that the chemiluminescence brightness and intensity are in positive correlation with the intracellular active oxygen level in the graph of the composition 1 for relieving the oxidative stress injury of the nerve cells of the aged mice (group 1, namely composition 1 experiment group), and the result shows that the composition 1 obviously reduces the active oxygen level in the nerve cells of the mice.

FIG. 2 animal in vivo imaging experiment to detect the remission of oxidative stress injury in aged mice by composition 1 (left: control group; right: group 1). In an animal living body imaging experiment, the fluorescence intensity shows the level of active oxygen in a mouse, and the result shows that the composition 1 can relieve the oxidative stress reaction in the mouse and eliminate the active oxygen.

Composition 2

The formula is as follows: and (3) ginkgo extract: lactobacillus reuteri: bifidobacterium adolescentis: lactobacillus rhamnosus: vitamin C: reduced glutathione = 1.5;

the purity of the ginkgo extract is 98 percent;

the purity of reduced glutathione is 99 percent;

table 4 effect of composition 2 on GSH in hippocampus, serum and liver of aged mice (n = 20)

Note: * Denotes p <0.05, indicates p <0.01. Table 5 effect of composition 2 on MDA in hippocampus, serum and liver of aged mice (n = 20)

Note: * Denotes p <0.01.

TABLE 6 Effect of composition 2 on SOD in hippocampus, serum and liver of aged mice (n = 20)

Note: * Denotes p <0.01, denotes p <0.001.

FIG. 3L-012 Probe test composition 2 for relief of oxidative stress injury to neurons in aged mice (group 2, composition 2 test group). The intensity and intensity of chemiluminescence was positively correlated with the level of reactive oxygen species in the cells, indicating that composition 2 significantly reduced the level of reactive oxygen species in mouse nerve cells.

FIG. 4 animal in vivo imaging experiment for detecting the remission of oxidative stress injury in aged mice by composition 2 (left: control group; right: subgroup 2). In an animal living body imaging experiment, the fluorescence intensity shows the level of active oxygen in a mouse, and the result shows that the composition 2 can obviously relieve the oxidative stress reaction in the mouse and eliminate the active oxygen.

Composition 3

The formula is as follows: dihydroquercetin: and (3) ginkgo extract: lactobacillus reuteri: bifidobacterium adolescentis: lactobacillus rhamnosus: vitamin C: reduced glutathione: phosphatidylserine: γ -aminobutyric acid = 4;

the purity of the dihydroquercetin is 98 percent;

the purity of the phosphatidylserine is 99 percent;

table 7. Effect of composition 3 on GSH in hippocampus, serum and liver of aged mice (n = 20)

Note: * Denotes p <0.001.

Table 8. Effect of composition 3 on MDA in hippocampus, serum and liver of aged mice (n = 20)

Note: * Denotes p <0.01, denotes p <0.001.

TABLE 9 Effect of composition 3 on SOD in hippocampus, serum and liver of aged mice (n = 20)

Note: * Denotes p <0.001.

FIG. 5L-012 Probe test composition 3 for its alleviation of oxidative stress injury to neurons in aging mice (group 3, composition 3 test group). The intensity and intensity of chemiluminescence was positively correlated with the level of reactive oxygen species in the cells, indicating that composition 3 significantly reduced the level of reactive oxygen species in mouse nerve cells.

FIG. 6 animal in vivo imaging experiment to detect the remission of oxidative stress injury in aged mice by composition 3 (right: control group; left: group 3). In an animal living body imaging experiment, the fluorescence intensity shows the level of active oxygen in a mouse body, and the result shows that the composition 3 can effectively relieve the oxidative stress reaction in the mouse body and eliminate the active oxygen.

Figure 7 in vivo animal imaging experiments tested the relief of oxidative stress injury in aged mice by

compositions

1, 2, 3 (left to right). In vivo animal imaging experiments, the fluorescence intensity shows the active oxygen level in mice.

And (4) conclusion:

lipid peroxidation is the degradation of lipids, which occurs as a result of oxidative damage, and is a useful marker of oxidative stress. Polyunsaturated lipids are susceptible to oxidative attack, usually reactive oxygen species, leading to a well-defined chain reaction with the final product, such as Malondialdehyde (MDA). Lipid peroxidation can lead to the pathology of a number of diseases including atherosclerosis, diabetes and alzheimer's disease.

