CN110664848A - Application of CCFM1025 in preparing food or medicine for relieving autism and adsorbing PCBs - Google Patents
Application of CCFM1025 in preparing food or medicine for relieving autism and adsorbing PCBs Download PDFInfo
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
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- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses application of CCFM1025 in preparation of food or medicine for relieving autism and adsorbing PCBs, wherein the Bifidobacterium breve CCFM1025 can improve 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 cognitive ability on new objects, improve hyperactivity symptom and effectively adsorb the polychlorinated biphenyls.
Description
Technical Field
The invention belongs to the technical field of functional microorganisms, and particularly relates to application of CCFM1025 in preparation of foods or medicines for relieving autism and adsorbing PCBs.
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.
As one aspect of the present invention, the present invention provides the use of bifidobacterium breve CCFM1025 in the preparation of a food or a medicament for alleviating autism and adsorbing PCBs, wherein: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines which can tolerate gastrointestinal fluids, relieve autism and adsorb PCBs.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines for improving autonomous behaviors caused by autism in strange environments and exploring behavior disorders.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines for improving social disorder caused by autism.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines for improving stereotypy caused by autism.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines for improving cognitive impairment of new objects caused by autism.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the bifidobacterium breve CCFM1025 can also be used for preparing food or medicines for improving hyperactivity caused by autism.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the polychlorinated biphenyl comprises a polychlorinated biphenyl Aroclor 1242.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the polychlorinated biphenyl comprises polychlorinated biphenyl Aroclor1254 and Aroclor 1260.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the food comprises fermented food.
As a preferred scheme of the application of the bifidobacterium breve CCFM1025 in preparing foods or medicines for relieving autism and adsorbing PCBs, the invention comprises the following steps: the fermented food comprises dairy products, bean products and fruit and vegetable products.
The invention has the beneficial effects that: the bifidobacterium breve CCFM1025 can improve 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 a new object, improve the reduction of limb coordination ability caused by nerve injury, improve the normal extension rate of a body, relieve myotonia and posture gait disorder caused by Parkinson's disease, and effectively adsorb PCBs.
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 breve CCFM 1025;
FIG. 2 is the effect of Bifidobacterium breve CCFM1025 on the autonomic exploration behavior of autistic rats;
FIG. 3 is the effect of Bifidobacterium breve CCFM1025 on social behavior in autistic rats;
FIG. 4 is a graph of the effect of Bifidobacterium breve CCFM1025 on repeat stereotypy in autistic rats;
FIG. 5 is the effect of Bifidobacterium breve CCFM1025 on cognitive ability in autistic rats;
FIG. 6 is the effect of Bifidobacterium breve CCFM1025 on the frequency of movement in autistic rats;
FIG. 7 shows the adsorption capacity of Bifidobacterium breve CCFM1025 for polychlorinated biphenyls.
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 breve CCFM1025(Bifidobacterium breve) is a published strain which is preserved in Guangdong province microbial strain collection center in 6 and 11 months in 2018, and the address is No. 59, No. 5, No. 100, Mr. school of Middleyau, Guangdong province microbial research institute, and the preservation number is GDMCC NO: 60386.
bifidobacterium breve CCFM1025 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 social disorder of the autistic rat can be obviously improved;
(7) the repeated stereotypy behavior of the rat with the autism can be relieved;
(8) the cognitive ability of the autistic rat on new objects can be relieved;
(9) the movement frequency of the rat with autism can be obviously reduced;
(10) has good adsorption capacity of PCBs.
Example 1: bifidobacterium breve CCFM1025 has good tolerance to simulated gastrointestinal fluid
Inoculating the frozen and preserved bifidobacterium breve CCFM1025 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 breve CCFM1025 with 9.0mL of artificial simulated gastric juice (the mMRS culture medium containing 1% pepsin and having a pH value 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 Bifidobacterium breve CCFM1025 culture solution 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 breve CCFM1025 has better tolerance to the artificial gastrointestinal fluids.
