CN110720512A - Application of CCFM16 in preparation of fungicide, food or medicine for relieving autism and adsorbing polychlorinated biphenyl - Google Patents
Application of CCFM16 in preparation of fungicide, food or medicine for relieving autism and adsorbing polychlorinated biphenyl Download PDFInfo
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- CN110720512A CN110720512A CN201911135840.8A CN201911135840A CN110720512A CN 110720512 A CN110720512 A CN 110720512A CN 201911135840 A CN201911135840 A CN 201911135840A CN 110720512 A CN110720512 A CN 110720512A
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- ccfm16
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- bifidobacterium bifidum
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses application of CCFM16 in preparation of a microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl, bifidobacterium bifidum CCFM16 can improve autonomous behavior and exploration behavior in a strange environment caused by autism, relieve social disorder caused by autism, obviously improve stereotypy behavior caused by autism, improve cognitive ability of new objects caused by autism, improve limb coordination ability reduction caused by nerve injury, improve normal body stretching rate, relieve myotonia and posture gait disorder caused by Parkinson's disease, and effectively adsorb polychlorinated biphenyl.
Description
Technical Field
The invention belongs to the technical field of functional microorganisms, and particularly relates to application of CCFM16 in preparation of a microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl.
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 biphenyl, also known as chlorinated biphenyl, is a synthetic organic compound, and is a chloride formed by substituting chlorine for hydrogen atoms on a 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.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, as one aspect of the invention, the invention overcomes the defects in the prior art and provides the application of bifidobacterium bifidum CCFM16 in preparing microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl.
In order to solve the technical problems, the invention provides the following technical scheme: application of bifidobacterium bifidum CCFM16 in preparing microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl, wherein: the bifidobacterium bifidum CCFM16 can be used for preparing microbial inoculum, food or medicine for tolerating gastrointestinal fluids, relieving autism and adsorbing polychlorinated biphenyl.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the bifidobacterium bifidum CCFM16 can be used for preparing microbial inoculum, food or medicine for improving autonomous behaviors caused by autism in strange environments and exploring behavior disorder.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the bifidobacterium bifidum CCFM16 can be used for preparing microbial inoculum, food or medicine for improving social disorder caused by autism.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the bifidobacterium bifidum CCFM16 can be used for preparing microbial agents, foods or medicines for improving stereotypy caused by autism.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the bifidobacterium bifidum CCFM16 can be used for preparing microbial inoculum, food or medicine for improving cognitive dysfunction caused by autism on new objects.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the bifidobacterium bifidum CCFM16 can also be used for preparing microbial inoculum, food or medicine for improving limb coordination capacity reduction caused by nerve injury.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the bifidobacterium bifidum CCFM16 can also be used for preparing microbial inoculum, food or medicine for relieving myotonia and gait disorder caused by Parkinson's disease.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the polychlorinated biphenyl comprises polychlorinated biphenyl Aroclor1242, Aroclor 1254 and Aroclor 1260.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the food comprises fermented food.
The preferred scheme of the application of the bifidobacterium bifidum CCFM16 in preparing the microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is as follows: the fermented food comprises dairy products, bean products and fruit and vegetable products.
The invention has the beneficial effects that: the bifidobacterium bifidum CCFM16 can improve the autonomous behavior and the exploration behavior in a strange environment caused by autism, relieve the social disorder caused by the autism, obviously improve the stereotypy behavior caused by the autism, improve the cognitive ability of the autism to a new object, improve the reduction of the coordination ability of limbs caused by nerve injury, improve the normal extension rate of a body, relieve the myotonia and the gait disorder of postures caused by the Parkinson disease and effectively adsorb polychlorinated biphenyl.
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 the colony morphology of Bifidobacterium bifidum CCFM 16;
FIG. 2 is the effect of Bifidobacterium bifidum CCFM16 on the autonomy of autistic rats;
FIG. 3 is a graph showing the effect of Bifidobacterium bifidum CCFM16 on the normal rate of elongation of the autistic rat body;
FIG. 4 is the effect of Bifidobacterium bifidum CCFM16 on social behavior in autistic rats;
FIG. 5 is the effect of Bifidobacterium bifidum CCFM16 on the repeated stereotypy of autistic rats;
FIG. 6 is the effect of Bifidobacterium bifidum CCFM16 on cognitive ability in autistic rats;
FIG. 7 shows the adsorption capacity of Bifidobacterium bifidum CCFM16 on 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 bifidum CCFM16(Bifidobacterium bifidum) is an open strain which is preserved in China general microbiological culture Collection Center (CCM) in 2017 at 17.1.7.with the address of No. 3 of Xilu No. 1 of Beijing Korean district, the institute of microbiology of China academy of sciences with the preservation number of CGMCC NO: 13632.
bifidobacterium bifidum CCFM16 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;
(6) the normal body elongation of the rats with autism 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;
(10) has good adsorption capacity of PCBs.
Example 1: bifidobacterium bifidum CCFM16 has good tolerance to simulated gastrointestinal fluid
Inoculating the frozen bifidobacterium bifidum CCFM16 in 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 medium of the bifidobacterium bifidum CCFM16 with 9.0mL of artificial simulated gastric juice (the mMRS culture medium containing 1% pepsin and having the 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 magar MRS 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 Bifidobacterium bifidum CCFM16 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 colonies, 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 bifidum CCFM16 has better tolerance to the artificial gastrointestinal fluid.
