Bifidobacterium lactis HC2786 for reducing harm of heavy metals in body and application thereof

Abstract

The invention relates to the technical field of screening and application of probiotics, in particular to bifidobacterium lactis HC2786 for reducing and removing harm of heavy metal in a body and application of the bifidobacterium lactis HC2786. The bifidobacterium anaticum subsp. Lactis HC2786 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 20222008, and the preservation date is December 23, 2022, the preservation number is CCTCC NO: M 20222008, the preservation number is CCTCC NO: M 20222008, and the preservation number is CCTCC NO: M 20222008. The strain has a relatively strong adsorption effect on heavy metals such as mercury, silver, cadmium and chromium, is not easy to desorb, can reduce the accumulation level of various heavy metals in a central nervous system, alleviates the toxic action of the heavy metals on the nervous system, effectively improves hyperactivity, anxiety and dysphoria, spatial memory disorder, attention deficit and other hyperactivity disorder related neurological behavior abnormalities, and has a good application prospect. The method has great application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotic screening and application, and particularly relates to a lactobacillus bifidus HC2786 for reducing the harm of heavy metals in the body and application thereof. BACKGROUND

[0002] Attention Deficit Hyperactivity Disorder (ADHD, also known as hyperactivity) is a neurodevelopmental and neurobehavioral disorder characterized by inattention, hyperactivity and impulsive behavior. It usually starts in childhood, and some patients' symptoms can last into adolescence or even adulthood. Hyperactivity not only has a long-term negative impact on patients' learning ability, behavior control ability and social adaptability, but also may be accompanied by anxiety and emotional instability. It has become an important problem that needs to be concerned and intervened in the field of public health.

[0003] Existing research shows that the mechanism of hyperactivity has significant multifactor characteristics, which is closely related to genetic susceptibility and significantly affected by environmental factors. Among them, exposure to adverse environmental factors during pregnancy or early childhood, especially heavy metal pollution, is considered to be one of the important risk factors for inducing or aggravating hyperactivity symptoms. Heavy metals such as lead, mercury, cadmium and chromium can enter the body through diet, water or environmental exposure, accumulate in the body for a long time, and may cross the blood-brain barrier and accumulate in the central nervous system, adversely affecting neuron development, neurotransmitter balance and synaptic plasticity, thereby causing or aggravating hyperactivity, attention deficit and emotional abnormalities and other neurobehavioral problems.

[0004] For the intervention of hyperactivity, the current clinical treatment mainly relies on central nervous system drugs for symptomatic treatment. This method usually takes adjusting neurotransmitter levels as the main mechanism of action, and although it can alleviate symptoms to some extent, it has limitations in the scope of application, controversies in long-term use safety, and lack of targeted intervention on environmental pathogenic factors. Especially for hyperactivity symptoms caused or aggravated by heavy metal exposure, simple intervention from the perspective of neurotransmitter regulation is difficult to reduce the body's heavy metal load from the source, and the potential toxic effects of heavy metals on the central nervous system may still exist.

[0005] In recent years, the role of probiotics in human health has gradually attracted attention. Some studies have reported that specific probiotics have certain potential in intestinal microecological regulation, metabolic regulation, and heavy metal adsorption. However, existing technologies on probiotics are mostly focused on intestinal health conditioning or heavy metal detoxification, and their applications are mainly limited to functional improvement of the digestive system. Although some probiotics have been reported to have the ability to adsorb one or several heavy metals, such technologies usually do not involve the distribution of heavy metals in the central nervous system and their effects on neural behavior. There is also no clear technical correlation between reducing heavy metal load in the body and improving hyperactivity, attention deficit, and anxiety-related neural behavior abnormalities. Hyperactivity is a typical neurobehavioral disorder, and its occurrence and development involve central nervous system dysfunction. Therefore, the existing technology still lacks a microecological intervention method that can effectively reduce the accumulation of heavy metals in the central nervous system under the premise of safe tolerance, alleviate the toxic effects of heavy metals on the nervous system, and prevent or assist in relieving hyperactivity-related behavioral abnormalities. SUMMARY

[0006] The existing intervention methods for hyperactivity disorder (ADHD) mainly focus on neurotransmitter regulation, which is difficult to reduce the toxic effects of heavy metals on the central nervous system from the source. The present application provides a Bifidobacterium lactis HC2786 for reducing the harm of heavy metals in the body and its application. The specific technical solutions are as follows: In a first aspect, the present application provides a Bifidobacterium lactis HC2786 for reducing the harm of heavy metals in the body. The Bifidobacterium lactis (Bifidobacterium lactis) Bifidobacterium animalis lactis subsp. Figure 1 ) HC2786 is deposited in the China Center for Type Culture Collection, with the accession number CCTCC NO: M 20222008, and the deposit date is December 23, 2022.

[0007] Further, the 16s rDNA sequence of the Bifidobacterium lactis HC2786 is SEQ ID NO: 3.

[0008] In a second aspect, the present application provides a use of the above-mentioned Bifidobacterium lactis HC2786 in the preparation of a product for reducing the harm of heavy metals in the body.

[0009] Further, the heavy metal is one or a combination of mercury, silver, cadmium, and chromium. Experiments have verified that the Bifidobacterium lactis HC2786 has strong adsorption effect on these heavy metals and is not easy to desorb, effectively shortening the residence time of heavy metals in the body.

[0010] In a third aspect, the present application further provides a use of the Bifidobacterium lactis HC2786 in the preparation of a functional food or a drug for preventing or assisting in improving hyperactivity-related behavioral abnormalities.

[0011] Further, the hyperactivity-related behavioral abnormalities include one or more of hyperactivity, anxiety, and restlessness.

[0012] Further, the hyperactivity-related behavioral abnormalities include spatial memory impairment and / or attention deficit.

[0013] Further, the functional food or the drug is a functional food or a drug for reducing the content of heavy metals in the nervous system.

[0014] The present application has the following beneficial effects: 1. The Bifidobacterium lactis HC2786 provided by the present application has strong adsorption effect on heavy metals such as mercury, silver, cadmium, and chromium, and is not easy to desorb, so that the residence time of heavy metals in the body can be effectively shortened. Through experiments, it has been verified that the strain can effectively improve the symptoms of hyperactivity, anxiety, restlessness, spatial memory impairment, and attention deficit of ADHD rats by reducing the accumulation level of various heavy metals in the central nervous system and reducing the toxic effects of heavy metals on the nervous system, and has great potential application value in preventing or assisting in relieving hyperactivity-related neurological behavioral abnormalities.