Glutathione is an important antioxidant in the body and can remove free radicals in the human body; since GSH is easily oxidized by some substances, it can protect sulfydryl in molecules such as many proteins and enzymes from being oxidized by harmful substances in vivo, thereby ensuring normal exertion of physiological functions of molecules such as proteins and enzymes; the content of glutathione in human erythrocytes is large, which has important significance for protecting the sulfhydryl of protein on erythrocyte membrane in reduction state and preventing hemolysis.

Superoxide dismutase (SOD) is an important free radical-superoxide radical O in vivo ² The natural scavenger of (a), being the main defense enzyme against free radical damage.

Experiments show that the composition 1 has an intervention effect on three indexes selected from hippocampus, serum and liver, but the intervention effect is not strong, and the composition 1 mainly acts on MDA and GSH of liver, but has a strong SOD regulation effect on hippocampus. Compared with the control group, the green fluorescence intensity of the composition 1 cell is weaker, which indicates that the Reactive Oxygen Species (ROS) level in the composition 1 cell is more stable, the ROS is less than that in the control cell, and the oxidative stress state is relieved. In vivo imaging experiments, the fluorescence intensity of the gavage composition 1 mice in vivo is significantly lower than that of the control group, and active oxygen scavenging and oxidative stress relieving are visually displayed on an individual level.

The composition 2 has intervention effect on three indexes of each tissue of a mouse, especially SOD, which shows that the composition can effectively relieve oxidative stress injury, thereby delaying senility. The green fluorescence intensity of the composition 2 cells is weaker compared with that of the control group, which indicates that the Reactive Oxygen Species (ROS) level in the composition 2 cells is stable, the ROS are less than that of the control group cells, and the oxidative stress state is relieved. In vivo imaging experiments, the fluorescence intensity of the gavage composition 2 in mice is obviously lower than that of a control group, active oxygen scavenging and oxidative stress relieving are intuitively displayed on an individual level, and free radicals in the mice are more obviously eliminated.

Compared with the first two compositions, the composition 3 has more obvious effect, shows obvious improvement on MDA, GSH and SOD in hippocampus, serum and liver, and has weaker green fluorescence intensity of cells of the composition 3 compared with a control group, which shows that the level of Reactive Oxygen Species (ROS) in the cells of the composition 3 is more stable, the ROS is less than that of the cells of the control group, and the oxidative stress state is relieved. In vivo imaging experiments, the fluorescence intensity of the gavage composition 3 mice in vivo is significantly lower than that of a control group, active oxygen scavenging and oxidative stress relieving are intuitively displayed on an individual level, and meanwhile, significant differences are found by comparing the in vivo oxidative stress levels of the three groups of compositions, specifically the composition 3

The effect is obviously better than that of the composition 2 and is obviously better than that of the composition 1.

The enzyme activity measuring kit adopts a spectrophotometry method, and uses a Glutathione (GSH) and Malondialdehyde (MDA) superoxide dismutase (SOD) activity detecting kit to measure the activity of GSH, MDA and SOD in hippocampus, serum and liver of mice.

GSH is the most important anti-oxidative sulfhydryl substance in cells, and plays an important role in anti-oxidation, protein sulfhydryl protection, amino acid transmembrane transport and the like. The reduced to oxidized ratio (GSH/GSSG) is the primary dynamic indicator of the redox state of a cell. Therefore, the content of GSH and GSSG and the ratio of GSHIGSSG in the cells are measured, and the redox state of the cells can be well reflected;

oxygen free radicals act on unsaturated fatty acids of lipids to generate lipid peroxides; the latter gradually decomposes into a series of complex compounds including Malondialdehyde (MDA). The level of lipid oxidation can be measured by measuring the level of MDA. Malondialdehyde (MDA) can be condensed with thiobarbituric acid (TBA) under acidic and high temperature conditions to produce a reddish-brown trimethylnaphthalene (3, 5-trimethyloxazole-2, 4-dione) having a maximum absorption wavelength of 532nm. The amount of MDA in the sample can be estimated after the colorimetric assay.

