Application of lactobacillus rhamnosus GR-1 in prevention of self-closing behavior

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to an application of lactobacillus rhamnosus GR-1 (Lactobacillus rhamnosus GR-1) in prevention of a self-closing behavior, in particular to an application of lactobacillus rhamnosus GR-1 in prevention of a self-closing behavior. The lactobacillus rhamnosus GR-1 is found to have the function of preventing the autistic behavior in the pregnancy period, the activity is very special, the activity is substantially reflected in that the intake period is completely before the birth of offspring, and the activity is not related to any period after the birth, and the lactobacillus rhamnosus GR-1 has a remarkable effect of preventing the autistic behavior. It is found for the first time that single-plant probiotics can prevent lonely behaviors possibly appearing in filial generations in a mother generation independent intake mode, and the method has important practical application value for expanding the application field of the probiotics and avoiding and reducing social behavior defects of organisms in the development period.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and specifically relates to application of Lactobacillus rhamnosus GR-1 in preventing autistic-like behaviors. Background Art

[0002] Autism spectrum disorder (also known as autism or autism) is a type of neurodevelopmental disease with a high incidence and widespread harm. The pervasive developmental disorders exhibited by autistic / autistic patients are mainly caused by brain biological factors; the causes of brain biological changes can be analyzed from several aspects such as medical biology, neuropsychology and ecology. Its pathogenic factors include genetic factors, immune factors, biochemical factors, pregnancy and delivery factors, etc.

[0003] Autistic behaviors are manifested as lack of social interaction, difficulties in social communication and interaction such as repetitive behaviors, and limited and stereotyped interests and behaviors. Various animal models such as the VPA pregnancy exposure model and the BTBRT~(+)tf / J (BTBR) spontaneous autism model are widely accepted and recognized by researchers and are reliable models of autistic behavioral characteristics. Despite the high incidence and great harm, the research and development of drugs and intervention substances for autism is seriously lagging behind. So far, there is no drug that has been widely recognized internationally for the core symptoms of autism. What is more noteworthy is that based on the principle of prevention-first intervention, the development of nutritional intervention substances with preventive effects is in a more prioritized and urgent position.

[0004] Probiotics are a type of living microorganisms that can impart beneficial health effects to the host. Probiotics promote nutrient absorption and maintain intestinal health by regulating the host's mucosal and systemic immune functions or regulating the balance of intestinal flora, thereby producing single microorganisms or mixed microorganisms with a clear composition that are beneficial to health. Studies have shown that probiotics maintain the host's intestinal health and promote nutrient absorption by regulating the composition of the host's internal flora or by regulating the host's immune function. The analysis and exploration of the spectrum of probiotic effects provides a theoretical basis for the research and development of probiotic preparations.

[0005] The probiotic effect of probiotics has significant strain specificity, and even different strains of the same species have significant differences in effect mode and action pathway. Lactobacillus rhamnosus GR-1 is a lactic acid bacterium isolated from the human urogenital tract, but the bacteria can also be taken orally to regulate intestinal balance, so it is a typical probiotic. At present, the analysis of the probiotic function of Lactobacillus rhamnosus GR-1 mainly lies in the competitive inhibition and immunomodulatory effects on pathogens. CN202210332169.1 discloses that Lactobacillus rhamnosus GR-1 has the effect of improving memory damage caused by lead. However, autistic-like behaviors based on complex social behaviors are significantly different from lead-induced learning and memory damage in terms of manifestation, brain areas of action, and mechanism pathways. Summary of the invention

[0006] When faced with the problem of improving autism spectrum disorders, the inventors have conducted long-term research and exploration and found that Lactobacillus rhamnosus GR-1 has the effect of improving autistic-like behaviors, especially through the intake of Lactobacillus rhamnosus GR-1 by pregnant mammals to improve the autistic-like behaviors of the offspring, thereby achieving the purpose of preventing autism spectrum disorders in the offspring of mammals.

[0007] In one aspect of the present invention, the present invention discloses the use of Lactobacillus rhamnosus GR-1 in the preparation of a composition for preventing autistic-like behavior in mammalian offspring. In the present invention, the autistic-like behavior is manifested as social behavior defects, language communication barriers with other people, stereotyped movements, obsessive interests, sensory sensitivity and emotional disorders, etc. In one embodiment of the present invention, the autistic-like behavior is manifested as autism spectrum disorder.

