CN110693919A - Application of bifidobacterium longum subspecies infantis - Google Patents
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Abstract
The invention discloses application of a bifidobacterium longum subspecies infantis, wherein the bifidobacterium longum subspecies infantis CCFM687 can be used for preparing food or medicine which can tolerate gastrointestinal fluid and relieve autism and adsorb PCBs, and the bifidobacterium longum subspecies infantis CCFM687 can improve autonomous behavior and exploration behavior in strange environment caused by autism, relieve social disorder caused by autism, obviously improve stereotypy behavior caused by autism, improve cognitive ability of autism on new objects, improve behavior despair (depression) state caused by autism and effectively adsorb PCBs.
Description
Technical Field
The invention belongs to the technical field of functional microorganisms, and particularly relates to an application of bifidobacterium longum subspecies neonatorum.
Background
Autism (Autism), also known as Autism, is recognized as a widespread neurodevelopmental disorder frequently occurring in infants, mainly manifested as social interaction disorder, communication (speech and non-speech) functional impairment and repetitive stereotypy, often accompanied with various clinical symptoms such as intellectual disturbance, epilepsy or hyperactivity, and generally occurring before the age of 3 years. According to the latest data published by the united states center for disease prevention and control (CDC), the prevalence of ASD was 1:59 in 2018, which increased by 15% over 1:68 in 2016, with the prevalence of males being 4-5 times greater than that of females.
ASD is a nerve development disease with high heterogeneity, genetic factors are main factors, but only 5% -25% of causes can be explained, and it is widely believed that ASD may be the result of dynamic development of interaction between susceptibility genes and internal and external environmental factors, thereby influencing development disorder in various aspects of individuals. In recent years, researches show that medicaments, virus infection, heavy metal poisoning and the like in pregnancy or perinatal period can increase the ASD risk of offspring. The population survey shows that the probability of the parents taking sodium valproate during pregnancy to breed ASD children is 7 times that of normal people, the risk of autism of children is increased by 3 times due to severe virus infection in the early pregnancy (the first three months), and the risk of autism of severe bacterial infection in the middle pregnancy (three to six months) is increased by 1.42 times. In addition, infants exposed to excessive amounts of neurotoxic insecticide pesticides (polychlorinated biphenyls (PCBs), organophosphates, etc.) have poor facial recognition ability, difficulty in concentrating attention, and reduced comprehensive intelligence compared to normal infants.
The exact pathophysiological basis for ASD is unclear and there is evidence to support that its major cause is a disorder of the neuroinflammatory system. The number of active microglia in the brain of an autistic patient is 2 times that of a normal person, and has regional differences. The sustained activity and proliferation of glial cells (astrocytes and microglia), with morphological changes, release of a variety of pro-inflammatory mediators (cytokines and chemokines) to coordinate immune responses and to modulate neuronal function and connectivity, and while such responses may serve neuroprotective effects (i.e., to help repair damaged tissue), long-term disturbances of CNS cytokines can lead to the destruction of neuronal function through their role in cognitive and synaptic regulation, including IL-6 and TNF- α, among others.
Polychlorinated biphenyls (PCBs), also known as chlorinated biphenyls, are a class of artificially synthesized organic compounds, which are chlorides formed by substituting chlorine for the hydrogen atom on the biphenyl ring. The wide industrial use of polychlorinated biphenyls has caused a problem of global environmental pollution. PCBs can be absorbed by the body through the skin, respiratory tract and digestive tract of animals, the absorptivity of the digestive tract is very high, and the PCBs poisoning of poultry and human caused by environmental pollution basically occurs after the PCBs are invaded by mouth and absorbed through the digestive tract.
The significance of screening out the probiotics which can regulate the intestinal function and effectively relieve the autism is great. The use of probiotic-related dietary intervention strategies and approaches also opens new approaches and solutions for the relief of autism.
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.
In one aspect of the invention, the invention provides the use of Bifidobacterium longum subsp.
