CN109055269B - Bifidobacterium longum subspecies infantis CCFM687, fermented food thereof and application thereof - Google Patents
Bifidobacterium longum subspecies infantis CCFM687, fermented food thereof and application thereof Download PDFInfo
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- CN109055269B CN109055269B CN201810963184.XA CN201810963184A CN109055269B CN 109055269 B CN109055269 B CN 109055269B CN 201810963184 A CN201810963184 A CN 201810963184A CN 109055269 B CN109055269 B CN 109055269B
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Abstract
The invention relates to bifidobacterium longum subspecies infantis CCFM687, a fermented food thereof and application thereof. The bifidobacterium longum subspecies CCFM687 can improve the depression-like behavior of depressed mice and improve the levels of 5-hydroxytryptamine, 5-hydroxytryptamine and brain-derived neurotrophic factors in the brains of the depressed mice; increasing the content of butyric acid in the intestinal tract of a depressed mouse; low abundance of desulfovibrio in intestinal tract, increased abundance of Bifidobacterium and S24-7 family, increased intestinal flora alpha-diversity, improved intestinal flora disturbance in depressed mice, and reduced occurrence of autism, inflammatory bowel disease, obesity, type I diabetes, etc.; increase the mRNA level of tryptophan hydroxylase 1 in the simulated enterochromaffin cells and increase the secretion amount of 5-hydroxytryptophan in the cells, thereby providing precursor substances for the synthesis of 5-hydroxytryptophan in the brain. Therefore, the method has wide application value.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bifidobacterium longum subspecies of infants CCFM687, fermented food thereof and application thereof.
Background
Depression is also known as depressive disorder and is characterized clinically by marked and persistent mood depression. Depression is actually a covered term, including major depressive disorder, bipolar disorder, seasonal affective disorder, postpartum depression, and the like. More than 3.5 million people worldwide currently suffer from depression, which is predicted to be the 1 st in global disease burden by 2030.
Depression is a heterogeneous disease and has an inconsistent response to treatment. The pathogenesis of depression is not yet clear, and the imbalance of monoamine neurotransmitters is the main research direction and is also confirmed by a large number of cases. The primary characteristic of monoamine neurotransmitter imbalance is the reduction in neurotransmitter levels in the brain's synaptic cleft, such as 5-hydroxytryptamine (5-HT), dopamine, norepinephrine, and the like. Another widely accepted mechanism is dysfunction of the hypothalamic-pituitary-adrenal axis (HPA), which is manifested by persistent activation of the HPA axis under chronic stress conditions, secreting excess Glucocorticoids (GC), with the persistent stress resulting in prolonged excessive GC attack of the hippocampus, which in turn exacerbates neuroendocrine abnormalities, and leads to mood changes.
Current antidepressants used in the clinic are mainly focused on increasing levels of monoamine neurotransmitters (mainly 5-HT) in the synaptic cleft of the brain. Wherein the selective 5-hydroxytryptamine reuptake inhibitor (SSRI) and the 5-hydroxytryptamine and norepinephrine reuptake inhibitor (SNRI) are the current first-line antidepressant drugs and account for more than 70 percent of clinical medication. However, the drug has the following two problems: first, there is a 2-4 week delay in the onset of drug action because, after administration of SSRI to increase the concentration of 5-HT in the synaptic cleft, 5-HT reacts against the presynaptic 5-HT1A receptor, resulting in negative feedback inhibition, leading to reduced presynaptic 5-HT synthesis and secretion. After 2-4 weeks of continuous administration, the SSRI can actually take effect only after desensitization of the 5-HT1A receptors. And the 2-4 weeks clinically leads to the reduction of the medication compliance of patients and greatly increases the suicide risk. Secondly, most antidepressant drugs have no preventive effect, and can cause side effects such as nonspecific abdominal pain, constipation, diarrhea, dyspepsia, flatulence and the like after long-term administration. Therefore, the exploration of a new antidepressant method is important, and the market potential is very wide.
The "brain-gut axis" is a new concept proposed in recent years, which regulates the function and behavior of the brain mainly through the neural, endocrine and immune pathways as a two-way communication system between intestinal bacteria and the brain. The intestine contains a large number of intestinal microorganisms (about 10)14-1015One), is the largest micro-ecosystem of the human body. Normal communication between the gut flora and its metabolites and the host is essential to maintain the health of the host. Disorders of intestinal microecology are associated with a number of diseases, including diabetes, obesity, inflammatory bowel disease, neurodegenerative diseases and tumors, among others. The regulation of intestinal flora by probiotics also becomes a new way for treating neurological diseases.