[0015] 2. The Bifidobacterium lactis HC2786 provided by the present application belongs to the genus Bifidobacterium, has high biological safety, and is superior to the commercially available Bifidobacterium lactis Bb-12 in terms of gastrointestinal fluid tolerance, intestinal colonization ability, antioxidant capacity, and cholesterol-lowering capacity, and can be used as an effective ingredient in functional foods or drugs for long-term use. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced as follows. Obviously, for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0017] Figure 2 It is a colony morphology diagram of the Bifidobacterium lactis HC2786.

[0018] Figure 3 It is a microscope photograph of the Bifidobacterium lactis HC2786.

[0019] Figure 4 It is a RAPD fingerprint of the Bifidobacterium lactis HC2786.

[0020] Figure 5 It is a rep-PCR fingerprint of the Bifidobacterium lactis HC2786.

[0021] Figure 6 Figure for the test results of heavy metal adsorption stability experiment in Example 6.

[0022] Figure 7 Figure for the test results of open field experiment in Example 11; wherein (a) is the total movement distance, (b) is the frequency of back and forth, (c) is the frequency of standing, (d) is the frequency of grooming.

[0023] Figure 8 Figure for the test results of Y maze experiment in Example 11; wherein (a) is the time of delaying entering the novel-odd arm, (b) is the exploration time of novel-odd arm, (c) is the total movement distance, (d) is the spontaneous alternation rate.

[0024] Figure 1 Figure for the test results of heavy metal content measurement of hippocampus tissue in Example 11; wherein (a) is the lead content of hippocampus tissue, (b) is the mercury content of hippocampus tissue, (c) is the cadmium content of hippocampus tissue, (d) is the chromium content of hippocampus tissue. DETAILED DESCRIPTION

[0025] In order to make the person skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings of the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the protection scope of the present application.

[0026] In the following examples, the preparation method of MRS solid culture medium is as follows: take 10 g of proteose peptone, 10 g of beef extract, 5 g of yeast extract, 5 g of sodium acetate, 5 g of glucose, 2 g of KH2PO4, 1 mL of Tween 80, 2 g of citric acid diamine, 20 g of CaCO3, 0.58 g of MgSO4·7H2O, 0.25 g of MnSO4·7H2O, and 15 g of agar, and then add pure water to 1000 mL, and adjust the pH to 6.2~6.5. The difference between MRS liquid culture medium and MRS solid culture medium is only that the agar is not added.

[0027] Example 1 Strain Isolation and Screening 1. Preliminary screening Take 10g of feces from a healthy 1-year-old child who has not consumed any probiotic products in the past six months, place it in a sampling bag containing 500mL of sterile normal saline, and repeatedly tap it with a tapping instrument for 5 minutes. Take 100μL of the sample liquid and spread it on MRS solid culture medium plates, let them stand for 10 minutes, then invert the plates in an anaerobic bag, and place the anaerobic bag in a 37°C constant temperature incubator for anaerobic culture for 48 hours. After the plates grow single colonies, pick up single clones for further streak culture. After 3 times of purification culture, pick up single clones for MALDI-TOF-MS strain rapid identification.

[0028] MALDI-TOF-MS strain rapid identification is operated according to the kit instructions, and the steps are as follows: the strain monoclone is evenly coated on the target plate in the form of a film, 1μL of lysate is added to cover the sample, and after drying, 1μL of matrix solution is added to cover the sample, and after drying, the sample target is placed in the mass spectrometer for identification. The co-crystallized thin film formed by the sample and the matrix is irradiated with laser, the proteins in the sample are ionized, and the ions are accelerated to fly through the flight tube under the action of a 10~20KV electric field. According to the different flight times of the ions reaching the detector, the molecular weight of the protein is detected. The ribosomal protein fingerprint is obtained by Autofms 1000 analysis software Autof Analyzer v1.0. According to the MALDI-TOF-MS identification results, a total of 40 strains of lactobacillus were screened out from the sample this time.

[0029] 2, strain re-screening 2.1 Lead tolerance screening (1) Prepare MRS liquid medium, cool to about 60°C, add dissolved lead acetate solution (filtered through a 0.22µm filter), and mix to make the final concentration of lead ions 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L and 10g / L. Shake well and cool to room temperature for standby use.

[0030] (2) Strain activation: inoculate the purified strain monoclone into 5mL MRS liquid medium test tube, and incubate the test tube in a 37°C constant temperature incubator overnight. After incubation, inoculate the bacterial liquid into MRS liquid medium at a 2% inoculation amount, and incubate again overnight to fully recover the activity of the strain. Collect the bacterial liquid for standby use.

[0031] (3) The minimum inhibitory concentration (MIC) method was used to determine the lead tolerance of lactic acid bacteria. The MIC is the minimum lead concentration that can completely inhibit the growth of the strain. The lead-containing medium was divided into 4 parts, each part was inoculated with 2.5 μL of bacterial solution, 4 parallel for each lead concentration, and MRS solid medium without lead was used as a blank control and inoculated with the same amount of bacterial solution. After 48 h of incubation at 37°C, the growth status of the strain was observed. The results showed that among the 40 strains of lactobacillus, 25 strains had MIC values of 1-3 g / L, 9 strains had MIC values of 4-5 g / L, and 5 strains had MIC values of 6 g / L. The strain numbered HC2786 had the largest MIC value and reached 7 g / L. This indicates that the HC2786 strain has the strongest tolerance to lead ions.

[0032] 2.2 Lead adsorption capacity screening The bacterial solution of 40 strains to be tested was obtained by using the strain activation method of step (2) of 2.1, then centrifuged at 4200 x g for 10 min at 4°C to remove the supernatant, and the bacterial body was washed with sterile ultrapure water, then vortexed and centrifuged again, repeated 2 times to obtain lactobacillus bacterial body. A certain amount of lead acetate was dissolved in ultrapure water to make the lead ion concentration 150 mg / L, and then filtered with a 0.22 μm filter for standby use. The lactobacillus bacterial body obtained by centrifugation was resuspended in the lead acetate solution to make the wet bacterial body concentration of lactobacillus 1 g / L, vortexed and mixed, then adjusted the pH to 5.0, placed in a constant temperature shaker at 37°C and 160 rpm for 1 h, then centrifuged at 4°C and 4200 x g for 10 min, and the supernatant was collected. The supernatant was diluted with 1% hydrochloric acid solution, and the lead ion concentration was detected by flame atomic absorption spectrometry, and the adsorption rate of the strain to lead was calculated. Each strain was done in triplicate.

[0033]

[0034] Wherein, C0 represents the lead ion concentration in the initial solution, and C1 represents the lead ion concentration after adsorption by lactobacillus.