In order to solve the technical problems, the invention provides the following technical scheme: use of bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis is bifidobacterium longum subspecies infantis CCFM687, and the bifidobacterium longum subspecies infantis CCFM687 can be used for preparing food or medicines which can tolerate gastrointestinal fluids, relieve autism and adsorb PCBs.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The bifidobacterium longum subsp. infantis CCFM687 can also be used for preparing food or medicines for improving autonomous behaviors caused by autism in a strange environment and exploring behavior disorders.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The bifidobacterium longum subsp.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis CCFM687 can be used for preparing food or medicines for improving stereotypy caused by autism.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis CCFM687 can be used for preparing food or medicines for improving cognitive dysfunction caused by autism on new objects.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis CCFM687 can also be used for preparing food or medicines for improving behavior despair state caused by autism.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The PCBs comprise Aroclor 1242.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The PCBs comprise Aroclor 1254 and Aroclor 1260.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The food comprises fermented food.
As a preferred embodiment of the use of the bifidobacterium longum subsp. The fermented food comprises dairy products, bean products and fruit and vegetable products.
The invention has the beneficial effects that: the bifidobacterium longum subspecies CCFM687 can improve the autonomous behavior and exploration behavior in a strange environment caused by autism, relieve social disorder caused by the autism, obviously improve stereotypy behavior caused by the autism, improve the cognitive ability of the autism on new objects, improve behavior despair (depression) state caused by the autism, and effectively adsorb PCBs.
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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 the colony morphology of Bifidobacterium longum subspecies infantis CCFM 687;
FIG. 2 is the effect of Bifidobacterium longum subspecies of infantis CCFM687 on the autonomic exploration behavior of autistic rats;
FIG. 3 is the effect of Bifidobacterium longum subspecies infantis CCFM687 on social behavior in autistic rats;
FIG. 4 is a graph of the effect of Bifidobacterium longum subspecies of infantis CCFM687 on the repeated stereotypical behavior of autistic rats;
FIG. 5 is the effect of Bifidobacterium longum subspecies infantis CCFM687 on cognitive ability in autistic rats;
FIG. 6 is the effect of Bifidobacterium longum subspecies CCFM687 on the behavioral despair (depressive) status of autistic rats.
FIG. 7 is the adsorption capacity of Bifidobacterium longum subspecies infantis CCFM687 for PCBs.
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.
Bifidobacterium longum subsp.infantis CCFM687(Bifidobacterium longum subsp. infantis) is an open strain, is preserved in Guangdong province microorganism strain preservation center in 6 and 11 months in 2018, is preserved at Guangzhou city Mr. Fujiu No. 100, No. 59, building, Guangdong province microorganism research institute, and has the preservation number of GDMCC No: 60387.
bifidobacterium longum subspecies infantis CCFM687 has the following biological properties:
(1) the characteristics of the thallus are as follows: gram-positive, non-sporulating, immotile bacteria.
(2) Colony characteristics: the anaerobic culture for 36 hours forms obvious colony with the diameter of 0.5-2mm, the front shape is round, the side shape is convex, the edge is neat, the color is milky white, the surface is moist and smooth, and no pigment is generated, see figure 1.
(3) Growth characteristics: the cells were cultured in mMRS medium at 37 ℃ under constant temperature anaerobic conditions for about 24 hours to the end of log.
(4) The compound has better tolerance to simulated gastrointestinal fluid;
(5) the open field autonomous exploration capability of the autism rat can be remarkably improved;
(7) the social disorder of the autistic rat can be obviously improved;
(8) the repeated stereotypy behavior of the rat with the autism can be relieved;
(9) the cognitive ability of the autistic rat on new objects can be relieved;
(6) the behavioral despair (depression) state of the autism rat caused by the autism can be obviously improved;
(10) has good adsorption capacity of PCBs.