Therefore, it is necessary to select a probiotic which can regulate intestinal flora and effectively relieve depression. Has very important significance for deeply digging the functions of the probiotics and developing the probiotics with higher health care value. And simultaneously, a new way and a solution are developed for relieving the depression by using a dietary strategy.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, in one aspect of the present invention, the present invention overcomes the disadvantages in the prior art, and provides a Bifidobacterium longum subsp. infantum CCFM687(Bifidobacterium longum subsp. infantis) which is deposited in the Guangdong province collection center at 11 th 6 th 2018, with the deposit address of the Guangzhou city mr. zhou No. 100, zhou hua 59, building 5, Guangdong province institute of microbiology, and the deposit number of the Bifidobacterium longum is GDMCC No. 60387.
As another aspect of the present invention, the present invention overcomes the disadvantages of the prior art and provides a fermented food.
In order to solve the technical problems, the invention provides the following technical scheme, wherein: the fermented food is prepared by fermenting Bifidobacterium longum subspecies of infants CCFM687, and the fermented food comprises solid food, liquid food, and semisolid food.
As a preferred method of the fermented food product of the present invention, wherein: the fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of the Bifidobacterium longum subspecies infantis CCFM687 in the preparation of in vivo colonization probiotics.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of Bifidobacterium longum subspecies infantis CCFM687 in preparing medicaments and health care products for resisting depression, autism, inflammatory bowel disease, obesity, type I diabetes and the like.
As a preferred scheme of the application of the bifidobacterium longum subsp. infantis CCFM687 in the preparation of in-vivo colonization probiotics, the method comprises the following steps: the Bifidobacterium longum subsp. infantis CCFM687 can improve the behavioral performance of depressed mice, increase the levels of 5-hydroxytryptamine (5-HT), 5-hydroxytryptamine (5-HTP) and brain-derived neurotrophic factor (BDNF) in the brains of the depressed mice, increase the content of butyric acid in the intestinal tracts of the depressed mice, reduce the abundance of Desulfovibrio (Desulfovibrio) in the intestinal tracts, increase the abundance of Bifidobacterium (Bifidobacterium) and S24-7 family, improve the alpha-diversity of intestinal flora, improve the disturbance of the intestinal flora of the depressed mice, and reduce the occurrence of autism, inflammatory bowel disease, obesity, type I diabetes and the like; bifidobacterium longum subspecies infantis CCFM687 can increase the level of tryptophan hydroxylase 1 mRNA in simulated enterochromaffin cells (RIN14B cells), increase the secretion of 5-hydroxytryptophan in the cells, and specifically provide precursor substances for the synthesis of 5-hydroxytryptophan in brain by stimulating the production of 5-hydroxytryptophan by enterochromaffin cells.
As another aspect of the present invention, the present invention provides the use of the fermented food according to claim 2 or 3 for preparing a functional food for anti-depression, anti-autism, anti-inflammatory bowel disease, anti-obesity, anti-type I diabetes, etc., which overcomes the disadvantages of the prior art.
As a preferable scheme of the application of the fermented food in preparing the anti-depression functional food, the fermented food comprises the following components: the Bifidobacterium longum subsp. infantis CCFM687 can improve the behavioral performance of depressed mice, improve the levels of 5-HT, 5-HTP and BDNF in the brains of the depressed mice, improve the content of butyric acid in the intestinal tracts of the depressed mice, reduce the abundance of Desulfovibrio (Desulfovibrio) in the intestinal tracts, improve the abundance of Bifidobacterium (Bifidobacterium) and S24-7 families, improve the alpha-diversity of intestinal flora, improve the intestinal flora disorder of the depressed mice, and reduce the occurrence of autism, inflammatory bowel disease, obesity, type I diabetes and the like; bifidobacterium longum subspecies infantis CCFM687 can increase the level of tryptophan hydroxylase 1 mRNA in simulated enterochromaffin cells (RIN14B cells), increase the secretion of 5-hydroxytryptophan in the cells, and specifically provide precursor substances for the synthesis of 5-hydroxytryptophan in brain by stimulating the production of 5-hydroxytryptophan by enterochromaffin cells.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention has the beneficial effects that: in a depression model mouse experiment, the administration of the bifidobacterium longum subspecies of infants CCFM687 can obviously relieve depression-like behaviors of mice, and evaluation indexes comprise a forced swimming experiment, a tail suspension experiment, a sugar water preference experiment and a diving platform experiment; the administration of CCFM687 can significantly increase the levels of 5-HTP and 5-HT in hippocampus of depressed mice, and also significantly increase the level of BDNF in the prefrontal cortex of the brain; the content of butyric acid in cecum of a depressed mouse can be obviously improved by taking CCFM 687; the administration of CCFM687 can improve intestinal flora disorder caused by depression, increase alpha-diversity of intestinal flora, reduce abundance of Desulfovibrio (Desulfovibrio), increase abundance of Bifidobacterium (Bifidobacterium) and S24-7 family, normalize intestinal flora, and reduce occurrence of autism, inflammatory bowel disease, obesity, type I diabetes, etc. In vitro experiments show that CCFM1025 can increase the level of mRNA of tryptophan hydroxylase 1 in a simulated enterochromaffin cell (RIN14B cell) and increase the secretion amount of 5-hydroxytryptophan in the cell, and can specifically stimulate the enterochromaffin cell to produce 5-hydroxytryptophan so as to provide precursor substances for the synthesis of 5-hydroxytryptophan in brain.
