Detailed Description
The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The disclosure provides an application of bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for preventing and treating mental disorder, wherein the bifidobacterium lactis is bifidobacterium lactis BL-11, and the preservation number of the bifidobacterium lactis BL-11 is CGMCC No. 20847.
Optionally, the psychiatric disorder comprises anxiety, depression, attention deficit hyperactivity disorder, autism, schizophrenia, hepatic encephalopathy, anorexia nervosa, tourette's syndrome, esberg syndrome.
Optionally, the food composition or pharmaceutical composition further comprises one or more of skimmed milk powder, trehalose, fructo-oligosaccharide, lactose, glucose, sucrose, sodium L-ascorbate, L-malic acid, L-lactic acid, etc.
Optionally, the food composition or pharmaceutical composition further comprises a flavoring agent, sweetener, thickener, stabilizer, surfactant, lubricant, acid neutralizer, dispersant, buffer or buffer, debittering agent, pH stabilizer, preservative, desugarizing agent and/or coloring agent, such as lactitol, sorbitol, maltitol, aspartame, stevia, luo han guo, sucralose, xylitol, vanilla, chocolate, fruit flavor, artificial flavor, or mixtures or combinations thereof.
Optionally, the food composition or pharmaceutical composition further comprises vitamins, minerals and/or dietary supplements or prebiotic nutrients, at least one prebiotic, wherein optionally the prebiotic comprises inulin, artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructo-oligosaccharides, galacto-oligosaccharides, isomalto-oligosaccharides, xylo-oligosaccharides, stachyose, oligomannose, arabino-oligosaccharides, resistant dextrins, resistant starches or mixtures or combinations thereof.
Optionally, the food composition or pharmaceutical composition further comprises ubiquinone (CoQ10), lycopene, beta-carotene, tryptophan, vitamin B6, vitamin B12, or mixtures or combinations thereof.
Optionally, the food composition or pharmaceutical composition further comprises a probiotic, wherein optionally the probiotic comprises a cultured or faecally extracted microorganism or bacterium or bacterial component, and optionally the bacterium or bacterial component comprises or is derived from Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium), escherichia coli (E coli), Prevotella (Prevotella), coprinus (Faecalibacterium), Blautia (Blautia), bacteroides (bacteroides), Firmicutes (Firmicutes) and equivalents, or mixtures or combinations thereof.
Optionally, the content of the bifidobacterium lactis BL-11 is 0.5 to 20 parts by weight, preferably 1 to 15 parts by weight, relative to 100 parts by weight of the food composition or the pharmaceutical composition; the bifidobacterium lactis BL-11 is used in the form of live thalli of the bifidobacterium lactis BL-11, inactivated thalli of the bifidobacterium lactis BL-11 or an extract of the bifidobacterium lactis BL-11; the viable count of the bifidobacterium lactis BL-11 is 1.0 multiplied by 106~1.5×1012CFU/g, preferably 3.0X 1010~5.0×1011CFU/g。
Optionally, the food composition is one or more of fermented milk, cheese, milk-containing beverage, solid beverage, powdered milk.
Optionally, the Bifidobacterium lactis BL-11 is administered in an amount of 2.0X 10 based on body weight9CFU~1.5×1011CFU/kg/day, preferably 3.0X 104CFU~8.0×1010CFU/kg/day.
Alternatively, the food or pharmaceutical composition may be a powder, lozenge, liquid, chewing gum, soft candy, tabletted candy, yoghurt, milk, cheese, ice cream, frozen food, health food, pharmaceutical or feed, using different delivery forms and carriers.
The bifidobacterium lactis BL-11 disclosed by the invention can improve intestinal permeability and reduce the level of LPS (low-cholesterol) and D-lactic acid in blood.
The bifidobacterium lactis BL-11 disclosed by the invention can be used for promoting autonomous active activities, improving the memory function of disposable avoidance response and improving the anhedonia caused by stress stimulation.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
Example 1
This example illustrates Bifidobacterium lactis BL-11 and its performance characteristics.