Chemiluminescence is a non-invasive, cheap and highly sensitive fluorescent substitute for studying biologically complex systems (including living animals)) Generation of active oxygen. The basic principle of chemiluminescence is the measurement of light emitted as a result of a reaction between chemical agents such as luminol or fluorescein and reactive oxygen species. L-012 is a more sensitive chemical analog than luminol, and shows higher luminescence yield than luminol and fluorescein when reacting with reactive oxygen species, so that the luminol analog L-012 is applied to the detection of Reactive Oxygen Species (ROS) as a chemiluminescent probe. L-012 has been exclusively used for detecting O from NOX NADPH oxidase ₂ And show a stable signal, even distribution in mice under pro-inflammatory conditions, including bacterial infection, LPS-induced sepsis and liver disease. L-012 is reported not to react with O in vitro ₂ Direct reaction but by HRP/H ₂ O ₂ Oxidation to the corresponding radical, which is then reacted with O ₂ The reaction forms endoperoxide, which decomposes into excited luminescence, giving off an intermediate state. Thus, the single electron oxidation of L-012 to its free radical will act as an "activation" for the probe and O ₂ React and produce chemiluminescence. How this chemical reaction translates into an in vivo situation is not clear. L-012 can also be used to measure certain reactive nitrogen species, particularly peroxynitrite, in the presence of peroxynitrite-derived radicals, and therefore it is commonly used to detect Reactive Oxygen and Nitrogen (RON). Regardless of the chemical nature of the oxidizing agent, the L-012 probe can detect a one-electron oxidizing agent (or peroxide activity) in a biological system. Probe L-012 is oxidized in the presence of ROS/RON, and the emission after absorption and scattering by the tissue can be detected and quantified by IVIS spectroscopy, thereby measuring ROS levels in vivo. Experiment is carried out by

The L-012 probe detects the active oxygen in the cells, aiming at detecting the interference effect of different compositions on the oxidative stress caused by cell aging through SH.SY 5Y cells.

The frozen tube containing SH.SY 5Y cells was gently shaken in a water bath at 37 ℃ and rapidly thawed (about 2 minutes). The contents of the frozen tube were transferred to a centrifuge tube containing 9.0mL of complete medium (PRMI-1640 medium supplemented with 10% fetal bovine serum and 1% double antibody). Centrifuge at 125 Xg for 7 minutes. Resuspending the cells with complete culture medium, mixing, placing the cells in the medium, and keeping the temperatureCulturing in an incubator at 37 deg.C, 5% ₂ . Cell adherence was observed after 8 hours. SH · SY5Y cells grow as a mixture of suspension cells and adherent cells, and the cells grow into a cluster of neuroblasts with a plurality of short and fine cell processes (neurites). Changing the culture solution every 3-4 days until the cells are fully paved on the bottom of the culture dish, and carrying out cell passage: sucking out the culture solution, adding PBS for light washing twice, adding 500 mu L of pancreatin for digestion, observing under a microscope, adding the same amount of culture solution after digestion is finished to stop digestion, transferring all the liquid into a centrifugal tube, centrifuging at 125 Xg for 5 minutes, removing the culture medium with enzyme solution, re-suspending the cells, and transferring the cells into a new culture dish for continuous culture. The cells with good growth status were counted and diluted to a final cell concentration of 1X 105/mL. The diluted cells were mixed well, 100. Mu.L of the mixture was added to a 96-well plate, and the mixture was cultured in a cell incubator for 48 hours. The PBS-solubilized composition was diluted with the culture medium, the culture medium was aspirated from a 96-well plate confluent with a monolayer of cells, 100. Mu.L of the solubilized composition was added to a final concentration of 400. Mu.M, 6 replicates per group, and 100. Mu.L of complete culture medium was added to the control group, which was placed in a cell incubator for 48h. Adding L-012 (dissolved in sterile double distilled water to 20mmol/L, and diluted with 500mmol/L Tris-HCl) with final concentration of 0.5mmol/L, incubating for 30min, and observing active oxygen condition by inverted fluorescence microscope.

The method comprises the following steps that (1) in vivo imaging of a mouse is carried out, an IVIS (integrated vision system) spectrum system is initialized, and a CCD (charge coupled device) camera is waited to be cooled if necessary; (2) The exposure time is initially set to 1 second, the F/stop is set to 1 and the binning is set to medium but this should be different depending on the image quality obtained; (3) Before imaging, the abdomen of the mouse is shaved or chemically depilated under anesthesia (4) to prepare a 2mg/mL L-012 fresh solution, which is dissolved in sterile water or physiological saline and injected intraperitoneally at a dose of 25mg/kg, in a manner of 0.1mL/10g body weight; (5) Anesthetizing a mouse in an induction chamber by using an induction dose of 4-5% of isoflurane and a maintenance dose of 2% of isoflurane (an oxygen flow meter is 1000-1500 mL/min), placing the mouse in a nose cone on a heating platform in a supine position after anesthesia, and fixing the lower limb by using a black adhesive tape; (6) Images were taken at different time points starting as soon as possible after the L-012 injection and at a higher frequency (5 min after injection) when the rate of change of the optical signal was expected to be relatively fast; (7) Checking the image, drawing a signal according to time, and determining an optimal time point, optimal exposure and boxing of imaging after injection; (8) Using these settings, non-injected mice were imaged to determine if a self-illuminating background could be detected at these settings.