The bifidobacterium longum subspecies CCFM687 can be used for preparing foods, health-care products and medicines with the functions of resisting depression, autism, inflammatory bowel disease, obesity, type I diabetes and the like, and has very wide application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a schematic diagram of the change of the behavior of the mouse after the strain intervenes in depressed mice for six weeks. (a) Forced swimming experiment; (b) tail suspension experiment; (c) a syrup preference experiment; (d) performing a diving platform experiment; wherein P <0.05, P <0.01, P <0.001(vs model group).
FIG. 2 is a graph showing the change in the levels of 5-HT (panel a) and 5-HTP (panel b) in hippocampal tissues, BDNF (panel c) in prefrontal cortex, six weeks after intervention in depressed mice by the present strain; wherein P <0.01, P <0.001(vs model group).
FIG. 3 is a schematic diagram showing the change of butyric acid content in intestinal tracts of mice after the strain intervenes in depressed mice for six weeks; wherein P <0.05, P <0.01(vs model group).
FIG. 4 is a schematic diagram of the alpha-diversity change of the intestinal flora of mice after the strain intervenes for six weeks in depressed mice; where P <0.05(vs model group).
FIG. 5 is a schematic diagram of the beta-diversity change of the intestinal flora of mice after the strain intervenes in depressed mice for six weeks.
FIG. 6 is a schematic diagram showing the changes of desulfurization vibrio (Desulfovibrio), Bifidobacterium (Bifidobacterium) and S24-7 family in intestinal tracts of mice after the present strain intervenes six weeks in depressed mice; wherein P <0.05, P < 0.01.
FIG. 7 is a graph showing the change in the intracellular mRNA level of tryptophan hydroxylase 1(TPH1) and the amount of 5-HTP in the cell supernatant after RIN14B cells were stimulated by the present strain; wherein P <0.05, P < 0.01.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The Bifidobacterium longum subsp. infantus CCFM687(Bifidobacterium longum subsp. infantis) is preserved in Guangdong province microorganism strain preservation center in 2018, 6 and 11 days, the preservation address is Guangzhou city Mr. Zhou Lu 100 college No. 59 building No. 5 building Guangdong province microorganism research institute, and the preservation number is GDMCC No. 60387.
Said bifidobacterium longum subspecies infantis CCFM687 has the following biological properties:
(1) the characteristics of the thallus are as follows: is gram-positive, does not form a brood, does not move.
(2) Colony characteristics: the bacterial colony is small, milky white and round, the edge is neat, the bacterial colony is slightly convex, the bacterial colony is opaque, and the surface is moist and smooth;
(3) growth characteristics: the minimum growth temperature of the strain is 15 ℃, the maximum growth temperature is 45 ℃, the strain grows optimally at the temperature of 35-37 ℃, the optimum growth pH is 6.5, and the strain enters a stable period after being cultured for 18 hours;
(4) the behavioral performance of the mouse can be obviously improved in a depressed mouse model;
(5) can increase the levels of 5-HT, 5-HTP and BDNF in the brain of a mouse in a depressed mouse model;
(6) the content of intestinal butyric acid in a depressed mouse model can be obviously improved;
(7) can reduce the abundance of Desulfovibrio (Desulfovibrio) in intestinal tracts of depressed mice, improve the abundance of Bifidobacterium (Bifidobacterium) and S24-7 families, improve the alpha-diversity of intestinal flora, improve the intestinal flora disorder of the depressed mice, and reduce the occurrence of autism, inflammatory bowel disease, obesity, type I diabetes and the like;
(8) can increase the level of mRNA of tryptophan hydroxylase 1 in a simulated enterochromaffin cell (RIN14B cell) and increase the secretion amount of 5-hydroxytryptophan in the cell.
The extraction method of the bifidobacterium longum subspecies infantis CCFM687 comprises the following steps:
(I) separation and screening of bifidobacteria:
(l) 1g of fresh feces of healthy newborn were taken. After gradient dilution, coating the solution on an mMRS solid culture medium, and culturing the medium for 72 hours at 37 ℃ in an anaerobic environment;
(2) observing and recording the colony morphology, selecting colonies, and streaking and purifying;
(3) the colonies were gram-stained in MRS liquid medium at 37 ℃ for 48 hours, and the morphology of the colonies was recorded.