[0008] In one aspect of the present invention, the purpose of the preparation of Lactobacillus rhamnosus GR-1 for preventing autistic-like behavior in mammalian offspring is achieved by the mother ingesting Lactobacillus rhamnosus GR-1 during pregnancy. In the present invention, the mammal can be a human, a primate, a cow, a sheep, a rat, or a mouse among rodents. In the present invention, the intake can be oral intake or other forms that are convenient for the mammal to obtain Lactobacillus rhamnosus GR-1. The oral intake dosage can be adjusted according to factors such as the size and weight of the animal. In one embodiment of the present invention, the amount of intake at one time is not less than 10 5 The frequency of intake is no less than 1 time / day, and the intake cycle is no less than 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 1 quarter, 2 quarters, 3 quarters, 4 quarters.

[0009] In one aspect of the present invention, the present invention discloses a composition for preventing autistic-like behavior in mammalian offspring, wherein the composition contains Lactobacillus rhamnosus GR-1. The composition product is in the form of tablets, powders, gels, drops, hard capsules, soft capsules, chewables, and oral liquid preparations. In the present invention, in addition to Lactobacillus rhamnosus GR-1, the composition product also contains excipients acceptable to food, health food, and medicine.

[0010] In one aspect of the present invention, the composition of the present invention may further contain other effective ingredients, including vitamins, minerals, animal and plant extracts, probiotics, prebiotics, and edible oils.

[0011] In one aspect of the present invention, the present invention discloses the use of a composition containing Lactobacillus rhamnosus GR-1 in the preparation of a product for preventing autistic-like behavior in offspring of mammals.

[0012] Beneficial Effects

[0013] The present invention utilizes Lactobacillus rhamnosus GR-1 to achieve the improvement of autistic-like behaviors, especially the above-mentioned effects can be achieved by exposure during pregnancy alone. The present invention is the first to discover that Lactobacillus rhamnosus GR-1 has the effect of preventing autistic-like behaviors, which has important technological advancement significance for the intervention of autistic-like defects, the development and use of effective drugs or nutritional intervention substances.

[0014] At the same time, based on the existence of strain specificity, Lactobacillus rhamnosus GR-1 can be used as a special psychoactive probiotic strain, providing a basis for the development of related postbiotics, parabiotics and fermentation-related products based on the strain itself. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 :Behavioral performance of mice in the social phase of the three-compartment behavioral experiment under the conditions of GR-1 and other strains. LGG is Lactobacillus rhamnosus GG; LRC14 is Lactobacillus reuteri RC14; GR-1S represents the supernatant of bacterial culture; prenatal represents prenatal exposure; VPA represents the animal model based on valproic acid; Vehicle represents the control group for solvent exposure. Starting from the 0 point of the horizontal axis, except for the blank control group (Empty), they are Control group, VPA+Vehicle group, VPA+prenatal LGR-1 group, Control+prenatal LGR-1 group, VPA+prenatal LGR-1S group, VPA+prenatal LRC14 group, and VPA+prenatal LGG group.

[0016] Figure 2:Behavioral performance of mice in the social exploration phase of the three-compartment behavioral experiment under the conditions of GR-1 and other strains. LGG is Lactobacillus rhamnosus GG; LRC14 is Lactobacillus reuteri RC14; GR-1S represents the supernatant of bacterial culture; prenatal represents prenatal exposure; VPA represents the animal model based on valproic acid. Starting from the 0 point of the horizontal axis, except for the blank control group (Empty), they are Control group, VPA+Vehicle group, VPA+prenatal LGR-1 group, Control+prenatal LGR-1 group, VPA+prenatal LGR-1S group, VPA+prenatal LRC14 group, and VPA+prenatal LGG group.

[0017] Figure 3 The behavioral performance of mice in the social phase of the three-chamber behavioral experiment under GR-1 treatment. BTBR is a spontaneous autistic behavior animal model. Starting from the 0 point of the horizontal axis, except for the blank group, there are BTBR+Vehicle group and BTBR+prenatal LGR-1 group.