1. Taxonomical characteristics of Bifidobacterium lactis BL-11:
bifidobacterium lactis BL-11 was observed under a microscope, and the results are shown in FIG. 1. The results of the physical and chemical tests are shown in tables 1 and 2.
TABLE 1
TABLE 2
+ represents metabolizable; -means unable to metabolize.
2. Tolerance of bifidobacterium lactis BL-11 to artificial gastric juice and intestinal juice:
bifidobacteria are genera that are generally not acid-fast. In this example, the tolerance of artificial gastric juice and intestinal juice of Bifidobacterium lactis BL-11 of the present invention was tested, while using Bifidobacterium lactis Bb-XX, which is excellent in acid resistance and can survive through the gastrointestinal tract, stored in the laboratory at present, as a comparison.
The survival rate detection result of the BL-11 strain in artificial gastric acid (pH 3) is shown in Table 3, the survival rate of the live bacteria is 44.7 percent when Bb-XX is treated in the artificial gastric acid for 1h, and the survival rate of the live bacteria is 29.5 percent when the Bb-XX is treated in the artificial gastric acid for 3 h; the survival rate of the live bacteria of the bifidobacterium lactis BL-11 treated by the method is 86.2 percent after 1 hour and 39.5 percent after 3 hours. The bifidobacterium lactis BL-11 disclosed by the invention has relatively good gastric acid resistance, and can reach the intestinal tract to play a probiotic role by passing through the stomach most smoothly.
The results of the survival rate of the BL-11 strain in artificial small intestine solution (pH 8) are shown in table 3. The data show that live bacteria survival by Bb-XX was 66.1% after 1 hour treatment in artificial intestinal fluid (pH 8); the survival rate of live bacteria of the bifidobacterium lactis BL-11 treated in the artificial intestinal juice for 1 hour is 67.5 percent, and the survival rate of the two bacteria after the artificial intestinal juice is treated for 3 hours is 49.4 percent and 32.1 percent respectively.
The above results show that bifidobacterium lactis BL-11 can still survive well after digestion with artificial gastric juice and intestinal juice (FIG. 2). The bifidobacterium lactis BL-11 has better digestive juice resistance compared with a reference bacterium, and can successfully survive and colonize in intestinal tracts.
TABLE 3
3. Bifidobacterium lactis BL-11 virulence experiment and safety detection:
inoculating the bifidobacterium lactis BL-11 of the invention into MRS liquid culture medium, carrying out anaerobic culture for 48 hours at 37 ℃, and counting the viable count of the bifidobacterium lactis BL-11 in the culture solution to be 3.7 multiplied by 109CFU/mL, the culture stock was gavaged continuously at a rate of 20.0mL/kg body weight for 3 days, followed by 7 days of observation. Healthy male BALB/C mice, 6-8 weeks old, 16-18g, maintainedRoom temperature (25 +/-2 ℃), relative humidity (55 +/-2)%, 12h/12h illumination, free food and water intake. The results show that: compared with the control group, the culture stock solution of the bifidobacterium lactis BL-11 has no toxic reaction or death observed in the two groups of tested mice, and the weight gain of the mice is not statistically different (p is more than 0.05).
The antibiotic sensitivity of the bifidobacterium lactis BL-11 is evaluated by adopting an SN/T1944-2007 method of determination of bacterial resistance in animals and products thereof. The evaluation results show that the bifidobacterium lactis BL-11 is sensitive to Ampicillin Ampicillin, penicillin G Penicillin G, Erythromycin Erythromycin, Chloramphenicol Chloramphenicol, Clindamycin Clindamycin, Vancomycin Vancomycin, Tetracycline and the like. Meets the requirements of European food Safety Committee (European food Safety Authority) on the evaluation specification of the resistance of the edible bacteria. The bifidobacterium lactis BL-11 does not contain exogenous antibiotic resistance genes and is safe to eat.
Example 2
This example illustrates the preparation of BL-11 lyophilized fungal powder.