Example 1: bifidobacterium longum subspecies infantis CCFM687 has good tolerance to simulated gastrointestinal fluid
Inoculating the cryopreserved bifidobacterium longum subspecies of babies CCFM687 into an mMRS culture medium (MRS culture medium + 0.05% cysteine hydrochloride), carrying out anaerobic culture at 37 ℃ for 48h, carrying out subculture for 2-3 times by using the mMRS culture medium, mixing 1mL of the culture solution of the bifidobacterium longum subspecies CCFM687 with 9.0mL of artificial simulated gastric juice (the mMRS culture medium containing 1% pepsin and having a pH of 2.5), carrying out anaerobic culture at 37 ℃, sampling at 0h, 0.5h, 1h and 2h respectively, carrying out pouring culture by using the mMRS agar culture medium, carrying out plate colony counting, measuring the viable count and calculating the survival rate.
The survival rate is the ratio of the logarithmic viable count at the sampling time to the logarithmic viable count at the 0h time in the culture solution, and is expressed by%. Adding 1mL of culture solution of Bifidobacterium longum subspecies of infant CCFM687 into 9mL of artificial simulated intestinal fluid (mMRS culture medium containing 0.3% of bovine bile salt, 1% of trypsin and pH 8.0), anaerobically culturing at 37 deg.C, sampling at 0h, 0.5h, 1h and 2h respectively, pouring and culturing with mMRS agar culture medium, counting plate colony, measuring viable count and calculating survival rate. The survival rate is the ratio of the logarithmic viable count at the sampling time to the logarithmic viable count at the 0h time in the culture solution, and is expressed by%. The results of the experiment are shown in tables 1 and 2. The result shows that the bifidobacterium longum subspecies of infants CCFM687 has better tolerance to the artificial gastrointestinal fluids.
TABLE 1 tolerance of Bifidobacterium longum subspecies infantis CCFM687 in simulated gastric fluid
TABLE 2 tolerance of Bifidobacterium longum subspecies infantis CCFM687 in artificial simulated intestinal fluid
Example 2: bifidobacterium longum subspecies of infant CCFM687 has no toxic and side effects on wistar rats
Suspending Bifidobacterium longum subspecies of infant CCFM687 in 10% skim milk to obtain a concentration of 3.0 × 108CFU/mL of bacterial suspension. Taking 6 male wistar autism rats with the weight of about 200g, adapting to the environment for one week, and then feeding bacterial suspension with the concentration into the stomach oneNext, one week of observation, death and body weight were recorded.
The results of these tests are listed in table 3. These results show that the feed concentration was 3.0X 108CFU/mL Bifidobacterium longum subspecies CCFM687 did not cause significant effect on rats, no significant change in body weight, and no death. The appearance of the rat has no obvious pathological symptoms.
TABLE 3 weight change and mortality in mice
Note: -: no death of rat
Example 3: bifidobacterium longum subspecies infantis CCFM687 can significantly improve the self-exploration capability of autistic rats
(1) Breeding of autistic young mouse
The method comprises the following steps of (1) breeding healthy male and female wistar rats in an adaptive environment for 1 week, closing the rats in a female-male ratio of 2:1 at 5 nights, observing vaginal embolus in 8 nights, performing vaginal smear if vaginal embolus is not observed, recording the 1 st day of pregnancy when sperm is observed, breeding the rats in cages after conception, and randomly dividing the rats into a model group and a control group. Administering 600mg/kg of sodium Valproate (VPA) (250 mg/mL solution prepared by normal saline) to the abdominal cavity on the 12.5 th day after pregnancy to serve as a model group; the control group pregnant mice are injected with the same amount of normal saline at the same period, and the young mice are weaned 21 days after birth.
(2) Autistic rat experimental grouping
Divided into a control group, a model group and an intervention group.