[0018] Figure 4 The behavioral performance of mice in the social exploration phase of the three-chamber behavioral experiment under GR-1 treatment. BTBR is a spontaneous autistic behavior animal model. Starting from the 0 point of the horizontal axis, except for the blank group, there are BTBR+Vehicle group and BTBR+prenatal LGR-1 group.

[0019] Figure 5 Behavioral performance of mice in the bead-burying test under GR-1 treatment conditions. BTBR is a spontaneous autistic behavior animal model; VPA represents a valproic acid-based animal model; Vehicle represents a solvent-exposed control group. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific embodiments.

[0021] The Lactobacillus rhamnosus GR-1 of the present invention and other comparative strains are all from commercial strain suppliers.

[0022] Example 1

[0023] (1) Activation of Lactobacillus rhamnosus GR-1: The prepared MRS liquid culture medium was sterilized in a high pressure steam sterilizer at 121°C for 20 min. After the liquid culture medium was cooled to room temperature, 50 μL of the frozen preserved bacteria was taken and inoculated into the liquid culture medium, and then the liquid culture medium was placed in a 37°C constant temperature incubator for overnight culture, and then 200 μL of the bacterial fermentation liquid was taken and inoculated into the corresponding liquid culture medium for subculture.

[0024] (2) Establishment of animal model

[0025] In addition to using the previously constructed BTBR spontaneous autistic behavior animal model, an environmental autistic model was established by intraperitoneal injection of valproic acid (400 mg / kg) into C57BL / 6J female mice at 12.5 days of gestation. The effectiveness and reliability of the strains were determined by the behavioral performance of the models from different sources.

[0026] (3) Intake of strains

[0027] After the activated GR-1 strain was collected, 8 The corresponding model mice were gavaged with a dose of / mouse / day. Gavage began one week after the female mice were caged with male mice and continued until before birth, that is, throughout the pregnancy. However, it is worth noting that all continuous exposures only lasted until before birth, strictly maintaining the actual "preventive" intervention paradigm. After the offspring were born, the mice were fed normally until the 6th to 8th week after birth and behavioral experiments began.

[0028] (4) Behavioral experiments

[0029] Autistic-like behaviors were assessed using the three-compartment test and the bead-burying test, which are widely accepted in the field and are used to characterize social behavior and repetitive behavior, respectively.

[0030] Prepare a 60*40*23 cm plexiglass container, which is divided into three equal-sized, interconnected chambers (left, middle, and right), and place two empty cages in the left and right chambers respectively. Place the mouse in the three boxes and first undergo a 10-minute adaptation period, during which it can freely explore the three-box device with empty cages; then put a mouse in the left cage, and measure social ability in the second 10-minute period, during which the experimental mouse can interact freely with an empty metal cage or a metal cage containing an age- and sex-matched stranger mouse of the same species. This part is used to test its basic sociability; in the third 10 minutes, put a new mouse in the right cage to measure its preference for social novelties. Use AnyMaze software to record the time of interaction (sniffing, climbing) with the empty cage or the stranger mouse in the cage, as well as the time spent in each chamber.

[0031] Prepare a 50*30 cm plexiglass container, lay 5 cm deep corn cob litter in the field, place 24 regularly arranged royal blue marbles on the surface, put the mouse in to explore freely for 10 minutes, then take the mouse out, and consider the marbles that are at least two-thirds of the depth covered by the litter as buried. Record the number of glass beads buried by each mouse during the test time and the proportion of total glass beads, and use this as a representation of repetitive stereotyped behavior.

[0032] result:

[0033] Statistical methods for behavioral experiments in the present invention: The sample sizes used in all relevant animal behavioral experiments are represented by the number of scattered points in the corresponding figures. Generally, the number of samples in all experimental groups is >6. The statistical methods are performed using one-way analysis of variance and two-way analysis of variance, where * indicates a p value less than 0.0; ** indicates a p value less than 0.01; *** indicates a p value less than 0.001. All of the above indicate that the corresponding statistical groups have statistically significant differences.