The second embodiment:

the difference between the present embodiment and the first embodiment is: the dihydroquercetin is replaced by fructus Pruni Pseudocerasi powder, and the reduced glutathione is replaced by any one of glutamic acid, cysteine and glycine.

Example three:

a quantitative adding device of functional probiotic composition is used for adding the probiotic composition, and comprises a machine head 1, more than nine charging barrels 6 with electromagnetic valves are arranged on the periphery of the upper side surface of the machine head 1, a cover is covered on the upper portion of the charging barrels 6, a quantitative cavity 17 and a blanking cavity 18 which are vertically distributed and communicated are arranged inside the machine head 1, the quantitative cavity 17 is positioned above the blanking cavity 18, after the electromagnetic valves on the charging barrels 6 are opened, the composition components filled inside fall into the quantitative cavity 17 downwards, a motor 8 and a lifting capacity adjusting structure are arranged on the upper portion of the machine head 1, that is, the motor 8 is fixedly connected to the middle part of the upper side of the machine head 1, the motor 8 is a low-speed motor, the rotating speed is 20-25rad \ min, the upper cavity is arranged in the machine head 1 and below the motor 8, the output end of the motor 8 is fixedly connected with the connecting shaft 23, the connecting shaft 23 is rotatably connected with the side wall of the machine head 1 above the upper cavity, the lower end of the connecting shaft 23 in the machine head 1 is fixedly connected with the push plate 22, the push plate 22 is fixedly connected to the outer side of the connecting shaft 23 at equal intervals, the thickness is 0.3-0.5mm, the lower side of the push plate 22 is flush with the lower end face of the connecting shaft 23, the lower part of the machine head 1 is conical and the taper is 1:5, two sides of one end of the push plate 22 far away from the connecting shaft 23 are fixedly connected with tapered strips, the included angle between the tapered strips and the contact lines of the push plate 22 is more than 50 degrees, namely the tangent plane of the contact line between one side of the tapered inner wall of the nose 1 far away from the connecting part of the push plate 22 and the tapered strip is clamped by more than 50 degrees with the push plate 22, so that the tapered strips and the push plate 22 can be prevented from being clamped at the connecting part of the tapered strips and the push plate 22, and the tapered strips connected with the push plate 22 and the tapered strip are contacted with the tapered inner wall of the nose 1, and the motor 8 drives the connecting shaft 23 to rotate at a low speed, so that the push plate 22 and the tapered strips connected with the push plate 22 can be driven to rotate slowly, and the area of the tapered strips and the push plate 22 towards the inner wall of the tapered part of the nose 1 is gradually reduced from top to bottom, i.e. an isosceles triangle is formed, how to efficiently push the composition raw materials attached to the tapered inner wall of the nose 1 during rotation, the tapered strips and the lower end surfaces of the push plate 22 are flat and are positioned at the center of the tapered part of the nose 1;

an inner cavity is arranged in the handpiece 1, the upper cavity is communicated with the inner cavity, the quantifying cavity 17 and the blanking cavity 18 are both positioned above the inner cavity, the connecting shaft 23 penetrates through the upper cavity downwards and extends into the inner cavity, the blanking cavity 18 is communicated with the inner cavity, the bottom of the blanking cavity 18 is fixedly provided with an inclined guide plate 20 inclined towards the outer side of the handpiece 1, the composition raw materials falling from the quantifying cavity 17 into the blanking cavity 18 can conveniently slide downwards towards the outer side of the handpiece 1 under the action of the inclined guide plate 20, the upper inner wall of the inner cavity and the edge positioned at the lower end of the inclined guide plate 20 are fixedly connected with an inner cavity shell 21, the composition raw materials sliding downwards from the inclined guide plate 20 cannot fall into the inner cavity shell 21, the upper part and the lower part of the inner cavity shell 21 are both conical, the connecting shaft 23 is arranged with an upward bulge at the bottom, the connecting shaft 23 is rotatably connected with the upward bulge of the inner cavity shell 21, the upward bulge of the inner cavity shell 21 is connected with an exhaust fan 4, the exhaust fan 22 is positioned below the inner cavity shell 21, the exhaust fan 4 is positioned outside the inner cavity 21, the lowest space between the upward bulge of the bottom of the exhaust fan 21 can reduce the air in the inner cavity, and the exhaust fan 22 is arranged in the inner cavity;