(4) Removing gram-negative bacteria strains and gram-positive cocci from the colonies, and selecting to obtain gram-positive bacilli.
(5) After catalase analysis, catalase-positive strains were discarded, and catalase-negative strains were retained.
(II) preliminary identification of Bifidobacterium: fructose-6-phosphate phosphoketolase assay
(1) Culturing the lactic acid bacteria obtained by screening in the step (I) in a liquid mMRS culture solution for 24h, and then centrifuging the lmL culture at 8000rpm for 2 min;
(2) washing twice with 0.05M KH2PO4 solution containing 0.05% (mass percentage) cysteine hydrochloride at pH 6.5;
(3) resuspending in 200. mu.L of the above phosphate buffer solution to which 0.25% (mass%) Triton X-100 was added;
(4) adding 50 mu L of mixed solution of 6mg/mL sodium fluoride and 10mg/mL sodium iodoacetate and 50 mu L of fructose-6-phosphate with the concentration of 80mg/mL, and incubating for 1h at 37 ℃;
(5) adding 300 μ L of hydroxylamine hydrochloride with concentration of 0.139g/mL and pH of 6.5, and standing at room temperature for 10 min;
(6) separately, 200. mu.L of 15% (mass percent) trichloroacetic acid and 4M HCI were added;
(7) when 200. mu.L of 0.1M HCI containing 5 mass% of ferric trichloride was added, the system rapidly turned red, which was positive for F6PPK, and it was preliminarily judged that it was a Bifidobacterium.
(III) molecular biological identification of bifidobacteria:
(l) Extracting a single-bacterium genome: culturing the bifidobacterium obtained by screening in the step (II) overnight, taking the overnight-cultured bacterium suspension lmL in a 1.5mL centrifuge tube, centrifuging for 2min at 10000rpm, and removing the supernatant to obtain thalli; purging the thallus with lmL sterile water, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain thallus; adding 200 μ L SDS lysate, and water bathing at 80 deg.C for 30 min; adding 200 μ L of phenol-chloroform solution into the thallus lysate, wherein the composition and volume ratio of the phenol-chloroform solution are Tris saturated phenol, chloroform and isoamylolTurning to 25:24:1, mixing, centrifuging at 12000rpm for 5-10min, and collecting supernatant 200 μ L; adding 400 μ L of glacial ethanol or glacial isopropanol into 200uL of supernatant, standing at-20 deg.C for 1h, centrifuging at 12000rpm for 5-10min, and discarding the supernatant; adding 500 μ L70% (volume percentage) of glacial ethanol, resuspending the precipitate, centrifuging at 12000rpm for 1-3min, and discarding the supernatant; oven drying at 60 deg.C, or naturally air drying; 50 μ L ddH2Re-dissolving the precipitate with O for PCR;
(2)16S rDNA PCR：
A. bacterial 16S rDNA 50 μ LPCR reaction:
10 × Taq buffer, 5 μ L; dNTP, 5. mu.L; 27F, 0.5 μ L; 1492R, 0.5 μ L; taq enzyme, 0.5. mu.L; template, 0.5 μ L; ddH2O，38μL。
PCR conditions:
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃ 2min；
C. preparing 1% agarose gel, mixing the PCR product with 10000 × loading buffer, loading 2 μ L, running at 120V for 30min, and performing gel imaging;
D. the obtained PCR product is sent to a professional sequencing company, and the obtained sequencing result is searched and compared with similarity in GeneBank by using BLAST to be identified as the Bifidobacterium longum subspecies infantis.
(3) Whole genome sequencing
The extracted whole genome is sent to a professional sequencing company, the whole genome of the strain is sequenced by using a second-generation sequencer, the obtained sequence result is searched and compared with similarity in GeneBank by using BLAST, and the sequencing result is identified as a newly discovered strain belonging to the Bifidobacterium longum subspecies infantis. The strain is preserved at-80 ℃ for later use.
Example 1: bifidobacterium longum subspecies infantis CCFM687 can significantly improve the behavioral indexes of depressed mice
32 male C57BL/6J mice at 6 weeks of age were selected, acclimated for one week, and then randomly divided into four groups according to body weight: control group, model group, fluoxetine dry-pre-group, CCFM687 dry-pre-group, each group containing 8 mice. Animal groups and treatment methods are shown in table 1.
TABLE 1 animal experiment grouping and processing method
Chronic unpredictable stress-depressed mouse model: the stimulation is randomly applied in 1-2 times every day, and the stimulation time is randomly determined, so that the circadian rhythm is avoided. Each method does not exceed three times, and takes five weeks. The stimulus factors include: (1) fasting for 24 h; (2) water is forbidden and the bottle is empty for 24 hours; (3) clamping tail for 3 min; (4) 24h for wet padding; (5) braking for 1-2 h; (6) the cage box is inclined for 24h at an angle of 45 degrees; (7) continuously illuminating for 24 h; (8) no padding is used for 24 hours; (9) forced swimming for 15 minutes; (10) and performing isolated culture for 24 hours.