[0035] The results showed that among the 40 strains of lactobacillus, 32 strains had an adsorption rate of 15.0% or less, 5 strains had an adsorption rate of 15.2%-35.6%, and 3 strains had an adsorption rate of 35.6% or more. Among them, the strain numbered HC2786 had the largest adsorption rate of lead ions, reaching 39.5%. Therefore, the strain numbered HC2786 was selected for further study.

[0036] Example 2 Strain identification 1. Colony morphology identification The HC2786 strain was inoculated on MRS solid medium and incubated at 37°C for 24 h. The colony photo is as follows: Figure 2As shown, the single colony of HC2786 strain appears milky white, with a colony size of 1.5-2.5 mm, smooth, convex, complete edge, shiny and soft texture. Under optical microscope, the HC2786 strain is observed as polymorphic bacilli, with short cell body, no flagellum, and arranged in clusters or dispersed. Bifidobacterium animalis lactis As shown, the single colony of HC2786 strain appears milky white, with a colony size of 1.5-2.5 mm, smooth, convex, complete edge, shiny and soft texture. Under optical microscope, the HC2786 strain is observed as polymorphic bacilli, with short cell body, no flagellum, and arranged in clusters or dispersed.

[0037] 2. Physiological and biochemical characteristics identification Preparation of HC2786 strain inoculum: the activated HC2786 strain is inoculated into MRS liquid medium at a 2% inoculation amount, and anaerobically cultured at 37°C for 24 h, until the viable bacterial amount reaches 10 9 CFU / mL.

[0038] 2.1 Temperature growth range experiment The HC2786 strain inoculum is inoculated into 10 mL MRS liquid medium at a 10% inoculation amount, and 10 mL MRS liquid medium without inoculation is used as a control, and placed in 0°C, 15°C, 30°C, 37°C, 45°C and 60°C constant temperature incubators for anaerobic culture for 48 h, and the turbidity of the culture is observed.

[0039] The results show that the culture medium remains clear after 48 h of culture in 0°C and 60°C constant temperature incubators, and is slightly turbid in 15°C and 45°C constant temperature incubators, while a large amount of bacterial cells are produced in 30°C and 37°C constant temperature incubators, and the maximum amount of bacterial cells is produced and the highest turbidity of the culture medium is obtained in 37°C constant temperature incubator. Therefore, the optimum growth temperature of HC2786 strain is 37°C.

[0040] 2.2 Salt tolerance test Under sterile conditions, the HC2786 strain inoculum is inoculated into 5 mL MRS liquid medium with a salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% (calculated as NaCl) at a 10% inoculation amount, and 5 mL MRS liquid medium without inoculation is used as a control, and placed in a 37°C constant temperature incubator for 48 h of constant temperature oscillation culture, and the turbidity of the culture is observed.

[0041] The results show that the culture medium with a salt concentration of 1%-3% becomes turbid, the culture medium with a salt concentration of 4% or above remains clear, and the turbidity of the culture medium with a salt concentration of 2% is the highest. Therefore, the highest tolerance salt concentration of HC2786 strain is 3%, and the optimum salt concentration is 2%.

[0042] 2.3 Glucose acid production and gas production test The medium formula used in the experiment is as follows: 0.5 g of proteose peptone, 0.3 g of yeast extract, 0.1 mL of Tween 80, 0.5 mL of salt solution A, 0.5 mL of salt solution B, 0.5 g of sodium acetate, 2.5 g of glucose, 0.05 mL of 2% (w / v) bromocresol green are mixed, and distilled water is used to make up to 100 mL, and the pH is adjusted to 6.8-7.0; wherein, the composition of salt solution A is as follows: 10 g of KH2PO4, 1.0 g of K2HPO4, dissolved in distilled water, and made up to 100 mL; the composition of salt solution B is as follows: 11.5 g of MgSO4·7H2O, 2.4 g of MnSO4·2H2O, 0.68 g of FeSO4·7H2O, dissolved in distilled water, and made up to 100 mL. The prepared medium is divided into large test tubes containing inverted small test tubes, 3 mL / tube, 121℃, high pressure sterilization for 15 min.

[0043] Under sterile conditions, the HC2786 strain inoculum is inoculated into the above-mentioned medium at a 10% inoculation amount, and the medium without inoculation is used as a control, then the top is sealed with 2 mL of sterile liquid paraffin, and placed at 37℃ for culture. Continuous culture for 6 days, observe the color change of the medium every day. The results show that after 6 days of culture at 37℃, the medium changes from green to yellow, and there is no gas in the small inverted tube, indicating that the HC1135 strain produces acid during glucose fermentation, but does not produce gas.

[0044] 3. Molecular biology identification Pick a single colony of HC2786 strain on the plate in MRS liquid medium, 37℃ culture for 24 hours, then take 800 μL of fermentation liquor, refer to TIANGEN ® The genomic DNA of the strain is extracted by the bacterial genomic DNA extraction kit (DP302) for the following molecular biology identification.

[0045] 3.1 16s rDNA gene sequence identification TIANGEN ® 2×Taq PCR premix kit is used to amplify the 16s rDNA gene of HC2786 strain, and the reaction system and reaction cycle setting are referred to the kit instruction.

[0046] The upstream primer is 27F: AGAGTTTGATCCTGGCTCA (SEQ ID NO: 1); The downstream primer is 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 2).

[0047] The electrophoresis verified that the size of the PCR amplification product was about 1500 bp, which met the requirements. The 16s rDNA gene sequencing results showed that the 16s rDNA sequence (SEQ ID NO: 3) of the HC2786 strain was as follows:

[0048] The above sequence was subjected to BALST comparison on the EzBioCloud website, and the similarity with Bifidobacterium lactis (Bifidobacterium animalis Bifidobacterium animalis lactis subsp. Figure 3 ) was the highest. Therefore, the HC2786 strain was identified as Bifidobacterium lactis (Bifidobacterium animalis Figure 3 subsp. Figure 4 ).

[0049] 3.2 RAPD fingerprint identification The HC2786 strain was amplified by using a TIANGEN ® 2×Taq PCR premix kit, and the reaction system and reaction cycle were set according to the instruction manual of the kit.

[0050] The M13 primer sequence was 5'-GAGGGTGGCGGTTCT-3' (SEQ ID NO: 4).

[0051] A 1.5% agarose gel plate was prepared, DL2000 DNA Marker was used as a result control, and stable voltage electrophoresis was performed at 100V for 80min. Finally, a gel imaging system was used to detect the electrophoretogram, and the RAPD fingerprint of the HC2786 strain was obtained, as shown in Figure 4 . After comparison, it was found that there was no RAPD fingerprint in the existing public reports that matched Bifidobacterium animalis lactis , so that the HC2786 strain is a new Bifidobacterium lactis strain.