[0034] See also Figure 1 , showing the behavioral performance of mice in the social stage of the three-compartment behavioral experiment under the conditions of GR-1 and other strains. Among them, LGG is Lactobacillus rhamnosus GG; LRC14 is Lactobacillus reuteri RC14; S represents the supernatant of bacterial culture; prenatal represents prenatal exposure; VPA represents the animal model based on valproic acid; Vehicle represents the control group for solvent exposure. According to the results of this figure, under the treatment of GR-1, the social defects of mice caused by VPA exposure were significantly repaired, while the supernatant (LGR-1S) or other similar strains such as LGG / RC14 could not achieve similar effects.

[0035] See also Figure 2 , showing the behavioral performance of mice in the social exploration phase of the three-compartment behavioral experiment under the conditions of GR-1 and other strains. LGG is Lactobacillus rhamnosus GG; LRC14 is Lactobacillus reuteri RC14; S represents the supernatant of bacterial culture; prenatal represents prenatal exposure; VPA represents the animal model based on valproic acid. According to the results of this figure, under the treatment of GR-1, the defects of social exploration behavior of mice caused by VPA exposure were significantly repaired, while the supernatant or other similar strains such as LGG / RC14 could not achieve similar effects.

[0036] See also Figure 3, showing the behavioral performance of mice in the social phase of the three-chamber behavioral experiment under GR-1 treatment. BTBR is a spontaneous autistic behavior animal model. According to the results of this figure, under the treatment of GR-1, the sociability of spontaneous social-deficient mice BTBR has been significantly restored, which is specifically reflected in the increased preference for one side of the animal in the three-chamber experiment.

[0037] See also Figure 4 , showing the behavioral performance of mice in the social exploration phase of the three-chamber behavioral experiment under GR-1 treatment. BTBR is a spontaneous autism-like behavior animal model. According to the results of this figure, under the treatment of GR-1, the social exploration behavior of spontaneous social-deficient mice BTBR has been significantly restored, which is specifically reflected in the preference for the new animal side and the enhancement of sociability in the three-chamber experiment.

[0038] See also Figure 5 , showing the behavioral performance of mice in the bead burying experiment under GR-1 treatment conditions. Among them, BTBR is a spontaneous autistic-like behavior animal model; VPA represents an animal model based on valproic acid; Vehicle represents a control group exposed to solvent. According to the results of this figure, under the treatment of GR-1, the repetitive-like behavior defects caused by VPA exposure have been significantly repaired, which is specifically reflected in the number and proportion of buried beads presented during the bead burying process compared with the VPA model group.

[0039] The present invention uses Lactobacillus rhamnosus GR-1 to improve autistic-like behaviors, especially the above-mentioned effect can be achieved by exposure during pregnancy alone, which is a proof of a new use with preventive function. This new use is the first time that Lactobacillus rhamnosus GR-1 has been found to have the effect of preventing autistic-like behaviors, and it is also the first case of using a single lactic acid strain to effectively prevent autistic-like behaviors. This has important technological advancement significance for the intervention of autistic-like defects, the development and use of effective drugs or nutritional intervention substances.

[0040] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. Use of Lactobacillus rhamnosus GR-1 in the preparation of a composition for preventing autistic-like behavior in mammalian offspring.

2. The use according to claim 1, characterized in that: The autistic-like behaviors manifest as autism spectrum disorder.

3. The use according to claim 1, characterized in that: The autistic-like behavior manifests itself as deficits in social behavior.

4. The use according to claim 1, characterized in that: The prevention is to reduce the risk of autistic-like behaviors in offspring produced by pregnant mammals.

5. The use according to claim 1, characterized in that: The prevention is achieved by the mammalian mother taking Lactobacillus rhamnosus GR-1 during pregnancy.

6. The use according to claim 3, characterized in that: The mammals are rodents, primates, and humans.

7. The use according to claim 1, characterized in that: The product form of the composition is tablet, powder, gel, drop, hard capsule, soft capsule, oral liquid preparation.

8. The use according to claim 1, characterized in that: The composition contains, in addition to Lactobacillus rhamnosus GR-1, excipients acceptable to foods, health foods and medicines.

9. Use of a composition containing Lactobacillus rhamnosus GR-1 in the preparation of a product for preventing autistic-like behavior in mammalian offspring.

10. The use according to claim 9, characterized in that: The product also includes one or more of vitamins, minerals, animal and plant extracts, probiotics, prebiotics, and edible oils.

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