Lactobacillus gastric lavage agent: collecting activated 2-generation Bacillus bifidus CCFM687, culturing at 37 deg.C for 24 hr, centrifuging at 4 deg.C and 8000r/min for 3min to collect thallus, discarding supernatant, and re-suspending with 5% sterile and defatted emulsion to reach lactobacillus concentration of 5 × 109CFU/mL. The gavage volume was 0.2 mL/day.
Starting in the fifth week, daily chronic unpredictable stress and drug and probiotic intervention was discontinued while all mice were behaviorally tested. Including forced swimming experiments, tail suspension experiments, sugar water preference experiments and diving platform experiments. The specific implementation method and results are as follows:
(1) forced swimming experiment:
the forced swimming test is a behavior despair test method and is a classic test model for evaluating the antidepressant action of the medicine. The experimental bucket is filled with clear water with the height of about 20cm, the water temperature is about 24 +/-1 degrees, and each mouse is subjected to swimming training test for 15 minutes 24 hours before the formal experiment. For the official experiment, each mouse was tested for 6 minutes. The whole course of the experiment was videotaped using a camera. Immobility (floating) time, i.e. immobility of the limbs or only slight hind limb movements, was recorded. The experimental results are shown in fig. 1a, the swimming time of the depressed mice in water is obviously reduced, and the behavior despair characteristics are shown. And the CCFM687 can obviously improve the phenomenon, which indicates that the depression symptom of the mice is relieved.
(2) Tail suspension experiment:
the tail suspension experiment is similar to the forced swimming experiment and is also a behavior despair model. Fixing the position 1/3 at the back of the tail of the mouse by using an adhesive tape, hanging the mouse on a bracket, and taking a picture with the head thereof being 30cm away from the table top, wherein the shooting background is obviously contrasted with the hair color of the mouse. And stopping after timing for 6min, and counting the immobility time of four minutes (3-6min) after the mice by using animal behavior analysis software. Wherein, the immobile state means that the animal abandons the active struggling and is in a completely immobile state. The experimental result is shown in figure 1b, the stationary time of the depressed mice in the tail suspension experiment is obviously increased, the stationary time can be reduced and the depressed state can be relieved by taking CCFM687, and the effect of the CCFM687 is better than that of the antidepressant drug fluoxetine.
(3) Sugar water preference experiment:
the carbohydrate preference test is a model for detecting anhedonia in depression. Before starting the test, two identical drinking bottles were placed in the cages and mice were acclimatized to drink for at least 3 days. After the adaptation was completed, one of the bottles of water was replaced with an aqueous solution containing 1% sucrose. The intake of water and sucrose solution was checked by weighing the bottle. The position of the two bottles is changed every day to reduce drinking preferences due to different water volumes. The sucrose preference calculation formula is: sucrose preference is V (sucrose solution)/[ V (sucrose solution) + V (water) ] x 100%, for a total of 3 days tested and averaged. The experimental result is shown in fig. 1c, the sugar water preference degree of the depressed mice is remarkably reduced, and after the mice take CCFM687, the mice recover to the normal sugar water preference degree, which indicates that the CCFM687 can relieve anhedonia caused by depression.
(4) A jump bench experiment:
depression is usually accompanied by a degree of anxiety and memory impairment. The diving platform experiment can reflect the memory degree of the mouse to the passive injury and the anxiety degree of the mouse during the memory reproduction. The first phase is the shock training phase. The mouse is placed on the platform of the diving platform reaction box, the electric grid at the bottom of the reaction box is electrified (38V), and when the mouse jumps down from the platform, the mouse is subjected to one-time electric shock stimulation. Training time is 3min, if individual mouse does not jump down the platform within 3min, then it is manually driven down to ensure all animals are hurt by electric shock, resulting in nociceptive memory. And after training, returning to the original cage for feeding. The second phase (24 h after shock training) is the memory reproduction test phase. The mouse is placed on a reaction box platform, the test time is 3 minutes, the power is not supplied to the test, the time from the platform on the station to the 1 st jump (the time of staying on the platform) of the mouse is recorded, namely the jump platform latency, and if the mouse does not jump down the platform all the time, the calculation is carried out for 180 s. The artificial interference to the animal behavior is avoided as much as possible in the whole experimental process. The experimental results show (fig. 1d) that the diving platform latency of depressed mice is significantly increased, representing an increase in the degree of depression and anxiety, while the diving platform latency of depressed mice can be significantly reduced by taking CCFM 687. The CCFM687 can relieve depression symptoms and improve anxiety symptoms accompanied by depression.