Recovering strains of BL-11 preserved bacteria liquid in a water bath kettle at 37 ℃ until all liquid in the freezing storage tube is melted; scribing on an MRS culture medium in a subarea manner, and carrying out anaerobic culture at 37 ℃ for 12-24 hours; selecting single colony, inoculating into an anaerobic culture tube filled with liquid culture medium, sealing, performing anaerobic culture at 37 deg.C for 6-12 hr, monitoring OD600 value of bacterial liquid, stopping growth, and stopping fermentation; carrying out strain fermentation production, carrying out stirring culture at constant temperature of 40 ℃ for 6-12 hours, maintaining the constant pH of the bacterial liquid to be 5.4 +/-0.5, and immediately stopping fermentation until the OD600 value of the bacterial liquid is monitored to stop increasing; centrifugally collecting bacterial sludge according to the following steps: adding a freeze-drying protective agent according to the volume ratio of 1: 1-10, stirring, uniformly mixing, and freeze-drying the bacterial powder in a freeze-drying machine; collecting the freeze-dried powder, crushing according to the quality requirement and packaging.
Example 3
This example illustrates the use of BL-11 lyophilized powder for the production of food products.
The freeze-dried bacterial powder prepared from the bifidobacterium lactis BL-11 can be used for common foods such as yoghourt, fermented milk, cheese and the likeOr a health food. Preferably, the amount of the bifidobacterium lactis BL-11 in the food for preparing the yoghurt is 1.0 x 106CFU~1.0×108CFU/kg, more preferably 1.2X 107CFU~1.5×108CFU/kg. The production mode is direct feeding or post-adding. When the raw milk is directly put into the fermentation tank, the raw milk is taken as a fermentation strain, after the raw milk is sterilized, the raw milk is added in proportion when the raw milk is reduced to a proper temperature, and then the raw milk is fermented for 10 to 48 hours at the temperature of 40 to 43 ℃. The mixture is stirred and compounded and then is subpackaged in a cup or a bag to be used as a finished product. When the post-addition mode is used for adding, after the fermentation of the yoghourt is finished, the yoghourt is added according to a certain proportion and then is uniformly stirred, and after the compounding is finished, the yoghourt is subpackaged to obtain a finished product.
Example 4
This example illustrates the use of BL-11 lyophilized bacterial powder for the production of dietary supplements, probiotics.
The freeze-dried powder prepared from the bifidobacterium lactis BL-11 provided by the invention can be used for producing probiotics. The probiotic freeze-dried powder comprises 0.5-30 parts of BL-11 freeze-dried powder, 5-20 parts of strains of lactobacillus fermentum, lactobacillus helveticus, lactobacillus reuteri, lactobacillus plantarum, bifidobacterium adolescentis, bifidobacterium breve, bifidobacterium longum and the like, 20-70 parts of prebiotics such as galacto-oligosaccharide, fructo-oligosaccharide, inulin and the like, 5-10 parts of nutrients such as GABA, tryptophan, lycopene, beta-carotene, vitamin B6, vitamin B12, coenzyme Q10, taurine, pectin, beta-glucan, fucose, carrageenan, guar gum, dietary fiber and the like, and also comprises 0.1-5 parts of antioxidants/anti-inflammatory substances such as tocopherol, carotenoid, ascorbic acid/vitamin C, ascorbyl palmitate, polyphenol, glutathione, superoxide dismutase and the like. The total number of viable bacteria of the probiotics is added as follows: the addition amount of BL-11 was 2.0X 106CFU~ 2.0×1010CFU/g, more preferably 3X 107CFU~3×1010CFU/g. The single bacterial additive amount of other bacterial is 1 × 106CFU is 3X 109 CFU/g. The preparation process comprises the following steps: weighing raw materials in proportion, mixing, subpackaging, quality inspection and the like.
Example 5
This example illustrates the effect of BL-11 lyophilized powder on improving intestinal permeability and behavior.