the lifting capacity adjusting structure comprises an electric push rod 7, a pressing frame 14, an inner connecting shell 24, an inner tube 31, an inner shell 32, a filling sleeve 33 and a top head 29, wherein a U-shaped supporting frame 34 is fixedly connected to the upper side surface of the machine head 1 and positioned above the motor 8, the electric push rod 7 is fixedly connected to the horizontal part of the upper part of the supporting frame 34, the output end of the electric push rod 7 is fixedly connected with the pressing frame 14, the pressing frame 14 is positioned at the inner side of the supporting frame 34, the pressing frame 14 is positioned at the outer side of the motor 8, the pressing frame 14 is U-shaped, two parallel vertical parts penetrate through the machine head 1 and downwards extend into the inner side of the inner cavity shell 21 and are fixedly connected with the inner connecting shell 24, one end of the inner tube 31 is fixed with the inner connecting shell 24, namely, the electric push rod 7 can drive the pressing frame 14 to enable the inner tube 31 connected with the inner connecting shell 24 to move up and down, the outside of interconnection shell 24 is connected with liquid nitrogen container 12 through conveyer pipe 11, after opening the valve that liquid nitrogen container 12 corresponds, nitrogen gas in liquid nitrogen container 12 can enter into conveyer pipe 11, later enter into the inside of interconnection shell 24, reenter in the inner tube 31, the other end of inner tube 31 is vertical upwards to pass oblique baffle 20 and stretch into in the inner shell 32, and end fixedly connected with inner limit ring 30, inner limit ring 30 sliding connection is in the inboard of top 29, the outside cover of inner limit ring 30 is equipped with first sealing washer, the middle part cavity of inner limit ring 30, nitrogen gas in the inner tube 31 can enter into the top 29 like this, side fixedly connected with toper sleeve 19 on the slope of oblique baffle 20, the effect through toper sleeve 19 leads to the motion of inner tube 31, the smooth and tapering of surface of toper sleeve 19 is 1:10, so that the composition raw materials can be greatly reduced from falling onto the conical sleeve 19, the conical sleeve 19 is matched with the vibration transmitted by the vibrator 9, so that the composition raw materials are basically not adhered to the conical sleeve 19, the difference between the outer diameter at the upper end of the conical sleeve 19 and the diameter of the inner pipe 31 is 0.2mm, the conical sleeve 19 is sleeved on the outer side of the inner pipe 31, the upper end of the inner shell 32 is fixedly connected with a spherical elastic head 28, the elastic head 28 and the filling sleeve 33 are both made of medical silica gel materials, when the filling sleeve 33 is arranged in the quantitative cavity 17, the size of the residual space in the quantitative cavity 17 can be changed by changing the size of the filling sleeve 33, so that the capacity in the quantitative cavity 17 can be controlled, the size of the filling sleeve 33 is designed according to the combined usage of the composition, the residual space in the quantitative cavity 17 and the density of each raw material, the filling sleeve can also be used for slowing down the speed of each raw material falling into the blanking cavity 18, the upper end of the top 29 is fixedly connected with the inner wall of the elastic head 28, the filling sleeve 33 is sleeved on the outer side of the lower part of the inner shell 32, the lower side of the inner shell 32 is limited to the inner side of the inner cavity 18, the bottom of the top 31 is in contact with the inner wall of the inner shell 29, and a second sealing ring 29 is embedded in the inner wall of the top head;