(3) Open field experiment of autistic rat
The open field experiment mainly aims to observe the autonomous behavior of the experimental animal in a new and different environment and explore the behavior and the tensity. The height of a rat open field reaction box is 30-40 cm, the bottom side is 100cm, the inner wall is black, a digital camera is arranged 2m above the rat open field reaction box, the visual field of the digital camera can cover the inside of the whole open field, the whole experiment process is recorded, each autism rat is tested for 6 minutes, the occurrence frequency and the duration of certain behaviors (the retention time, the movement speed, the movement distance and the like of the animal in the central area and the marginal area in unit time) of the experimental animal in a novel environment are used for reflecting the autonomous behavior and the exploration behavior of the experimental animal in a strange environment. The experimental results are shown in fig. 2, compared with the blank group of rats, the search accumulated time of the autism rats in the central area of the open field shows that the autonomous search ability in the strange environment is reduced, and the administration of the bifidobacterium longum subspecies CCFM687 can obviously improve the autonomous search ability caused by the autism symptoms, which shows that the screened bifidobacterium longum subspecies CCFM687 can improve the autonomous behavior and the search behavior in the strange environment.
Example 4: bifidobacterium longum subspecies infantis CCFM687 can significantly improve the social ability of autistic rats
The experimental modeling, grouping and processing methods are the same as example 3.
Three-box animal social behavior experiments are the most commonly used experiments to assess autistic social disturbance. The detection mice are all male, the birth time difference is not more than 1d, the body mass difference is less than 15g, and the mice are raised in cages. The detection was performed in a transparent three-chamber box with a size of 60cm x60cm x60 cm. 1d before the experiment, all the detected mice are placed in a detection room to adapt to the environment. In the experiment, 1 mouse to be tested is put into a middle chamber and is firstly adapted for 10 min. Then, a strange rat was placed in the left small chamber and covered with an empty wire cage, and the same rat was placed in the right small chamber and covered with the same wire cage. Opening a channel through which the middle small chamber enters the left small chamber and the right small chamber, recording the behavior of the detected mouse within 10min by shooting, and evaluating the social behavior ability of the animal by calculating the time, the contact times and other indexes of the detected animal approaching a certain steel wire cage through software. The experimental result is shown in fig. 3, the contact time between the autism rat and the strange rat is obviously shorter than that of the rats in the blank group, and the result shows that the bifidobacterium longum subspecies infantis CCFM687 screened by the invention can improve the social ability of the autism rat.
Example 5: bifidobacterium longum subspecies CCFM687 can significantly improve repeated stereotypy behavior of rats with autism
The experimental modeling, grouping and processing methods are the same as example 3.
The marble bead burying experiment is a classic experiment for evaluating repeated stereotypy of autism, and the corn cob packing material with the thickness of 5cm is paved in a rat cage box, and is laid flat, 20 glass beads (the diameter is 15mm) are placed in the rat cage box and are divided into five rows, and each row is divided into 4, and the corn cob packing material is regularly arranged. The mice were placed in a cage for 15min and the number of embedded glass beads was counted (embedded volume greater than 50%). The experimental result is shown in fig. 4, the number of the embedded beads of the autism rat is obviously higher than that of the rats in the blank group, and the result shows that the bifidobacterium longum subspecies CCFM687 screened by the invention can effectively relieve the repeated stereotypy behavior of the autism rat.
Example 6: bifidobacterium longum subspecies infantis CCFM687 can significantly improve cognitive ability of autistic rats
The experimental modeling, grouping and processing methods are the same as example 3.
The basic procedure for rat object identification experiments consists of mainly 3 stages: (1) during the adaptation period, rats are sequentially placed in an experimental device without any object, so that the rats can be freely explored to adapt to the experimental environment, and the irritability of animals during the experiment is reduced as much as possible. (2) During the familiarity period, two identical objects (A1 and A2) were placed adjacent to or opposite the floor of the experimental setup, the rats were placed in the experimental setup with the two objects facing away from each other, and 10min later the animals were removed and placed back in the animal cages. After the corresponding time interval, test period experiments were performed. (3) The test session and the familiarity session are performed similarly, except that one of the two identical objects is exchanged for a different object, and the objects in the test session are referred to as the familiar object (A3) and the novelty object (B), respectively, with respect to the two objects in the familiarity session. The cognitive ability of the animal is evaluated by calculating the time and times of the tested animal approaching the familiar object and the novel object through software. The experimental results are shown in fig. 5, the residence time and frequency of the autism rats in the novelty body are obviously lower than those of the blank group and the CCFM687 feeding group, and the bifidobacterium longum subspecies CCFM687 can better relieve the cognitive ability of the autism rats.