Example 2: CCFM687 can significantly improve the level of neurotransmitter and precursor in the brain of depressed mice
Mice in example 2 were euthanized at the end of the sixth week, and mouse brain tissue was taken and hippocampus and prefrontal cortex were isolated on ice. Respectively taking a certain mass of fresh hippocampal and prefrontal cortex tissues (the weight is not less than 50mg), adding 9 times of sterile PBS buffer solution (equivalent to 1g of tissues and 9ml of homogenate), homogenizing by using a tissue homogenizer, centrifuging the tissue solution for 3000g and 20min, taking supernatant, and detecting the content of 5-HT and BDNF by using an ELISA kit. Adding 5% perchloric acid precipitated protein with the same volume into the hippocampal tissue fluid supernatant, centrifuging for 10min at 10000g, sucking the supernatant, filtering by a 0.22 mu m water system filter membrane, and performing content determination of 5-HTP by using a high performance liquid chromatography-fluorescence detection method (HPLC-FLD). The column used was Shimadzu Intertsil ODS-3(5 μm, 4.6 mm. times.250 mm) and the mobile phase A was 0.1mol/L NaAc (which contained 0.1mmol/L EDTA-2Na), pH 5.1. Mobile phase B was methanol, mobile phase a: and B is 85:15, the flow rate is 1.0mL/min, the fluorescence detection excitation wavelength is 290nm, the emission wavelength is 330nm, and the sample injection amount is 10 mu L. The results are shown in fig. 2 and indicate that administration of CCFM687 significantly reversed stress-induced reductions in 5-HT, 5-HTP in hippocampus and BDNF in prefrontal cortex. Wherein, the improvement degree of CCFM687 on hippocampal 5-HTP and 5-HT is equivalent to that of fluoxetine, and the improvement effect on BDNF in prefrontal cortex is obviously superior to that of fluoxetine.
Example 3: bifidobacterium longum subspecies infantis CCFM687 can increase the content of butyric acid in intestinal tract of mice
The mice in example 2 were euthanized at the end of the sixth week and the cecal contents of the mice were collected. Weighing 50mg of the cecum content subjected to vacuum freeze drying to an EP tube, adding 500uL of saturated NaCl, and shaking uniformly; adding 40uL 10% sulfuric acid, and shaking uniformly; adding 1ml of diethyl ether, and shaking uniformly; 18000g, centrifugate for 15min at 4 deg.C, take the supernatant to 2ml EP tube, add 0.25g anhydrous sodium sulfate. 18000g, and centrifuging at 4 ℃ for 15 min. 500uL of the supernatant was taken to a gas phase vial and the content of short chain fatty acids (acetic acid, propionic acid, isobutyric acid, butyric acid and isovaleric acid) was detected by GC-MS. The GC-MS adopts an Rtx-Wax column, the length of the column is 30m, and the inner diameter is 0.25 mu m; the carrier gas is He, and the flow rate is 2 mL/min; the sample injection volume is 1 mu L, and the split ratio is 10: 1; the sample introduction temperature is set to 240 ℃, and the temperature is increased according to the following procedures: the initial temperature is 100 ℃, the temperature is increased to 140 ℃ according to the speed of 7.5 ℃/min, then the temperature is increased to 200 ℃ according to the speed of 60 ℃/min, the temperature is kept for 3min, and the ionization temperature is 220 ℃; analysis Using full scan mode, external standard method to calculate the concentration of each short chain fatty acid (. mu. mol/g). The results are shown in fig. 3, where the butyric acid content in the cecum of depressed mice is significantly reduced. The intestinal short-chain fatty acid (especially butyric acid) not only can oxidize and supply energy to intestinal epithelial cells, but also has the important functions of maintaining water electrolyte balance, resisting pathogenic microorganisms, resisting inflammation, regulating intestinal flora balance, improving intestinal functions, regulating immunity, resisting tumors, regulating gene expression and the like. The administration of Bifidobacterium longum subspecies of infants CCFM687 can improve the reduction of butyric acid level caused by depression, and has significant beneficial effect on intestinal microecological balance. The result shows that fluoxetine has no regulating effect on the content of the intestinal butyric acid, and the CCFM687 also has the function of regulating the intestinal physiology on the basis of the antidepressant function and has greater application value.