[0052] 3.3 rep-PCR fingerprint identification The HC2786 strain was amplified by using a TIANGEN ® 2×Taq PCR premix kit, and the reaction system and reaction cycle were set according to the instruction manual of the kit.

[0053] The rep-PCR primer sequence was 5'-GTGGTGGTGGTGGTG-3' (SEQ ID NO: 5).

[0054] A 1.5% agarose gel plate was prepared, DL2000 DNA Marker was used as a result control, and stable voltage electrophoresis was performed at 100V for 80min. The amplification result was detected, and the rep-PCR fingerprint of the HC2786 strain was as shown in Bifidobacterium animalis lactis . After comparison, it was found that there was no rep-PCR fingerprint in the existing public reports that matched Figure 5 , so that the HC2786 strain screened by the present application is a new Bifidobacterium lactis strain.

[0055] The results of physiological and biochemical characteristics experiments and molecular biology experiments show that the HC2786 strain belongs to Bifidobacterium lactis Figure 6 subsp. Figure 6 , and is different from the currently reported Bifidobacterium lactis, and is a new strain of Bifidobacterium lactis. The Bifidobacterium lactis HC2786 is preserved in the China Center for Type Culture Collection, and is classified and named as Bifidobacterium lactis Figure 6 subsp. Figure 7 , and the preservation number is CCTCC NO: M20222008, and the preservation date is December 23, 2022.

[0056] Example 3 Gastrointestinal fluid tolerance test of Bifidobacterium lactis HC2786 In 1L distilled water, 5g peptone, 2.5g yeast extract, 1g glucose and 2g NaCl were added in turn, and after fully mixing, the pH of the system was adjusted to 3.0, and sterilized at 115℃ for 20 minutes. After sterilization, the solution was cooled to a suitable temperature, and before the test, 3.2g of porcine mucosa pepsin powder was added, and the container was shaken to mix the powder thoroughly to obtain the artificial simulated gastric juice. In 1L distilled water, 5g peptone, 2.5g yeast extract, 1g glucose, 6.8g potassium dihydrogen phosphate and 3.0g choline salt were added in turn, and after stirring and dissolving, 77mL of NaOH solution was added, and the pH of the system was adjusted to 6.8±0.1, and then sterilized at 115℃ for 20 minutes. Before the test, 1.0g of trypsin powder was added to the cooled sterilized solution, and shaken to mix thoroughly to obtain the artificial simulated intestinal juice.

[0057] The activated Bifidobacterium lactis HC2786 bacterial solution was adjusted to OD600 value of 1.5. 1mL of the bacterial solution was centrifuged at 6000×g for 10min at 4℃, and the supernatant was discarded, and the bacterial body was resuspended in 1mL of simulated gastric juice, and cultured in an anaerobic environment at 37℃ for 3h, and the sample was plated for colony counting at the beginning (0h) and end (3h) of the culture. Subsequently, the bacterial solution cultured in the simulated gastric juice for 3h was again centrifuged at 6000×g for 10min at 4℃, and the supernatant was discarded, and the bacterial body was resuspended in an equal volume of simulated intestinal juice, and continued to be cultured at 37℃ for 2h, and then plated for colony counting. Bifidobacterium lactis Bb-12 in a commercially available product was used as a control strain. The survival rate of the strain was calculated according to the following formula:

[0058] In the formula, N0 and N1 are the number of surviving bacteria (CFU / mL) before and after the strain is treated with simulated gastric juice or simulated intestinal juice, respectively.

[0059] Table 1 Number of viable bacteria and survival rate after artificial gastric and intestinal juice digestion

[0060] As shown in Table 1, the Bifidobacterium lactis HC2786 has high tolerance to simulated artificial gastric juice and intestinal juice, and the survival rate of the viable bacteria is 88.92%±1.70% after digestion by simulated gastric juice, while the survival rate of the viable bacteria of the control strain Bifidobacterium lactis Bb-12 is only 38.50%±8.93%. This indicates that the Bifidobacterium lactis HC2786 can tolerate the harsh gastric acid environment in the human body and successfully enter the small intestine. After digestion by simulated intestinal juice, the survival rate of the viable bacteria of the Bifidobacterium lactis HC2786 is still 82.86%±3.80%, while the survival rate of the viable bacteria of the control strain Bifidobacterium lactis Bb-12 is only 26.96%±4.66%. This indicates that the Bifidobacterium lactis HC2786 can not only tolerate the harsh gastric acid environment in the human body, but also tolerate the intestinal bile salt environment.

[0061] Example 4 Antibiotic tolerance and hemolytic experiment of Bifidobacterium lactis HC2786 1. Antibiotic tolerance experiment The minimum inhibitory concentration (MIC value) of antibiotics on the HC2786 strain was determined by micro-broth dilution method. The antibiotics were prepared as follows: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were prepared into a storage solution of 2048 μg / mL, and stored at -20℃ for standby use. When used, the storage solution was diluted into a use solution by 2-fold serial gradient dilution with MRS liquid medium, and the gradient dilution concentration was 1~1024 μg / mL.

[0062] 190 μL of MRS liquid medium without antibiotics was added into 8 empty holes of the first column of the 96-well plate as negative controls, 190 μL of MRS liquid medium with antibiotic gradient dilution concentration of 1~1024 μg / mL was added into 11 holes of the second to twelfth columns, and then 10 μL of inoculum was inoculated. Each group of experiments was repeated 4 times, and the un-inoculated holes were used as blank controls. 50 μL of sterilized paraffin oil was added into each hole to prevent water evaporation during the culture. The 96-well plate was incubated at 37℃, and the OD600 value was measured every 5 min, the MIC value of different strains was calculated, and the specific results are shown in Table 2.

[0063] Table 2 MIC value of HC2786 strain antibiotics

[0064] Note: The unit of MIC value is μg / mL; R represents drug resistance; S represents sensitivity.

[0065] As shown in Table 2, the HC2786 strain provided by the present application is sensitive to common antibiotics such as erythromycin, streptomycin, clindamycin, tetracycline, gentamicin, and ampicillin, and has good biological safety.

[0066] 2. Hemolytic experiment Weigh the various components of the TBS basal medium: tryptone 17.0 g, NaCl 5.0 g, soybean peptone 3.0 g, KH2PO4 2.5 g, glucose 2.5 g, dissolved in 1 L of distilled water, autoclaved at 121°C for 15 min, after the medium is cooled to 50°C, add 5% sterile defibrillated sheep blood, mix well and pour into plates. The HC2786 strain is streaked onto the prepared blood cell plates and cultured in a 37°C incubator for 24-48 h, and whether the HC2786 strain has hemolytic phenomenon is observed.