Intestinal microorganisms and the gut-brain axis (GBA) exert two-way communication in stress regulation. Microorganisms communicate with the gut-brain axis by different mechanisms. Interact directly with mucosal cells, affect brain development and behavior by immune cells and by contacting nerve endings. Brain stress can also affect gut microbiota, which is responsible for gastrointestinal dysfunction and disorders, by GBA. Stress also affects the synthesis of microbial by-products and precursors that enter the brain through the blood and hindbrain regions, release cytokines through mucosal immune cells, release gut hormones, such as 5-hydroxytryptamine (5-HT), through enteroendocrine cells, or through afferent pathways, including the enteric nervous system.
Preparing a probiotic preparation for intervention: the probiotic preparation is lyophilized powder containing BL-11, beta-carotene, vitamin B6, vitamin B12, coenzyme Q10 and maltodextrin, the control group is maltodextrin, and the intragastric administration dosage of experimental mice is 100 hundred million CFU/mouse/day.
20C 57BL/6J mice, 6 weeks old, were housed in groups of 5 mice per cage, with free access to food and water. Food and water intake were recorded twice weekly. Mice were divided into control and probiotic 2 groups of 10 mice each. After the experimental mice are adapted to the environment for one week, the experimental mice are randomly grouped: model group, probiotic intervention group. The experimental mouse has the following growth environmental conditions: ambient temperature (23 + -2) ° c, relative humidity (50 + -10)%, light pattern (12h dark/12 h light).
The physical stimulation is considered to cause chronic stress model, and the specific stimulation mode comprises the following steps: (1) fasting and water deprivation are carried out for 24 hours; (2) continuously illuminating for 24 h; (3) clamping the tail of the iron clamp at a position of 1cm from the tail tip for 3 min/time; (4) restraint stress, circular sleeve restraint action 2 h/day. 1-2 different kinds of stress stimulation are adopted every day, the stimulation time is not fixed every time, and the molding time is kept for 4 weeks.
And (3) stopping stress stimulation from the sixth week, carrying out a sugar water preference experiment, a diving platform experiment and an open field experiment, and measuring the physical and chemical indexes of each group of mice after the behavior experiment is finished. Data analysis the data obtained were processed using SPSS statistical software, expressed as mean ± standard deviation, and compared between groups using independent sample tests, with P <0.05 considered statistically significant.
1) Sweet water preference experiment
Before the experiment, two identical water bottles are placed on a cage simultaneously, wherein one bottle is filled with pure water, the other bottle is filled with cane sugar water with the concentration of 1%, and the experimental mouse is allowed to adapt to the sugar water for 24 hours. In order to avoid the interference caused by the water drinking habit of the laboratory mouse, the position of the water bottle is changed every 1 h. After the adaptation is finished, the experimental mice are fasted and are forbidden to drink water for 24 hours. One bottle of pure water and one bottle of sucrose solution of 1% concentration were placed on each cage before the start of the experiment, and the consumption of the syrup and pure water was recorded every 3 hours.
The sugar water preference degree is equal to sugar water consumption/(sugar water consumption + pure water consumption) × 100%.
The experimental results showed that the control group had a reduced preference for sugar water (49.63% ± 15.79), whereas after probiotic supplementation, the preference for sugar water was increased (68.79% ± 12.34), indicating that the probiotic was able to ameliorate anorgasmia caused by stress stimulation.
2) Diving platform experiment
Diving tower instrument experimental box (DTT-2 type mouse diving tower, institute of medicine of Chinese academy of medical science). The diving platform box is about 120cm long, 12cm wide and 30cm high and is made of organic glass materials. The total number of the chambers is 6, and each chamber is 12cm in length, 12cm in width and 30cm in height. The experimental box allowed 6 mice to be tested simultaneously. Copper grids are laid on the bottom surface of the experiment box, the copper grids are connected with a power supply through electric wires, and the current voltage is set to be 36V. An insulating table (a pentagonal wood block with the long diameter of 5.7cm, the short diameter of 4.5cm and the height of 4.8 cm) is arranged on the copper grid in the experimental box and close to one corner in the box to serve as a safety area for animals to avoid electric shock. The testing device is connected with the automatic recording system of the computer. During the experiment, a mouse is placed in a diving platform instrument experimental box to adapt to the environment for 5min, then the mouse is lightly placed on a diving platform, the copper grid is electrified, the mouse can be shocked when four limbs of the mouse jump down from the diving platform and contact the copper grid, the normal avoidance response is that the mouse jumps up and returns to a safety zone to avoid the shock, the mouse is learned for 5min, and the electric shock frequency (error frequency) within 5min is recorded, so that the mouse is taken as the learning achievement. And (3) after 24h, carrying out a memory capability test, placing the mouse on a diving platform, and recording the time of the mouse receiving an electric shock from staying on the diving platform to the 1 st jump, namely the latency and the error frequency within 5min (the frequency of electric shocks when the four limbs of the mouse simultaneously contact the copper grid), so as to be used as an evaluation index of the memory function.