Example 4: bifidobacterium longum subspecies infantis CCFM687 regulating effect on intestinal flora of depressed mice
Fresh feces of mice at the end of the sixth week in example 2 were taken and total DNA in the feces samples of the mice was extracted using the feces kit of MP. The specific operation steps are mainly carried out according to the kit instructions. Mouse fecal genome as templateThe upstream primer 520F (5 '-AYTGGGYDTAAAGNG-3') and the downstream primer 802R (5 '-TACNVGGGTATCTAATCC-3') are used as primers to amplify a 16S rDNA V3-V4 region fragment, and the length of the target fragment is about 247 bp. And after the PCR reaction is finished, performing electrophoresis again on all the PCR samples with the observed target bands, preparing 2.0% agarose gel, performing electrophoresis for 40min under the condition of 120V, and after the gel is run, rapidly cutting the target bands under an ultraviolet lamp. The recovery of the target band Gel was carried out according to the QIAquick Gel Extraction Kit Gel recovery Kit instructions. The DNA concentration of the Sample is detected according to a Qubit DNA3.0 Kit, then a library is constructed according to a TurSeq DNA LT Sample Preparation Kit and the description thereof, and finally the concentration is determined on an Illumina Miseq sequencer according to a MiSeq Regent Kit and the description thereof. After the sequencing is finished, single sequences with the sequence length less than 200bp, primer sequences and non-splicing sequences are removed, and the single sequences are overlapped according to the basic groups>10bp standard splicing sequence without mismatch. Defining the sequence with similarity greater than 97% as a classification Unit (OTU), by Ribosomal Database Project (RDP)
Bayessclasifier to determine species. Calculating the alpha-diversity and the beta-diversity of the sample to evaluate the flora diversity of the sample. The α -diversity was characterized by the chao1 and PD white tree indices and the results showed (fig. 4) that the intestinal flora in depressed mice was reduced, indicating that depression was accompanied by a degree of disturbance of the intestinal flora. The CCFM687 can remarkably up-regulate the alpha-diversity of the intestinal flora and improve the species abundance degree of the intestinal flora. The beta-diversity was evaluated by principal coordinate analysis (PCoA) (FIG. 5), and the results showed that the intestinal flora of depressed mice was significantly different from that of normal mice, and after the mice took CCFM687, the intestinal flora of the mice was separated from that of depressed mice to some extent, and had a tendency to transform normal mice flora. The Desulfovibrio (Desulfovibrio) has certain relevance with intestinal diseases, particularly the sulfate reducing bacteria which is the dominant bacteria group can generate endogenous hydrogen sulfide, has neurotoxicity, is found to be up-regulated in abundance in a plurality of diseases such as infantile autism and is associated with the occurrence of infantile autismThe diseases have close relationship. The administration of CCFM687 can significantly reduce the abundance of Desulfuron in the intestinal tract of depressed mice (figure 6), thus having the functions of relieving depression and relieving autism. Meanwhile, CCFM687 can increase the abundance of Bifidobacterium (beneficial bacterium) which is an intestinal beneficial bacterium (fig. 6), and Bifidobacterium has a plurality of important physiological functions of biological barrier, nutrition, tumor resistance, immunity enhancement, gastrointestinal tract function improvement, aging resistance and the like, is a physiological beneficial bacterium, and can further inhibit the growth of harmful bacteria through competition. In addition, CCFM687 can increase the abundance of S24-7 family. S24-7 belongs to Bacteroides, and contains acetyl CoA, so that butyric acid can be produced. The butyric acid in the intestinal tract not only can supply energy for the oxidation of intestinal epithelial cells and improve the barrier function of the intestinal tract, but also can play an anti-Inflammatory role through two signal paths of a G protein-coupled receptors (GPCRs) activation path and a Histone Deacetylase (HDACs) inhibition path, and has a remarkable improvement effect on Inflammatory Bowel Disease (IBD) and obesity. In addition, the research finds that the abundance of S24-7 in the intestinal tract of the type I diabetic mice is remarkably reduced, and the correlation exists with the reduction of the number of spleen Foxp3/CD4 Treg cells. Therefore, the increase in abundance of S24-7 family caused by administration of CCFM687 also shows potential for prevention and alleviation of type I diabetes. The results show that the CCFM687 can regulate the intestinal flora and reduce the risk of intestinal tract and other metabolic diseases on the basis of the antidepressant function.
Example 5: effect of Bifidobacterium CCFM687 on 5-HTP Synthesis by RIN14B cells
Determination of 5-HTP in cell supernatants: RIN14B cells at 4X 105Density seed/mL in 24 well plates, incubate for 72 h. The medium was discarded, cells were washed with HBSS (1mL) containing 0.1% Bovine Serum Albumin (BSA) and 2. mu.M fluoxetine, 1mL HBSS suspension containing CCFM687 (control group was HBSS without bacteria) was added, incubation was carried out at 37 ℃ for 20min, the supernatant was collected, centrifuged at 6000g for 5min to remove the precipitate, and the supernatant was frozen at-80 ℃ for assay. HPLC-FLD detected 5-HTP in the cell supernatant (cf. example 3).