[0067] The results show that the blood cell plate has no change, and the HC2786 strain cannot grow on the blood cell plate, indicating that the HC2786 strain does not produce hemolysin and cannot lyse blood cells, and has good biosafety.

[0068] Example 5 Determination of antioxidant function of Bifidobacterium lactis HC2786 In this example, the strain culture and preparation of fermentation supernatant and strain lysate are as follows: the HC2786 strain after 3 generations of activation is cultured in MRS liquid medium at 37°C for 24 h, then centrifuged at 6000 rpm for 10 min, and the supernatant is collected as the fermentation supernatant. The HC2786 strain after 3 generations of activation is cultured in MRS liquid medium at 37°C for 24 h, and then directly subjected to ultrasonic crushing for 20 min using an ultrasonic crusher to obtain the strain lysate. The commercially available Bifidobacterium lactis Bb-12 is used as a control strain, and the preparation method of its fermentation supernatant and strain lysate is the same as that of the HC2786 strain.

[0069] 1. Determination of the ability of the strain to scavenge DPPH (1,1-diphenyl-2-trinitrobenzene hydrazine) free radicals Take 1 mL of the fermentation supernatant or strain lysate of the strain to be tested, add 1 mL of 0.4 mM freshly prepared DPPH free radical solution, mix well, and then place in a room temperature light-shielded reaction for 30 min. Then measure the absorbance A of the fermentation supernatant and strain lysate sample at a wavelength of 517 nm. 样品 Take an equal volume of deionized water and DPPH free radical solution as the control group, measure the absorbance A 控制 , and take an equal volume of fermentation supernatant or strain lysate and DPPH solvent mixture as the blank zero, measure the absorbance A 空白 . Each group is measured in triplicate, and then the DPPH free radical scavenging rate is calculated according to the following formula, and the results are shown in Table 3.

[0070]

[0071] Table 3 DPPH free radical scavenging rate

[0072] According to the results in Table 3, the DPPH radical scavenging rate of the fermentation supernatant and the strain lysate of Bifidobacterium lactis HC2786 was significantly better than that of Bifidobacterium lactis Bb-12.

[0073] 2. Determination of the ability of the strain to scavenge hydroxyl radicals 100 μL of a 5 mM sodium salicylate-ethanol solution, 100 μL of a 5 mM ferrous sulfate, 500 μL of deionized water and 200 μL of the fermentation supernatant or strain lysate to be tested were mixed, 100 μL of hydrogen peroxide solution (3 mM) was added, and the absorbance A was measured at 510 nm after 15 min of incubation at 37°C. 样品 The absorbance A was measured after the same treatment except that deionized water was used instead of the sample to be tested. 控制 The absorbance A was measured after the same treatment except that deionized water was used instead of the sample to be tested and the hydrogen peroxide solution. 空白 The hydroxyl radical scavenging rate was calculated according to the following formula, and the results are shown in Table 4.

[0074]

[0075] Table 4 Hydroxyl radical scavenging rate

[0076] As shown in Table 4, the fermentation supernatant and the lysate of Bifidobacterium lactis HC2786 had a significantly better HRS radical scavenging rate than the control strain Bifidobacterium lactis Bb-12.

[0077] 3. Identification of the anti-lipid peroxidation of the strain Linoleic acid emulsion was prepared by mixing 0.1 mL of linoleic acid, 0.2 mL of Tween 20 and 19.7 mL of deionized water. To 0.5 mL of PBS solution, 1 mL of linoleic acid emulsion and 1 mL of FeSO4 (1%) were added, and then 0.5 mL of the sample to be tested was added. The mixture was incubated at 37°C for 1.5 h, 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) were added, and the mixture was incubated at 100°C for 30 min. After rapid cooling, the mixture was centrifuged at 4000 rpm for 15 min, and the supernatant was collected and the absorbance was measured at 532 nm. The absorbance of the control group, in which 0.5 mL of distilled water was used instead of the sample, was A0. The lipid peroxidation inhibition rate was calculated according to the following formula, and the results are shown in Table 5.

[0078]

[0079] Table 5 Anti-lipid peroxidation inhibition rate

[0080] Example 6 Test of the hydrophobic cell surface of Bifidobacterium lactis HC2786 1. Preparation of the test bacteria solution: pick the purified B. lactis HC2786 colony and inoculate it into a freshly prepared MRS liquid medium, and shake culture at 40°C for 24-48 h. Then inoculate 1% (V / V) of the culture into the MRS liquid medium and continue to shake culture at 40°C for 24-48 h. Centrifuge at 6000 x g for 10 min, collect the bacterial cells, rinse twice with sterile normal saline, resuspend the bacterial cells with 1 mL of sterilized 0.1 M KNO3 solution, and use the solution as the test bacteria solution.

[0081] 2. Measurement of surface hydrophobicity: take 50 μL of the bacterial suspension and add it into 2450 μL of 0.1 M KNO3 solution, and record OD 600 A0. Take 1.5 mL of the bacterial suspension, mix it with 500 μL of xylene, and stand still at room temperature for 10 min (at this time, a two-phase system is formed). Shake the two-phase system for 2 min, and stand still for 20 min to re-form the water phase and the organic phase. Carefully take the water phase (without sucking the organic phase) and measure the absorbance A1 at 600 nm. The cell hydrophobicity is calculated according to the following formula, and the average value of three tests is taken.

[0082]

[0083] The results show that the cell surface hydrophobicity of B. lactis HC2786 is 57.77% ± 8.24%, and the cell surface hydrophobicity of B. lactis Bb-12 is 6.24% ± 4.12%, indicating that B. lactis HC2786 has a greater adhesion potential.

[0084] Example 7. Intestinal epithelial cell adhesion test of B. lactis HC2786 Take the Caco-2 cells from the liquid nitrogen tank, recover and subculture, and expand the number of cells to the required amount. Observe the cell growth under an inverted microscope, and when the cells are nearly 80% adherent, the subsequent experiment can be performed. Discard the original culture medium in the cell culture bottle, rinse twice with PBS, and add an appropriate amount of trypsin to digest the cells. After adding trypsin, put the cells back into the CO2 incubator, and observe the cells under the naked eye until they are completely detached. Then add 2-3 times the volume of culture medium to terminate the digestion, and blow and beat the cells repeatedly for about 10 times. Under a microscope, observe the cells to be in a single cell state as much as possible. Take the single cell suspension into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently tap the cell pellet and add an appropriate amount of fresh culture medium to resuspend the cells. Count the cells using a hemocytometer, dilute the cell suspension with PBS, and plate 2 x 10 6 cells per well in a 6-well plate, and add 2 mL of culture medium per well. After placing the 6-well plate in the CO2 incubator for 24 h, the subsequent cell adhesion experiment can be performed.