Example 7: bifidobacterium longum subspecies infantis CCFM687 can significantly improve behavioral despair (depression) state of autistic rat
The experimental modeling, grouping and processing methods are the same as example 3.
The forced swimming test is a behavior despair test method. Is a classical experimental model for evaluating the antidepressant action of a medicament. The experimental barrel is filled with clear water with the height of about 40cm, the water temperature is about 24 +/-1 ℃, and each rat carries out swimming training test for 15min 24h before the formal experiment. In the official experiment, each rat was tested for 6 min. 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. 6, the swimming time of the autistic rat in water is obviously reduced, the immobility time is more, and the behavior despair characteristic is shown. Bifidobacterium longum subspecies infantis CCFM687 has more swimming time and less immobility time, which indicates that Bifidobacterium longum subspecies infantis CCFM687 can relieve depression caused by autism.
Example 8: bifidobacterium longum subspecies of infant CCFM687 has good adsorption effect on PCBs
3mL (1.0X 10) of a bacterial solution of Bifidobacterium longum subspecies CCFM687 in late logarithmic growth stage10CFU/mL) at 4 ℃ for 15min at 8000r/min, removing supernatant, washing bacterial sludge with a synthetic liquid culture medium containing no carbon source, further centrifuging, taking bacterial sludge, adding 3mL of a mixture of aroclone 1242/1254/1260(1:1:1), wherein the concentration of PCBs is 3.0mg/L, incubating at 37 ℃ for 24h, and detecting the residual quantity of PCBs by liquid phase, wherein the experimental result is shown in figure 7, and the results show that the degrading capacities of bifidobacterium longum subsp. infantis CCFM687 to aroclone 1242, 1254 and 1260 are 73.3%, 57.2% and 25.1% respectively.
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 (10)
1. Use of bifidobacterium longum subspecies infantis, characterized in that: the bifidobacterium longum subspecies infantis is bifidobacterium longum subspecies infantis CCFM687, and the bifidobacterium longum subspecies infantis CCFM687 can be used for preparing food or medicines which can tolerate gastrointestinal fluids, relieve autism and adsorb PCBs.
2. Use of bifidobacterium longum subsp. The bifidobacterium longum subsp. infantis CCFM687 can also be used for preparing food or medicines for improving autonomous behaviors caused by autism in a strange environment and exploring behavior disorders.
3. Use of bifidobacterium longum subsp. The bifidobacterium longum subsp.
4. Use of bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis CCFM687 can be used for preparing food or medicines for improving stereotypy caused by autism.
5. Use of bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis CCFM687 can be used for preparing food or medicines for improving cognitive dysfunction caused by autism on new objects.
6. Use of bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis CCFM687 can also be used for preparing food or medicines for improving behavior despair state caused by autism.
7. Use of bifidobacterium longum subsp. The PCBs comprise Aroclor 1254.
8. Use of bifidobacterium longum subsp. The PCBs comprise Aroclor1242 and Aroclor 1260.
9. Use of bifidobacterium longum subsp. The food comprises fermented food.
10. Use of bifidobacterium longum subsp. The fermented food comprises dairy products, bean products and fruit and vegetable products.
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| CN111073828A (en) * | 2019-11-19 | 2020-04-28 | 江南大学 | Bifidobacterium longum subspecies longum and application thereof |
| CN111073828B (en) * | 2019-11-19 | 2022-04-15 | 江南大学 | Bifidobacterium longum subspecies longum and application thereof |
| WO2023098540A1 (en) * | 2021-11-30 | 2023-06-08 | 内蒙古伊利实业集团股份有限公司 | New use of bifidobacterium infantis ylgb-1496 in anti-aging and improvement of innate immunity |
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