the quantitative cavity 17 is connected with the charging barrel 6 upwards, the upper part of the quantitative cavity is fixedly connected with a silica gel pad 25 and a thin steel sheet 27 which are distributed up and down, the silica gel pad 25 is made of medical silica gel materials, and the composition entering the quantitative cavity 17The raw materials fall onto the silica gel pad 25 firstly, the middle part of the silica gel pad 25 is raised upwards, through holes are densely distributed at the outer side of the raised part of the silica gel pad 25, and the arrangement characteristics of the through holes are 15 \ cm ² The thin steel sheet 27 is fixedly connected with a guide pipe 26 inserted into the through hole, the guide pipe 26 is communicated with the space above the silica gel pad 25 and below the thin steel sheet 27, the elastic head 28 is upwards fixedly connected with the thin steel sheet 27, the middle part of the thin steel sheet 27 is of a convex structure and is made of a spring steel sheet with the thickness of 0.3mm, the thin steel sheet 27 is not propped against the elastic head 28, namely when the thin steel sheet 27 is in a concave state at the middle part, the deformation of the thin steel sheet 27 is pulled down and the extrusion effect of the silica gel pad 25 is realized, so that the upper end of the tube 26 is blocked by the silica gel pad 25, and the composition raw material above the silica gel pad 25 cannot further fall in the quantitative cavity 17;

the outer side surface of the handpiece 1 is fixedly connected with an upper support plate 5 at the transition position of a quantitative cavity 17 and a blanking cavity 18, the lower side surface of the upper support plate 5 is fixedly connected with a graphene pad 15, the inner side of the graphene pad 15 is provided with a vibrating piece 16, the vibrating piece 16 is made of a spring steel sheet with the thickness of 0.5mm and the width of 2mm, the lower side surface of the graphene pad 15 is fixedly connected with a vibrator 9 for pressing the vibrating piece 16, the vibration of the vibrator 9 is reduced through the graphene pad 15 and then transmitted to the support plate 5, and then transmitted to the handpiece 1 through the support plate 5, and the vibration of the vibrator 9 is also transmitted to the vibrating piece 16, so that the vibration frequencies of the vibrating piece 16 and the graphene pad 15 are different, the vibrating piece 16 horizontally penetrates through the upper inner side surface of the blanking cavity 18 from the handpiece 1, the middle part of the vibrating piece 16 is in a suspended state in the handpiece 1, and after the lower part of the inner shell 32 correspondingly limited in the blanking cavity 18 moves downwards to contact with the vibrating piece 16, the vibration of the inner shell 32 is transmitted to the inner shell 32, so that the composition raw material adhered to the surface of the elastic head 28 is further vibrated, and the residual on the surface of the inner shell 32 and the elastic head 28 is reduced.

Conveyer pipe 11 is made by steel wire hose and stainless steel pipe, steel wire hose and stainless steel pipe are connected, steel wire hose's the even shell 24 of other end fixed connection, liquid nitrogen container 12 is connected to the stainless steel pipe other end, liquid nitrogen container 12 is located the outside of aircraft nose 1, stainless steel pipe passes aircraft nose 1 and stretches into in inner chamber shell 21 at the lateral wall in the inner chamber outside, steel wire hose is located the inboard in inner chamber shell 21, can guarantee even shell 24 and press the nitrogen gas of being connected with stainless steel pipe when driving from top to bottom of frame 14 to pass through and can not be obstructed.

The connecting shaft 23 passes through the middle of the inner connecting shell 24 and is rotatably connected with the inner connecting shell 24, and the inner connecting shell 24 is positioned on the inner side of the inner cavity shell 21.

Outside fixedly connected with bottom suspension fagging 2 of 1 toper part of aircraft nose, fixedly connected with bracing piece 10 between bottom suspension fagging 2 and the last backup pad 5, the bight department of bottom suspension fagging 2 is provided with the mounting hole, be convenient for be connected with the processing equipment on next step of combination medicine, for example, the chewable tablet pelleter is connected, the lower extreme department of 1 toper part of aircraft nose is ejection of compact head 13, ejection of compact head 13 intercommunication inner chamber, the side department of going up of bottom suspension fagging 2 is installed to air exhauster 4, the side department of going up of bottom suspension fagging 2 is provided with controller 3, air exhauster 4, electric putter 7, vibrator 9, the solenoid valve that feed cylinder 6 corresponds all with controller 3 electric connection.