Compared with a normal control group, the incubation period of the probiotic group in the jump test is obviously shortened (P is less than 0.05), the error frequency of the jump test has no obvious difference, but the probiotic group shows the trend of reducing the error frequency. Mice in the probiotic group were shown to improve the memory dysfunction in the one-time avoidance response (see table 4).
TABLE 4 Effect of probiotics on avoidance response memory function
|
Number of mistakes (times)
|
Incubation period (S)
|
Control group
|
1.45±0.82
|
268.32±50.63
|
Probiotic group
|
1.07±0.87
|
248.05±82.33
|
P value
|
0.0348
|
0.0782 |
3) Open field experiment
The open field experiment analysis system is used for observing and researching neuropsychiatric changes of experimental animals and various behaviors after the experimental animals enter an open environment, such as fear of the animals to the new open environment, activities mainly in a peripheral area and less activities in a central area, but the exploration characteristics of the animals also promote the animals to generate motivation for activities in the central area, and anxiety psychology generated by the motivation can also be observed. For assessing voluntary activity and anxiety levels in animals.
The mice were moved to the open field experimental test room 60min before the experiment and adapted to the environment in advance. During experiment, the mouse is taken out of the cage and placed in an open field experimental device (the length, the width and the height of the cage are 100cm, x 40cm, the inner surface and the bottom surface are blue, a camera is arranged right above the central area, after the experiment is started, the mouse is placed in the fixed position of the central area, the head is placed towards one side, a shading cover curtain is quickly pulled up, the recording system is opened after the serial number, the date and the state of the mouse are recorded in operation software, a Sudoku mode is selected, the central area proportion is 0.5, the activity condition of the mouse is measured for 5min by a camera above the open field device and a monitor connected with the camera, the measurement indexes comprise the motion time, the total distance, the central area residence time percentage (the central area residence time LPMM ═ s), the central area horizontal motion percentage (the central area horizontal motion distance L horizontal motion distance), the four side horizontal motion distance (the four side horizontal motion distance L horizontal motion distance) and four corners Area horizontal movement percentage (horizontal movement L horizontal movement distance of four corner areas). The number of erections and the number of hair managements were recorded, and then the inside environment was cleaned with 75% alcohol, and after the alcohol was evaporated and smelled, the next 1 operation was performed. The results are shown in Table 5.
Compared with the control group, the probiotic group mice have significantly higher central zone entry frequency and central zone residence time than the control group (P < 0.05). The number of erections and grooming was significantly increased (P <0.05), while the central zone entry number was not very different. The rest open-field ethology results have no obvious difference.
TABLE 5 Experimental results of the open-field behavior of mice
4) Intestinal permeability detection
To evaluate intestinal permeability in vivo, D-lactic acid in serum was measured, and LPS content was used to evaluate intestinal permeability.
D-lactic acid is a metabolite of bacterial fermentation, produced by a variety of bacteria in the intestinal tract, and is normally poorly absorbed into the blood even after ingestion from food, and mammals do not have an enzyme system to rapidly degrade it. Therefore, when the permeability of the intestinal mucosa increases, a large amount of D-lactic acid produced by bacteria in the intestinal tract enters the blood through the damaged mucosa, so that the level of D-lactic acid in the blood is increased.