TABLE 1 tolerance of Bifidobacterium breve CCFM1025 in simulated gastric juice
TABLE 2 tolerance of Bifidobacterium breve CCFM1025 in artificially simulated intestinal fluids
Example 2: bifidobacterium breve CCFM1025 has no toxic and side effects on wistar rats
Bifidobacterium breve CCFM1025 strainResuspending in 10% skim milk to a concentration of 3.0X 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 108CFU/mL Bifidobacterium breve CCFM1025 has no obvious effect on rats, no obvious change of 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 breve CCFM1025 can remarkably improve the autonomous exploration capability of autism 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. As shown in fig. 2, compared with the blank group of rats, the cumulative search time of the autistic rats in the central area of the open field shows that the autonomous search ability is reduced in the strange environment, and the administration of the bifidobacterium breve CCFM1025 can significantly improve the autonomous search ability reduction caused by the autism symptom and can also reduce the moving frequency of the autistic rats in the open field (fig. 6), which indicates that the bifidobacterium breve CCFM1025 screened by the present invention can reduce the occurrence of diseases such as hyperactivity.
Example 4: bifidobacterium breve CCFM1025 can remarkably improve the social ability of autism 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 bifidobacterium breve CCFM1025 screened by the invention can improve the social ability of the autism rat.
Example 5: bifidobacterium breve CCFM1025 can remarkably 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. 4, 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 breve CCFM1025 screened by the invention can effectively relieve the repeated stereotypy of the autistic rat.
Example 6: bifidobacterium breve CCFM1025 can remarkably 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 autistic rats in the novelty body are obviously lower than those of the blank group and the CCFM1025 feeding group, and the bifidobacterium breve CCFM1025 can better relieve the cognitive ability of the autistic rats.
Example 7: bifidobacterium breve CCFM1025 has good adsorption effect on PCBs
3mL (1.0X 10) of a culture solution of Bifidobacterium breve CCFM1025 in late logarithmic growth stage is taken10CFU/mL) at 4 ℃ at 8000r/min for 15min, removing supernatant, washing bacterial sludge with synthetic liquid culture medium containing no carbon source, further centrifuging, collecting bacterial sludge, adding 3mL of aroclone 1242/1254/1260(1:1:1) mixture with PCBs concentration of 3.0mg/L, incubating at 37 ℃ for 24h, detecting the residual PCBs by liquid phase, and the experimental result shows that the average degradation capacities of Bifidobacterium breve CCFM1025 to aroclone 1242, 1254, 1260 are 78.1%, 35.5%, and 19.4%, respectively (FIG. 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. The application of the bifidobacterium breve CCFM1025 in preparing the food or the medicine for relieving the autism and adsorbing the PCBs is characterized in that: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines which can tolerate gastrointestinal fluids, relieve autism and adsorb PCBs.
2. Use of bifidobacterium breve CCFM1025 in the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 1, wherein: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines for improving autonomous behaviors caused by autism in strange environments and exploring behavior disorders.
3. Use of bifidobacterium breve CCFM1025 for the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 1 or 2, wherein: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines for improving social disorder caused by autism.
4. Use of bifidobacterium breve CCFM1025 for the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 1 or 2, wherein: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines for improving stereotypy caused by autism.
5. Use of bifidobacterium breve CCFM1025 for the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 1 or 2, wherein: the bifidobacterium breve CCFM1025 can be used for preparing food or medicines for improving cognitive impairment of new objects caused by autism.
6. Use of bifidobacterium breve CCFM1025 for the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 1 or 2, wherein: the bifidobacterium breve CCFM1025 can also be used for preparing food or medicines for improving hyperactivity caused by autism.
7. Use of bifidobacterium breve CCFM1025 for the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 1 or 2, wherein: the polychlorinated biphenyl comprises a polychlorinated biphenyl Aroclor 1242.
8. Use of bifidobacterium breve CCFM1025 for the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 1 or 2, wherein: the polychlorinated biphenyl comprises polychlorinated biphenyl Aroclor1254 and Aroclor 1260.
9. Use of bifidobacterium breve CCFM1025 for the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 1 or 2, wherein: the food comprises fermented food.
10. Use of bifidobacterium breve CCFM1025 in the manufacture of a food or a medicament for alleviating autism and adsorbing PCBs according to claim 9, wherein: the fermented food comprises dairy products, bean products and fruit and vegetable products.
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