[0085] Bifidobacterium lactis HC2786 and Bifidobacterium lactis Bb-12 in logarithmic growth phase were resuspended in MRS liquid medium to 5 x 10 7 CFU / mL (OD 600 Around 0.4). The adhered Caco-2 monolayer in 6-well plates was washed twice with PBS, 1 mL of anti-free cell culture fluid and 1 mL of the above 5 x 10 7 CFU / mL bacterial suspension were added and incubated in a CO2 incubator for 2 h. After incubation, the Caco-2 cells were washed with PBS for 3 times to remove the unadhered bacteria, and the PBS buffer was slowly added along the wall to avoid the cell layer being washed up. After 500 μL of trypsin was added for 3 min and then 1.5 mL of cell culture fluid was added to terminate the digestion, the solution was repeatedly blown and collected into a sterile EP tube. The collected solution was gradiently diluted by 10 times, 100 times, 1000 times and 10000 times, and the number of adhered bacteria was calculated by plate counting. The adhesion ability of the test strain was calculated according to the following formula.

[0086]

[0087] The results showed that the adhesion ability of Bifidobacterium lactis HC2786 to intestinal epithelial cells Caco-2 was 71.66 ± 3.13 (CFU / cell), and the adhesion ability of Bifidobacterium lactis Bb-12 was 13.71 ± 4.80 (CFU / cell), indicating that the adhesion ability of Bifidobacterium lactis HC2786 to intestinal epithelial cells Caco-2 was much higher than that of Bifidobacterium lactis Bb-12, and it had strong intestinal colonization ability.

[0088] Example 8 Cholesterol degradation test in vitro of Bifidobacterium lactis HC2786 (1) Preparation of cholesterol solution: 1 g of cholesterol was accurately weighed, dissolved in anhydrous ethanol, and made up to 100 mL. Under sterile conditions, a 0.22 μm microporous filter membrane was used to filter and sterilize to obtain a cholesterol solution.

[0089] (2) 10.0 g of proteose peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 1.0 mL of Tween 80, 5.0 g of sodium acetate, 0.1 g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0 g of potassium hydrogen phosphate, 1000 mL of distilled water were weighed, dissolved and adjusted to pH 7.3, sterilized at 115°C for 30 min, and then the cholesterol solution was added after the medium was cooled to make the final concentration of cholesterol 0.1%, to obtain a liquid medium containing cholesterol.

[0090] (3) The activated Bifidobacterium lactis HC2786 and Bifidobacterium lactis Bb-12 were inoculated into the liquid medium containing cholesterol prepared in step (2) at an inoculation amount of 0.1%, and statically cultured at 37°C for 48 hours. Then, 0.2 mL of the culture solution was taken, 1.8 mL of anhydrous ethanol was added, mixed, statically cultured for 10 minutes, centrifuged at 3000 rpm for 5 min, and the supernatant was taken. The cholesterol content in the supernatant was determined according to the method specified in GB 5009.128-2016 "Determination of Cholesterol in Food" of the National Food Safety Standard, and the cholesterol degradation rate was calculated.

[0091] The results show that the cholesterol degradation rate of Bifidobacterium lactis HC2786 is 65.40%, which is much higher than that of the control strain Bifidobacterium lactis Bb-12, indicating that the strain helps to reduce the cholesterol level in the blood and maintain cardiovascular health.

[0092] Example 9 Determination of the adsorption capacity of Bifidobacterium lactis HC2786 to mercury, cadmium and chromium According to the lead adsorption capacity screening scheme in Reference Example 1 2.2, the adsorption capacity of Bifidobacterium lactis HC2786 to mercury, cadmium and chromium was studied, and Bifidobacterium lactis Bb-12 in the market was used as a control strain.

[0093] Table 6 Adsorption rate of Bifidobacterium lactis HC2786 to mercury, cadmium and chromium

[0094] The results are shown in Table 6, which shows that Bifidobacterium lactis HC2786 has strong adsorption capacity to mercury, cadmium and chromium, and the effect is better than that of the control strain Bb-12. The use of Bifidobacterium lactis HC2786 helps to alleviate the toxic effects of mercury, cadmium and chromium on the body.

[0095] Example 10 Study on the adsorption stability of Bifidobacterium lactis HC2786 to lead, mercury, cadmium and chromium 0.1 mM and 1.0 mM EDTA solutions and 1.5 mM and 15 mM HNO3 solutions were prepared for desorption experiments. According to the operation steps of lead, mercury, cadmium and chromium adsorption test in Example 1 and Example 9, the bacterial bodies after adsorption were obtained by centrifugation, and the adsorption amount of heavy metal ions on the bacterial bodies was calculated. Different concentrations of EDTA solution and HNO3 solution were added to the supernatant in equal volume for desorption, incubated at room temperature for 10 min, then centrifuged at 5000 x g for 15 min at 4°C, the supernatant was taken and the heavy metal content was determined, each desorption solution was repeated 3 times in parallel, and the change of heavy metal ion content on the bacterial body was observed. The proportion of heavy metal ions not eluted on the bacterial body was calculated according to the following formula.

[0096]

[0097] wherein C i represents the content of heavy metal ions on the bacterial body at the end of adsorption, and Ct represents the content of heavy metal ions in the supernatant.

[0098] Table 7 Rate of heavy metal ions not eluted

[0099] The results are shown in Table 6 and Table 7. Figure 7 As shown in Table 6 and Table 7, B. lactis HC2786 has stable adsorption capacity for lead, mercury, cadmium and chromium. Only under the elution of 1.0 mM EDTA, the adsorption rate of cadmium decreased to below 50%, and the adsorption rate of the rest of the different heavy metal elements and different eluents was above 54%. B. lactis HC2786 is not easy to desorb after adsorbing heavy metals, which is conducive to reducing the residence time of lead, mercury, cadmium and chromium heavy metals in the body.

[0100] Example 11 Study on B. lactis HC2786 improving hyperactivity-related behavioral abnormalities In this embodiment, the preparation method of B. lactis HC2786 bacterial liquid for gavage is as follows: B. lactis HC2786 strain is inoculated into MRS liquid medium, and cultured at 37°C under anaerobic conditions until the stable growth phase. The bacterial body is collected by centrifugation, washed twice with PBS, and resuspended with 0.9% sterile normal saline to make the viable bacterial concentration of the bacterial liquid 1×10 9 CFU / mL.