The lactic acid has D type and L type, only L-lactic acid is normally generated by human body, and D lactic acid can be generated by microorganisms such as bacteria. Monitoring the level of D-lactic acid in blood can reflect the damage degree and permeability change of intestinal mucosa in time. Can be used for auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory reaction, repeated fever, emesis, etc.
Lipopolysaccharide (LPS), also known as bacterial endotoxin, is a component of the cell wall of gram-negative bacteria, LPS being a toxic substance to animals. The structure of LPS can be divided into 3 parts: glycolipid domain-lipid a, short chain of sugar residues-core oligosaccharide, hypervariable polysaccharide domain-O antigen. The structure of LPS determines its agonist/antagonist action on TLR 4. In vivo, LPS binds to the TLR4/MD-2 receptor complex, activating different signaling pathways through Myd 88-dependent or TRIF-dependent pathways; different parts of intestinal epithelial cell TLR expression levels are different, inflammatory response caused by LPS can be prevented, and pathogenic bacteria can be resisted.
Bacterial translocation is the passage of viable intestinal bacteria from the intestine across the epithelial mucosa into the body. Bacteria can enter the lymphatic system via mesenteric lymph nodes and circulate throughout the body. Bacteria can also enter the blood circulation, leading to bacteremia, and can also be located in tissues. Bacterial translocation can cause bacterial overgrowth in the small intestine, intestinal injury and shock. Any stress response, both psychological and physiological, that results in intestinal permeability can potentially lead to bacterial translocation.
LPS is involved in the pathogenesis of various diseases, such as IBD, enterocolitis and other intestinal diseases, and even Parkinson's disease and Alzheimer's disease. LPS can not only enter the blood, but also enter and remain in the brain for a lifetime, possibly causing alzheimer's disease.
The level of LPS in the blood reflects the permeability of the intestinal tract, LPS is not allowed to enter the normal intestinal barrier, and the higher level of LPS in the blood indicates that intestinal bacteria or LPS are shifted to the blood, which indicates that the permeability of the intestinal tract is increased and the probability of intestinal leakage symptoms is increased. The content of LPS in blood can indicate inflammatory reaction and stress state, and excessive LPS can cause the abnormality of human immune system, and cause chronic or acute inflammatory reaction, and acute inflammation such as fever, pain, etc. Can be used for auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory reaction, repeated fever, emesis, mental disease, stress reaction, etc.
After the experiment was completed, blood was collected from the tail vein terminal. Blood was centrifuged at 3000g for 15 minutes. The content of D-lactic acid and LPS in serum is detected by adopting an intestinal barrier function analysis system (JY-DLT, Beijing Zhongshengjin domain diagnostic technique GmbH) according to an operation instruction.
The results showed a significant reduction in LPS, D-lactic acid levels in the probiotic group compared to the control group (P < 0.05). The stress stimulation is shown to result in the increase of intestinal permeability, and the probiotics can reduce the intestinal permeability and reduce the risks of endotoxemia, systemic inflammatory reaction and the like.
Example 6
This example illustrates the effect of BL-11 lyophilized bacterial powder on the composition of intestinal microorganisms.
After the above behavioural experiments were completed, the collected cecal contents were stored at-80 ℃. Two groups of mouse feces were collected and DNA of fecal flora was extracted using TIANMap fecal DNA kit (TIANGEN, catalog number DP 328). The extracted DNA was quantitatively detected using a Qubit instrument. Detection using 1% agarose gel electrophoresis: the voltage is 100V and 40 min. Photographing recording by a UVI gel imaging system: the DNA electrophoresis has no miscellaneous band and tailing, which indicates that the DNA fragment has good purity and no obvious degradation. An appropriate amount of sample was taken in a centrifuge tube, and the sample was diluted to 1ng/ul with sterile water. The DNA was stored in a freezer at-20 ℃ until use.