The working principle is as follows: after raw materials of each composition are placed in each material cylinder 6 and covered with a cover, an electric push rod 7 is controlled from a controller 3 to drive a pressing frame 14 to move upwards, then the pressing frame 14 drives an inner connecting shell 24 to move upwards, so that the inner connecting shell 24 can drive an inner pipe 31 to move upwards, an inner limiting ring 30 of the inner pipe 31 moves upwards in a top head 29 in the process, then an elastic head 28 is pushed, so that the elastic head 28 drives an inner shell 32 to move upwards, the lower part of the inner shell 32 moves upwards to the limit position of a blanking cavity 18, the communication position of the blanking cavity 18 and a quantitative cavity 17 is blocked, and the elastic head 28 is pushed upwards by the top head 29 in the process, so that a guide pipe 26 corresponding to a thin steel sheet 27 extends upwards to the upper side surface of a silica gel pad 25, and the two ends of the guide pipe 26 are communicated;

then the electromagnetic valves of the exhaust fan 4, the vibrator 9 and the material cylinders 6 are controlled to be opened from the controller 3, under the vibration transmission of the vibrator 9, the raw materials of the corresponding compositions in each material cylinder 6 enter the quantitative cavity 17 through the conduit 26 and are tightly filled under the vibration, in the process, the liquid nitrogen tank 12 is opened, nitrogen enters the inner connecting shell 24 through the delivery pipe 11 and then enters the inner pipe 31 from the inner connecting shell 24, then the nitrogen goes out from the middle part of the inner limiting ring 30 to the top head 29, and then the temperature of the inner shell 32 is reduced, so that the raw materials of the compositions are tightly filled, and in addition, the raw materials of the compositions can be prevented from being deteriorated;

after the electric push rod 7 is controlled to drive the connecting structure to descend and the motor 8 is controlled to be opened, the electric push rod 7 drives the connecting structure to move downwards, the ejector 29 is further pulled downwards from the inner limiting ring 30 corresponding to the inner tube 31, the ejector 29 pulls the elastic head 28 downwards, so that a gap is exposed at the uppermost position of the blanking cavity 18 at the lower part of the inner shell 32, the composition raw materials in each quantitative cavity 17 fall into the blanking cavity 18 under the action of vibration, then the composition raw materials are guided by the inclined guide plate 20 and act on the conical part at the upper part of the inner cavity shell 21, the falling composition raw materials can slowly fall into the conical inner part of the handpiece 1, the falling composition raw materials are slowed down under the action of the filling sleeve 33, the elastic head 28 does not support the thin steel sheet 27, the elastic deformation recovery of the thin steel sheet 27 and the action of the silica gel pad 25 block the upper end of the guide tube 26, so that the raw materials above the silica gel pad 25 cannot continuously fall, the motor 8 drives the conical strip connected with the push plate 22 after the push plate 22 to rotate, the slowly falling composition raw materials are blocked at the upper end of the conical strip connected with the inner cavity, the conical strip is driven by the motor 8, the conical strip is driven by the conical shell to rotate, and the conical shell 21, the conical shell is not combined with the air extractor, and the air extractor 4, and the air extractor is combined effect of the conical shell is promoted to integrally reduced.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A functional probiotic composition, characterized by: comprises dihydroquercetin, semen Ginkgo extract, lactobacillus reuteri, bifidobacterium adolescentis, lactobacillus rhamnosus, vitamin C, reduced glutathione, phosphatidylserine and gamma-aminobutyric acid.

2. A functional probiotic composition according to claim 1, characterized in that: the corresponding mass ratio of the dihydroquercetin, the ginkgo biloba extract, the lactobacillus reuteri, the bifidobacterium adolescentis, the lactobacillus rhamnosus, the vitamin C, the reduced glutathione, the phosphatidylserine and the gamma-aminobutyric acid is as follows: 4:1.5:2:1:1:2:1:2:3.

3. A functional probiotic composition according to claim 1 or 2, characterized in that: the dihydroquercetin is replaced by cherry fruit powder, and the reduced glutathione is replaced by any one of glutamic acid, cysteine and glycine.

4. A functional probiotic composition according to claim 3, characterized in that: also comprises one or more pharmaceutically acceptable excipients or carriers.

5. A functional probiotic composition according to claims 2 or 4, characterized in that: the probiotic composition is applied to a medicament for treating and/or preventing aging.