TABLE 1 tolerance of Bifidobacterium bifidum CCFM16 in simulated gastric juice
TABLE 2 tolerance of Bifidobacterium bifidum CCFM16 in artificially simulated intestinal fluids
Example 2: bifidobacterium bifidum CCFM16 has no toxic and side effects on wistar rats
Suspending Bifidobacterium bifidum CCFM16 in 10% skimmed milk to obtain a suspension with a concentration of 3.0 × 108CFU/mL of bacterial suspension. Taking 6 male wistar autism rats with the weight of about 200g, after adapting to the environment for one week, feeding the bacterial suspension with the concentration once a day for intragastric administration, observing for one week, and recording the death and weight conditions.
The results of these tests are listed in table 3. These results show that the feed concentration was 3.0X 108The CFU/mL bifidobacterium bifidum CCFM16 has no obvious influence on rats, no obvious change in body weight and no death phenomenon. 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 bifidum CCFM16 can significantly improve the self-exploration capability of rats with autism
(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. As shown in fig. 2, compared with the rats in the blank group, the cumulative time of exploration in the central area of the open field of the rats with autism shows that the autonomous exploration ability is reduced in the strange environment, and the administration of the bifidobacterium bifidum CCFM16 can remarkably improve the autonomous exploration ability reduction caused by the autism symptom and can also improve the normal body elongation rate (fig. 3), which indicates that the bifidobacterium bifidum CCFM16 screened by the invention can reduce the occurrence of behavioral disturbance diseases such as parkinson and the like.
Example 4: bifidobacterium bifidum CCFM16 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. 4, 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 bifidobacterium bifidum CCFM16 screened by the invention can improve the social ability of the autism rat.
Example 5: bifidobacterium bifidum CCFM16 can significantly improve repeated stereotypy 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. 5, the number of the embedded beads of the autistic rat is obviously higher than that of the rats in the blank group, and the bifidobacterium bifidum CCFM16 screened by the invention can effectively relieve the repeated stereotypy of the autistic rat.
Example 6: bifidobacterium bifidum CCFM16 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. 6, the residence time and frequency of the autism rats in the novelty body are obviously lower than those of the blank group and the group fed with CCFM16, and the bifidobacterium bifidum CCFM16 is shown to be capable of relieving the cognitive ability of the autism rats.
Example 7: bifidobacterium bifidum CCFM16 has good adsorption effect on PCBs
3mL (1.0X 10) of a bacterial solution of Bifidobacterium bifidum CCFM16 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 Aroclor1242/1254/1260(1:1:1), incubating at 37 ℃ for 24h, detecting the residual quantity of PCBs through a liquid phase, and the experimental result shows that the degradation capacities of Bifidobacterium bifidum CCFM16 on Aroclor1242, 1254 and 1260 are respectively 80.1%, 61.6% and 32% (figure 7).
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. Application of bifidobacterium bifidum CCFM16 in preparing microbial inoculum, food or medicine for relieving autism and adsorbing polychlorinated biphenyl is characterized in that: the bifidobacterium bifidum CCFM16 can be used for preparing microbial inoculum, food or medicine for tolerating gastrointestinal fluids, relieving autism and adsorbing polychlorinated biphenyl.
2. Use of bifidobacterium bifidum CCFM16 in the preparation of bacterial agents, food or drugs for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 1 wherein: the bifidobacterium bifidum CCFM16 can also be used for preparing microbial inoculum, food or medicine for improving autonomous behaviors caused by autism in strange environments and exploring behavior disorder.
3. Use of bifidobacterium bifidum CCFM16 in the preparation of bacterial agents, food or drugs for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 1 or 2, wherein: the bifidobacterium bifidum CCFM16 can also be used for preparing microbial inoculum, food or medicine for improving social disorder caused by autism.
4. Use of bifidobacterium bifidum CCFM16 in the preparation of bacterial agents, food or drugs for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 1 or 2, wherein: the bifidobacterium bifidum CCFM16 can also be used for preparing microbial agents, foods or medicines for improving stereotypy caused by autism.
5. Use of bifidobacterium bifidum CCFM16 in the preparation of bacterial agents, food or drugs for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 1 or 2, wherein: the bifidobacterium bifidum CCFM16 can also be used for preparing microbial inoculum, food or medicine for improving cognitive dysfunction caused by autism on new objects.
6. Use of bifidobacterium bifidum CCFM16 in the preparation of bacterial agents, food or drugs for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 1 or 2, wherein: the bifidobacterium bifidum CCFM16 can also be used for preparing microbial inoculum, food or medicine for improving limb coordination capacity reduction caused by nerve injury.
7. Use of bifidobacterium bifidum CCFM16 in the preparation of bacterial agents, food or drugs for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 1 or 2, wherein: the bifidobacterium bifidum CCFM16 can also be used for preparing microbial inoculum, food or medicine for relieving myotonia and gait disorder caused by Parkinson's disease.
8. Use of bifidobacterium bifidum CCFM16 in the preparation of bacterial agents, food or drugs for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 1 or 2, wherein: the polychlorinated biphenyl comprises polychlorinated biphenyl Aroclor1242, Aroclor 1254 and Aroclor 1260.
9. Use of bifidobacterium bifidum CCFM16 in the preparation of bacterial agents, food or drugs for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 1 or 2, wherein: the food comprises fermented food.
10. Use of bifidobacterium bifidum CCFM16 in the preparation of a bacterial agent, food or medicament for alleviating autism and adsorbing polychlorinated biphenyl as claimed in claim 9 wherein: the fermented food comprises dairy products, bean products and fruit and vegetable products.
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