[0101] 1. Modeling and grouping of ADHD rats Healthy female SD rats were selected and adaptively fed in a SPF level standard animal feeding environment for 1 week. The environmental temperature was maintained at 22±2°C, the relative humidity was controlled at 40%~50%, the light condition was 12h light and dark alternation, and standard feed was provided, and the rats were allowed to freely eat and drink. After the adaptive feeding, female SD rats with a body weight of 200~220g were randomly selected, caged with male SD rats according to a certain ratio for natural mating, and pregnancy was confirmed by vaginal smear or vaginal plug observation. After confirming pregnancy, the rats were caged and fed, and the mixed heavy metal exposure modeling was started.

[0102] The mixed heavy metal solution was prepared by dissolving lead, mercury, cadmium and chromium in distilled water, wherein the final concentration of each heavy metal was: lead 0.010 mg / L, mercury 0.001 mg / L, cadmium 0.005 mg / L and chromium 0.015 mg / L.

[0103] During the experiment, the blank group rats drank ordinary distilled water; the modeling group rats and the probiotic intervention group rats drank the mixed heavy metal solution described above. At the same time, the probiotic intervention group rats were given Bifidobacterium lactis HC2786 liquid by gavage at a dose of 0.2 mL / 10 g of body weight once a day from the gestation period; the blank group and the modeling group rats were given sterile normal saline by gavage at the same dose at the same time. After the rats gave birth, the corresponding heavy metal exposure treatment was continued, and the pups indirectly ingested heavy metals through the breast milk route. After weaning (21 days after birth, PDN21), the pups began to eat and drink the solution containing mixed heavy metals independently. When the SD rats grew to 5 weeks old, they were subjected to behavioral tests to assess hyperactivity and attention deficit-related behavior, and their heavy metal content was simultaneously detected to verify the model construction effect.

[0104] 2. Test scheme 2.1 Open field test The open field test is used to evaluate the rat's autonomous activity, exploration behavior and emotional state. The experiment is carried out at 8 a.m. every day to reduce the influence of the animal's behavior on the circadian rhythm, and the whole experiment is completed in a quiet, light-proof environment. Three days before the formal experiment, adaptive training was carried out, and the rats were placed in the black open field device one by one every day, and freely moved for 10 min to reduce the stress response to the new environment. The open field device is a square box with an open cover, with a size of 100 cm x 100 cm x 40 cm, and the inner walls on all four sides are black. The bottom is evenly divided into 25 20 cm x 20 cm squares, of which the 16 squares near the four walls are defined as the peripheral area, and the remaining 9 squares are defined as the central area. An infrared camera device is fixed about 2 m above the device and connected to a computer for real-time recording and collection of rat movement trajectories and behavior data.

[0105] At the beginning of the formal experiment, the rats were gently placed in the center of the open field device, and the timing started at the same time. The free activity of the rats within 5 min was recorded under the condition of no human interference. A blind observation method was used, and the rat's front paw completely off the ground standing as the upright behavior determination standard was recorded. The number of rats grooming and the number of rats standing during the test were recorded. Each rat was tested once a day for 7 consecutive days. After each rat was tested, it was removed from the device in time, and the inner walls and bottom of the open field device were cleaned with alcohol to eliminate the influence of the odor information left by the previous rat on the subsequent test animals.

[0106] 2.2 Y maze test Y-maze test was used to evaluate the spatial working memory and attention level of rats. The Y-maze device consisted of three arms with an angle of 120° between the arms, each arm was 30 cm x 8 cm x 15 cm in size, and a movable partition was provided at the center of each arm. Before the experiment, the three arms were randomly set as the starting arm, the novel arm and the other arm, and the arm position remained unchanged during the experiment. The experiment was conducted in a quiet and light-proof compartment to minimize the influence of external factors such as the movement of the experimenter. A camera was installed about 2 m above the device to record the activity trajectory of the rats for subsequent analysis.

[0107] The first three days before the formal test were the training phase, and the novel arm was closed by the partition. Each day, the rats were placed in the starting arm and allowed to freely explore the two open arms for 10 min to adapt to the maze environment. On the fourth day, the formal behavioral test was conducted, the partition of the novel arm was removed, and the rats were still placed in the starting arm and allowed to freely explore for 5 min. Each rat was tested only once. The criterion for a rat entering an arm was that all four limbs were completely in the arm. After the test, the inner walls and bottom of each arm of the maze were cleaned with alcohol to avoid the influence of odor residues on the behavior of the next rat. The attention and spatial working memory ability of rats in the Y-maze were evaluated by spontaneous alternation behavior. A valid alternation behavior was defined as the rat entering three different arms in succession (e.g. ABC, ACB, BCA, BAC, CAB or CBA), and the spontaneous alternation rate was calculated according to the following formula.

[0108]

[0109] 2.3 Determination of heavy metal content in hippocampal tissue After the behavioral test, the rats were sacrificed and the hippocampal tissue was quickly isolated. After accurately weighing the mass of the hippocampal tissue using an analytical balance, the sample was transferred to a nitration tube, 4 mL of high-purity concentrated nitric acid and 2 mL of analytical-grade 30% hydrogen peroxide were added, and the sample was soaked overnight. Then the sample was placed in a microwave digestion furnace for heating and digestion, the digestion temperature was raised to 200°C and maintained for 30 min, until the sample was completely nitrated and no visible suspended solids were present. After digestion, the sample was treated with acid to concentrate the volume to less than 1 mL. Then the sample was transferred to a volumetric flask and diluted to 10 mL with deionized water. The concentrations of lead, mercury, cadmium and chromium in the sample were determined using a flame atomic absorption spectrometer. According to the measured concentrations of heavy metals and the volume of the sample, the total mass of each heavy metal was calculated, and then divided by the mass of the corresponding hippocampal tissue to obtain the content of different heavy metals in the hippocampal tissue.

[0110] 3. Test results 3.1 Open field test Hyperactivity is one of the significant behavioral characteristics of attention deficit hyperactivity disorder (ADHD), and the open field test is commonly used to evaluate the spontaneous activity level and emotional state of rats in animal models of ADHD. For example, Figure 7As shown in (a), the average total movement distance of the control group rats was 7.90±1.02 m. Compared with the control group, the total movement distance of the modeling group rats increased significantly (21.11±3.02 m, P<0.01), indicating that the rats exhibited obvious hyperactivity after modeling, demonstrating the successful establishment of the ADHD rat model. Compared with the modeling group, the total movement distance of the probiotic group rats decreased significantly by 30.90% (14.59±3.71 m, P<0.01), suggesting that intervention with Bifidobacterium lactis HC2786 can effectively reduce the hyperactivity level of rats.