6. A dosing device for functional probiotic compositions for adding the probiotic composition of claim 5, characterized in that: the automatic feeding machine comprises a machine head (1), wherein a plurality of charging barrels (6) with electromagnetic valves are arranged on the periphery of the upper side surface of the machine head (1), a quantitative cavity (17) and a blanking cavity (18) which are vertically distributed and communicated are arranged in the machine head (1), a motor (8) and a lifting capacity adjusting structure are arranged on the upper portion of the machine head (1), a connecting shaft (23) is fixedly connected to the output end of the motor (8), and a push plate (22) is fixedly connected to the lower end of the connecting shaft (23) in the machine head (1);

an inner cavity is formed in the machine head (1), the blanking cavity (18) is communicated with the inner cavity, an inclined guide plate (20) inclined towards the outer side of the machine head (1) is fixed at the bottom of the blanking cavity (18), and an inner cavity shell (21) is fixedly connected to the upper inner wall of the inner cavity and the edge of the lower end of the inclined guide plate (20);

the lifting capacity adjusting structure comprises an electric push rod (7), a pressing frame (14), an inner connecting shell (24), an inner pipe (31), an inner shell (32), a filling sleeve (33) and a top head (29), wherein the output end of the electric push rod (7) is fixedly connected with the pressing frame (14), the pressing frame (14) vertically penetrates through a machine head (1) and downwards extends into the inner side of an inner cavity shell (21) and is fixedly connected with the inner connecting shell (24), the outer side of the inner connecting shell (24) is connected with a liquid nitrogen tank (12) through a conveying pipe (11), one end of the inner pipe (31) is fixed with the inner connecting shell (24), the other end of the inner pipe vertically upwards penetrates through an inclined guide plate (20) and extends into the inner shell (32), an end part of the inner connecting shell is fixedly connected with an inner limiting ring (30), the inner limiting ring (30) is slidably connected to the inner side of the top head (29), the upper end of the inner shell (32) is fixedly connected with a spherical elastic head (28), the upper end of the top head (29) is fixedly connected with the inner wall of the elastic head (28), the inner shell (33) is sleeved on the outer side of the inner shell (32), and the lower end of a blanking cavity (18) is limited at the lower end of the inner side of the inner shell (32);

the quantitative cavity (17) is upwards connected with the charging barrel (6), the upper part of the quantitative cavity is fixedly connected with a silica gel pad (25) and a thin steel sheet (27) which are vertically distributed, through holes are densely distributed at the outer edge of the silica gel pad (25), a guide pipe (26) inserted into the through holes is fixedly connected onto the thin steel sheet (27), the elastic head (28) is upwards fixedly connected with the thin steel sheet (27), and the middle part of the thin steel sheet (27) is of a convex structure;

the utility model discloses a vibrating plate, including the transition department fixedly connected with in ration chamber (17) and unloading chamber (18) of lateral surface of aircraft nose (1), go up backup pad (5), the downside fixedly connected with graphite alkene pad (15) of going up backup pad (5), the inboard of graphite alkene pad (15) is provided with trembler (16), the downside side fixedly connected with of graphite alkene pad (15) pushes down vibrator (9) of trembler (16), trembler (16) are from the upper portion medial surface department that the level passed unloading chamber (18) in aircraft nose (1), the middle part of trembler (16) is in unsettled state in aircraft nose (1).

7. A dosing device of a functional probiotic composition according to claim 6, characterized in that: the lower part of aircraft nose (1) is the toper, both sides fixedly connected with toper strip of the one end of connecting axle (23) is kept away from in push pedal (22), toper strip and push pedal (22) all contact with the toper inner wall of aircraft nose (1).

8. A dosing device of a functional probiotic composition according to claim 6, characterized in that: inner chamber shell (21) are the toper for upper and lower part, and the bellied setting that makes progress in bottom, connecting axle (23) rotate with the bellying that makes progress of inner chamber shell (21) and are connected, and the bellying union coupling that makes progress of inner chamber shell (21) has air exhauster (4), push pedal (22) are located the below of inner chamber shell (21).

9. The dosing device of a functional probiotic composition, according to claim 6, characterized in that: conveyer pipe (11) are made by wire hose and stainless steel pipe, wire hose and stainless steel pipe are connected, even shell (24) in wire hose's the other end fixed connection, liquid nitrogen container (12) are connected to the stainless steel pipe other end, connecting axle (23) are passed from the middle part of interconnection shell (24), even shell (24) are located the inboard of inner chamber shell (21).

10. A dosing device of a functional probiotic composition according to claim 9, characterised in that: the outer side of the conical part of the machine head (1) is fixedly connected with a lower supporting plate (2), a supporting rod (10) is fixedly connected between the lower supporting plate (2) and the upper supporting plate (5), and the lower end of the conical part of the machine head (1) is provided with a discharging head (13).