Bacterial 16S rRNA gene amplification: the V3-V4 region of the bacterial 16S rRNA gene was amplified using bacterial universal primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) using Barcode specific primers based on the selection of the sequenced region using diluted genomic DNA as a template. 100ng of the extracted DNA was subjected to PCR at 56 ℃ for strand renaturation, and denaturation at 94 ℃ was started for 4 minutes, followed by 30 cycles of 94 ℃ reduction for 30 seconds, 56 ℃ reduction for 30 seconds, and 72 ℃ reduction for 1 minute.
Sequencing of amplicon gene: constructing a Library by using a Library construction Kit of TruSeq DNA PCR-Free Library Preparation Kit of Illumina company, carrying out Qubit quantification and Library detection on the constructed Library, and carrying out flora sequencing by using an Illumina HiSeq2500 PE250 sequencing platform after the constructed Library is qualified.
Processing and analysis of sequencing data: the raw data from the colony sequencing was imported into QIIME (2019.4), denoised with DADA2 to obtain representative amplicon variants (ASVs) and constructed into phylogenetic trees. After quality control, the filtered ASVs are used
The layer classifier (NBC) method was aligned and species annotated to gene sequences in the Greenenges (V-13.5) database. In performing Alpha and Beta diversity analysis, the depth of resampling was 10000 sequences per sample to ensure sufficient sequence. In order to reduce the influence of excessive species on the results, the results are corrected by calculating False Discovery Rate (FDR).
The results are shown in FIGS. 4 to 7 and Table 6. The results show that at the genus level, the control group and the probiotic intestinal flora are obviously different. The control group had a significantly lower bifidobacterium content than the probiotic group. Furthermore, the alpha diversity index was also higher in the probiotic group than in the control group, with insignificant difference in the indices (Shannon index P-0.9118 (Mann-Whitney stability); Simpson index P-0.35268 (Mann-Whitney stability)). The overall gut flora composition of the control and probiotic groups differed on the beta diversity analysis, with the two groups being clearly distinguishable on the PCoA analysis (F-value 2.4268; R-squared 0.1188; p 0.009 (PERMANOVA)).
At the genus level, there are more specific species of the difference, with coprobacterium increasing significantly after probiotic use (FDR < 0.001).
TABLE 6 analysis of differences in the genus-horizontal intestinal flora between the two groups
|
Control group
|
Probiotic group
|
Pvalues
|
FDR
|
D_5__Coprobacillus
|
38.183
|
55075
|
2.17E-07
|
1.93E-05
|
D_5__Ruminiclostridium
|
9582.9
|
467.42
|
2.69E-06
|
0.00011987
|
D_5__Butyricicoccus
|
4825.7
|
1522.6
|
9.28E-06
|
0.00018877
|
D_5__Ruminococcaceae_UCG_003
|
9141.7
|
2175.5
|
9.41E-06
|
0.00018877
|
D_5__Clostridium_innocuum_group
|
296.9
|
79573
|
1.06E-05
|
0.00018877
|
D_5__Erysipelatoclostridium
|
1063.5
|
33313
|
3.11E-05
|
0.00046104
|
D_5__Alistipes
|
280680
|
64967
|
3.89E-05
|
0.00049419
|
D_5__Parasutterella
|
24852
|
58661
|
0.00016202
|
0.001442
|
D_5__Ruminococcus_1
|
51491
|
19309
|
0.00025718
|
0.0017088
|
D_5__Dorea
|
1648.8
|
322.35
|
0.00030703
|
0.0017088
|
D_5__Odoribacter
|
3703.3
|
917.7
|
0.00037746
|
0.0018663
|
D_5__Rikenella
|
106830
|
18411
|
0.00054891
|
0.0020444
|
D_5__Helicobacter
|
41385
|
16121
|
0.00057426
|
0.0020444
|
D_5__Roseburia
|
44371
|
27314
|
0.0010615
|
0.0033072
|
D_5__Faecalibacterium
|
766540
|
300440
|
0.0012887
|
0.0036999
|
D_5__Prevotella_9
|
434970
|
22910
|
0.0025961
|
0.0061604
|