[0111] When rats are placed in the central area of ​​an open field, their exploratory instincts cause them to frequently shuttle back and forth between the peripheral and central areas. This frequency of back-and-forth movement can be used to characterize the rats' hyperactive behavior. Figure 7 As shown in (b), the frequency of back-and-forth movements in the control group was 1.63±0.43. Compared with the control group, the frequency of back-and-forth movements in the modeling group was significantly increased (8.00±2.00, P<0.01), further verifying the presence of significant hyperactivity in the model animals. Compared with the modeling group, the frequency of back-and-forth movements in the probiotic group decreased by 45.12% (4.39±1.88, P<0.01), indicating that HC2786 intervention can significantly improve hyperactivity.

[0112] Furthermore, ADHD is often accompanied by emotional abnormalities such as anxiety and irritability, which can be evaluated in open field experiments through the upright posture and grooming behavior of rats. Figure 8 As shown in (c) and (d), the frequency of standing and grooming in the control group rats was 7.49±1.38 and 3.37±1.91, respectively. Compared with the control group, the frequency of standing and grooming in the modeling group rats was significantly increased (30.42±6.11, P<0.01; 9.97±1.41, P<0.01), indicating that the model animals exhibited significant anxiety and irritability. Compared with the modeling group, the frequency of standing and grooming in the probiotic group rats decreased by 52.74% and 36.78%, respectively (14.38±4.33, P<0.01; 6.31±2.03, P<0.01), showing that the intervention of the strain had a significant effect on alleviating mood abnormalities.

[0113] In conclusion, the open field experiment results indicate that intervention with Bifidobacterium lactis HC2786 can significantly improve the hyperactivity behavior of heavy metal-induced ADHD rats and alleviate the accompanying anxiety and irritability symptoms to some extent.

[0114] 3.2 Y-maze experiment Inattention and memory impairment are another typical symptom of attention deficit hyperactivity disorder (ADHD). The Y-maze test is often used to evaluate spatial recognition and memory abilities and attention levels, and can also reflect the animal's activity status. By measuring the time to enter the novel arm, the exploration time of the novel arm, the total distance traveled, and the rate of spontaneous alternation in rats, behavioral differences between different treatment groups were analyzed.

[0115] like ​ As shown in (a), the time to enter the neo-arm was 1.31 ± 0.20 s in the control group. Compared with the control group, the time to enter the neo-arm was significantly prolonged in the modeling group, increasing by 196.95% (3.89 ± 0.57 s, P < 0.01), indicating that heavy metal exposure significantly impaired the spatial recognition memory ability of rats. Compared with the modeling group, the time to enter the neo-arm was significantly shortened in the probiotic group, decreasing by 33.32% (2.59 ± 0.48 s, P < 0.01), suggesting that intervention with Bifidobacterium lactis HC2786 can improve impaired spatial recognition memory.

[0116] like ​ As shown in (b), the exploration time of the control group rats in the novel arm was 189.22 ± 16.86 s. Compared with the control group, the exploration time of the modeling group rats in the novel arm was significantly reduced by 58.86% (77.85 ± 18.84 s, P < 0.01), indicating a significant decline in their spatial recognition and memory abilities. Compared with the modeling group, the exploration time of the probiotic group rats in the novel arm was significantly increased by 52.14% (118.43 ± 26.42 s, P < 0.01), indicating that HC2786 intervention can effectively promote the exploration behavior of rats in novel environments.

[0117] like ​ As shown in (c), the total movement distance of the control group rats in the Y maze was 8.51±1.27m. Compared with the control group, the total movement distance of the modeling group rats increased significantly, by 125.17% (19.17±2.24m, P<0.01), indicating that the model animals exhibited obvious hyperactivity. Compared with the modeling group, the total movement distance of the probiotic group rats decreased by 17.58% (15.80±4.84m, P<0.05), indicating that HC2786 intervention can alleviate hyperactivity to some extent.

[0118] like ​As shown in (d), the spontaneous alternation rate of rats in the control group was 74.79% ± 13.44%. Compared with the control group, the spontaneous alternation rate of rats in the modeling group was significantly reduced by 32.26% (50.66% ± 12.04%, P < 0.01), indicating that their attention level and spatial working memory function were significantly impaired. Compared with the modeling group, the spontaneous alternation rate of rats in the probiotic group was significantly increased by 30.30% (66.01% ± 13.31%, P < 0.05), suggesting that HC2786 intervention can effectively improve attention deficit and spatial memory impairment.

[0119] In conclusion, the Y-maze test results indicate that intervention with Bifidobacterium lactis HC2786 can significantly improve spatial recognition and memory impairment and attention deficit in ADHD rats induced by heavy metal exposure, and to some extent alleviate their hyperactivity.

[0120] 3.3 Heavy metal content in hippocampal tissue Table 8 Heavy metal content in rat hippocampus

[0121] The results of heavy metal content determination in hippocampal tissue of different groups of rats are shown in Table 8 and ​ As shown in the figure, compared with the control group, the levels of four heavy metals (lead, mercury, cadmium, and chromium) in the hippocampus of rats in the modeling group were significantly increased (P < 0.01), indicating that under mixed heavy metal gavage, heavy metals can accumulate significantly in the hippocampus of rats, causing a toxic burden on the central nervous system. The established heavy metal induction model has good stability and reproducibility. Compared with the modeling group, after intervention with Bifidobacterium lactis HC2786, the levels of lead, mercury, cadmium, and chromium in the hippocampus of rats decreased by 26.28%, 20.36%, 16.54%, and 15.20%, respectively, and the differences were statistically significant (P < 0.05). This indicates that Bifidobacterium lactis HC2786 can effectively reduce the accumulation level of multiple heavy metals in the central nervous system and alleviate the toxic effects of heavy metals on the nervous system.

[0122] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A Bifidobacterium lactis HC2786 strain that reduces the harmful effects of heavy metals in the body, characterized in that, Bifidobacterium lactis ( Bifidobacterium animalis subsp. lactis HC2786 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20222008 and deposit date of December 23, 2022.

2. The Bifidobacterium lactis HC2786 as described in claim 1, characterized in that, Its 16S rDNA sequence is SEQ ID NO:

3.

3. The use of Bifidobacterium lactis HC2786 as described in claim 1 in the preparation of products that reduce the harm of heavy metals in the body.

4. The application as described in claim 3, characterized in that, Heavy metals are one or more combinations of mercury, silver, cadmium, and chromium.

5. The use of Bifidobacterium lactis HC2786 as described in claim 1 in the preparation of functional foods or medicines for the prevention or assistance in improving ADHD-related behavioral abnormalities.

6. The application as described in claim 5, characterized in that, ADHD-related behavioral abnormalities include one or more of the following: hyperactivity, anxiety, and irritability.

7. The application as described in claim 5 or 6, characterized in that, ADHD-related behavioral abnormalities include spatial memory impairment and / or attention deficit.

8. The application as described in claim 5, characterized in that, Functional foods or drugs are those that reduce the content of heavy metals in the nervous system.

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