mRNA assay for tryptophan hydroxylase 1(TPH 1): adherence in the above stepCells were washed three times with HBSS, 1mL of Trizol was added, incubated on ice for 5-10min, blown to shed cells, and lysates transferred to enzyme-free EP tubes. Total cellular RNA was extracted by a conventional method and cDNA was synthesized according to the instructions of a reverse transcription Kit (Prime Script RT reagent Kit gDNAeraser, Takara). The synthesized cDNA sample is tested for concentration and purity (A260/A280) by a ultramicro spectrophotometer (NanoDrop 2000C), and is stored at-80 ℃ for later use. The samples were mixed with the fluorescent dye SYBR Green super mix (Qiagen, Germany) in a PCR system of 5. mu.L mix, 1. mu.L cDNA, 1. mu.L forward and reverse primers, supplemented with dd water to a total volume of 10. mu.L. In real-time fluorescent quantitative gene amplification instrument CFX96TMThe detection was performed on the Real-Time System (Bio-Rad, USA), 3 parallel wells were set for each sample, and housekeeping gene β -Actin was used as an internal reference, and the results were obtained using 2-ΔΔCqThe method of (1) for analysis; the primer sequences used are shown in Table 2.
TABLE 2qPCR primer sequences
The results show that after CCFM687 stimulates RIN14B cells, the mRNA level of TPH1 in the cells is obviously improved. Accordingly, the amount of 5-HTP secreted by the cells is also significantly increased. The 5-HTP secreted by enterochromaffin cells can enter blood circulation and pass through the blood brain barrier to provide precursor substances for the synthesis of 5-HT in the brain. Therefore, CCFM687 can specifically stimulate enterochromaffin cells to secrete 5-HTP, thereby promoting the synthesis of 5-HT in the brain and realizing the antidepressant function.
Example 6: fermented food containing bifidobacterium longum subspecies of infants CCFM687 prepared by the method
Selecting fresh vegetables, cleaning, juicing, instantly sterilizing at high temperature, sterilizing at 140 deg.C for 2 s, immediately cooling to 37 deg.C, and inoculating Bifidobacterium longum prepared by the methodBacillus and baby subspecies CCFM687 microbial inoculum leaven, the concentration of which reaches 106More than CFU/mL, and storing at 4 deg.C under refrigeration to obtain fruit and vegetable beverage containing viable bacteria of Bifidobacterium longum subspecies of infantis CCFM687 of the invention.
The invention can be used for fermenting and producing other fermented foods by using the bifidobacterium longum CCFM687, wherein the fermented foods comprise solid foods, liquid foods and semi-solid foods. The fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
The fermented food can improve the depressive behavior of depressed mice, increase the content (5-HT, 5-HTP and BDNF) of neurotransmitter and precursor in brain tissues of depressed mice, reduce the level of corticosterone in serum of depressed mice, reduce the abundance of Desulfovibrio (Desulfovibrio) in intestinal tracts, increase the abundance of Bifidobacterium (Bifidobacterium) and S24-7 families, improve the alpha-diversity of intestinal flora, improve the disturbance of the intestinal flora of depressed mice, and reduce the occurrence of autism, inflammatory bowel disease, obesity, type I diabetes and the like; can increase the level of mRNA of tryptophan hydroxylase 1 in simulated enterochromaffin cells (RIN14B cells), increase the secretion amount of 5-hydroxytryptophan in the cells, and specifically stimulate the enterochromaffin cells to produce 5-hydroxytryptophan so as to provide precursor substances for the synthesis of 5-hydroxytryptophan in the brain.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (6)
1. Bifidobacterium longum subspecies infantis (Bifidobacterium longumsubsp. infantis) CCFM687, which was deposited at 11.6.2018 in Guangdong province of microbial culture Collection with the deposit address of "Xieli Zhonglu 100" in Guangzhou CityThe institute of microorganism, Guangdong province, No. 59 building, No. 5 building of the university, with the collection number GDMCC No. 60387.
2. A fermented food product characterized by: the fermented food is prepared by fermenting the bifidobacterium longum subsp.
3. The fermented food product according to claim 2, wherein: the fermented food is dairy products, bean products and fruit and vegetable products, and the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
4. Use of a bifidobacterium longum subsp. infantis CCFM687 as claimed in claim 1 for the preparation of in vivo colonizing probiotics.
5. Application of Bifidobacterium longum subspecies infantis CCFM687 in preparing antidepressant, autism, anti-inflammatory bowel disease, anti-obesity, anti-type I diabetes medicine and health product; wherein Bifidobacterium longum subspecies infantis (Bifidobacterium longumsubsp. infantis) CCFM687, which was deposited at 11.6.2018 in Guangdong province of microbial cultures collection center with the deposit number GDMCC No.60387 at the institute of microbiology of Guangdong province, No. 59, No. 5, No. 59, Zhou Lu, Mieli, Guangzhou, Middy.
6. Use of a fermented food product fermented with bifidobacterium longum subsp.
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