D_5__Sutterella
|
84556
|
31719
|
0.0026303
|
0.0061604
|
D_5__Dialister
|
12917
|
5567.9
|
0.0027349
|
0.0062412
|
D_5__Oscillibacter
|
1843.5
|
892.9
|
0.0032466
|
0.0068797
|
D_5__Bifidobacterium
|
23109
|
477820
|
0.018848
|
0.029955
|
D_5__Desulfovibrio
|
18676
|
36259
|
0.027401
|
0.041334
|
D_5__Enterococcus
|
182820
|
47499
|
0.034995
|
0.051058
|
D_5__Escherichia_Shigella
|
5240.1
|
223620
|
0.04823
|
0.068135
|
D_5__Streptococcus
|
5183.5
|
16444
|
0.049637
|
0.069026
|
D_5__Prevotellaceae_NK3B31_group
|
17987
|
3146.2
|
0.11963
|
0.15211
|
D_5__Ruminococcaceae_UCG_014
|
50049
|
18180
|
0.13508
|
0.16932
|
D_5__Lactobacillus
|
2479500
|
884430
|
0.16394
|
0.19717
|
D_5__Akkermansia
|
91396
|
419080
|
0.28756
|
0.33238
|
D_5__Ruminococcus_torques_group
|
21859
|
24878
|
0.36456
|
0.40557
|
D_5__Blautia
|
312350
|
580010
|
0.43421
|
0.4771
|
D_5__Staphylococcus
|
26482
|
5180.9
|
0.47398
|
0.51444
|
D_5__Prevotellaceae_UCG_001
|
96887
|
135950
|
0.53159
|
0.56323
|
D_5__Bacteroides
|
1039200
|
1192500
|
0.57971
|
0.59993 |
Example 7
This example serves to illustrate the results of probiotic intervention on a 3 year old 6 month child diagnosed with Autism Spectrum Disorder (ASD) by a probiotic consisting of BL-11 bacteria. In the form of oral lyophilized powder, the administration period is 90 days, and the dosage is 2 times per day, 500 hundred million CFU per time. After the medicine is taken, the defecation habit of the children is changed, and the defecation frequency is changed from one time in 3-5 days to one time in 1-2 days; parents feed back the vocabulary expression ability of children, socialization and the frequency of eye-to-eye communication are increased, and the spoken vocabulary is increased by 3-5 words. The self-sensing frequency of the active feedback is increased, and the number of times of actively proposing the demand is increased. Less repetitive motion than before intervention. The parent chooses to continue taking and still observes and records the continuous improvement.
Example 8
This example illustrates the improvement of Attention Deficit Hyperactivity Disorder (ADHD) by probiotics consisting of BL-11 bacteria.
Cases were obtained from the hospital for children in Hebei province, and physicians diagnosed according to ADHD criteria in the diagnostic and statistical manual of mental disorders, aged 6.5 years, and were of the hyperactive-impulsive type, excluding schizophrenia, affective disorders, epilepsy, and other organic diseases. It also has chronic constipation and abdominal discomfort.
The treatment mode adopts oral probiotic freeze-dried powder for treatment, and the dosage is as follows: 2 times a day, 600 hundred million CFU each time. Taken with warm water after breakfast and supper. Treatment was continued for 12 weeks. Evaluation was performed using the Conners parental questionnaire, including behavioral, somatic, anxiety, learning, hyperactivity, and other indicators. The results showed that the children had decreased impulsive, anxious and hyperkinetic scores, improved defecation function, changed from once every 3-4 days to 1-2 times a day, sometimes 2-3 days with 1 defecation, increased stool volume and gradually disappeared abdominal distension and pain. The overall spirit improvement is obvious. The parent is satisfied with the result.
TABLE 7 Conners parental questionnaire results before and after treatment
|
Behavior
|
Study of
|
Heart body
|
Impulse
|
Anxiety disorder
|
Multiple action
|
Before treatment
|
1.6
|
2.1
|
0.35
|
1.34
|
0.52
|
1.38
|
After treatment
|
1.2
|
1.8
|
0.29
|
1.32
|
0.35
|
1.23 |
The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.