Bifidobacterium pseudominor chain for high-yield short-chain fatty acid and application of bifidobacterium pseudominor chain

Abstract

The invention relates to the technical field of functional probiotic screening and application, and particularly provides a strain of Bifidobacterium pseudocatenatum (Bifidobacterium pseudocatenatum) and application of the strain of Bifidobacterium pseudocatenatum (Bifidobacterium pseudocatenatum). The bifidobacterium pseudocatenuicola is screened from excrement of healthy infants, the preservation number is CGMCC No.28940, and the bifidobacterium pseudocatenuicola can produce short-chain fatty acid at high yield, has relatively strong adhesion and antibacterial ability, has a certain regulation effect on flora diversity of constipation patients, can be widely applied to production of foods, health care products or medicines, and has a wide prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic screening and application, and particularly relates to a strain of Bifidobacterium pseudocatenulatum capable of highly producing short-chain fatty acids and an application thereof. Background Art

[0002] The human intestine is densely populated with various microorganisms, and the intestinal microbiome has become an important frontier for understanding human homeostasis and disease development. Studies have shown that when intestinal microorganisms are out of balance, it can cause the occurrence of a variety of intestinal diseases. Constipation is one of the most common functional gastrointestinal diseases, characterized by recurrent abdominal pain or discomfort, which seriously affects the quality of life and also seriously threatens physical health. In the past decade, intestinal microbiome regulation has developed into a new therapeutic strategy, and metabolites derived from intestinal flora have also become important targets for the treatment of many intestinal diseases. Studies have shown that the main characteristics of constipation patients are a relative decrease in beneficial bacterial genera, a relative increase in potential pathogenic bacteria, and a decrease in species richness. Intestinal microorganisms can affect intestinal motility and the intestinal environment through metabolites produced by bacterial fermentation.

[0003] Bifidobacterium, a typical Gram-positive polymorphic bacillus, is an obligate anaerobe with an optimal growth temperature of 37°C to 41°C. It can be shaped like a Y, V, curved, or spatula. The colonies are smooth, convex, with intact edges and a creamy white color. As one of the most representative members of the intestinal microbiome, Bifidobacterium is also an important probiotic with multiple important physiological functions. For example, Bifidobacterium can improve intestinal diseases caused by immune disorders; produce nutrients needed by the intestines, promote the growth and repair of the intestinal mucosa, effectively regulate intestinal function and resist the invasion of foreign bacteria; scavenge free radicals and increase the activity of the host's antioxidant enzymes, thereby alleviating oxidative damage to the body and delaying aging; and improve constipation and diarrhea caused by intestinal flora imbalance, which is closely related to human health.

[0004] By systematically developing and utilizing probiotic strain resources, we can provide more comprehensive and precise solutions for human health, while promoting related scientific research and industrial development. Summary of the Invention

[0005] The present invention aims to provide a strain of Bifidobacterium pseudocatenulatum and its use. The strain is screened from the feces of healthy infants and young children, can produce high levels of short-chain fatty acids, has strong adhesion and antibacterial properties, has a certain regulatory effect on the flora diversity of constipation patients, and has broad application prospects.

[0006] On the one hand, the present invention relates to a Bifidobacterium pseudocatenulatum, named Bifidobacterium pseudocatenulatum BP-18 strain, which was deposited in the General Microbiological Culture Collection Center of the China Culture Collection Administration on November 10, 2023, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 28940.

[0007] The present invention also relates to the use of Bifidobacterium pseudocatenulatum BP-18 strain in the preparation of food or health products.

[0008] The present invention also relates to the use of Bifidobacterium pseudocatenulatum BP-18 strain in the preparation of medicines for preventing or treating constipation.

[0009] The present invention also relates to the application of Bifidobacterium pseudocatenulatum BP-18 strain in the production of short-chain fatty acids.

[0010] The present invention also relates to a probiotic preparation comprising Bifidobacterium pseudocatenulatum BP-18 strain.

[0011] The present invention also relates to a probiotic yogurt, which is prepared by using Bifidobacterium pseudocatenulatum BP-18 strain as a fermentation strain.

[0012] The preparation method of the probiotic yogurt is:

[0013] (1) Sterilization of raw materials: Mix the raw materials according to the proportion, stir for 20 minutes, mix evenly, sterilize, and homogenize;

[0014] (2) Inoculation: Inoculate the activated bacterial solution of Bifidobacterium pseudocatenulatum BP-18 into the above raw materials at a volume ratio of 3-4%, and stir thoroughly;

[0015] (3) Fermentation: Ferment at 42°C for at least 6 hours. When the acidity is below 4.5, the fermentation can be stopped and the temperature can be quickly lowered to 10°C.

[0016] (4) Filling: Aseptic filling to obtain probiotic yogurt.

[0017] The components of the raw materials in step (1) and their weight parts are respectively: 90 parts of fresh milk, 0.8 parts of whole milk powder, 2 parts of concentrated whey protein, 3 parts of xylitol, 2 parts of food additives, and 3 parts of inulin.

[0018] The food additives include sodium hydroxymethyl cellulose, citric acid, sodium citrate, pectin and natural vitamins.

[0019] The present invention screened out Bifidobacterium pseudocatenulatum BP-18 from the feces of healthy infants and young children, has good biosafety, strong acid production ability, and a calcium-dissolving zone diameter of 5.6±0.04 mm, which is significantly higher than that of the control bacteria; the ability to produce short-chain fatty acids is stronger, among which the production of acetic acid, propionic acid, and butyric acid is the highest, reaching 28.00±0.67μg / mL, 10.91±0.80μg / mL, and 14.54±0.15μg / mL, respectively; the total acid production reaches 61.77±2.87μg / mL, which is 120% higher than that of the control bacteria, achieving unexpected technical effects, helping to regulate intestinal balance and improve intestinal function.

[0020] Bifidobacterium pseudocatenulatum BP-18 has strong self-aggregation ability and hydrophobic properties, with a hydrophobicity of 81%±0.08%. The self-aggregation ability is proportional to the culture time and can reach 85%±0.04% after 24 hours of culture. It can effectively adhere to the intestinal mucosa and play a prebiotic role.

[0021] Bifidobacterium pseudocatenulatum BP-18 has a strong antibacterial ability and has a significant inhibitory effect on Escherichia coli, Salmonella and Staphylococcus aureus, with the maximum inhibition zone diameter reaching 19.7mm.

[0022] Bifidobacterium pseudocatenulatum BP-18 can maintain good stability and survival ability in the digestive tract environment. After being treated in artificial gastric juice for 2 hours, its survival rate is as high as 85%, which is significantly higher than that of the control bacteria; after being treated in artificial intestinal fluid environment for 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours, its survival rate is significantly higher than that of the control bacteria; after being treated in artificial gastrointestinal fluid for 24 hours, Bifidobacterium pseudocatenulatum BP-18 still has a survival rate of 30%, which is conducive to its colonization in the intestine and exerting a prebiotic effect.

[0023] Bifidobacterium pseudocatenulatum BP-18 can increase the content of short-chain fatty acids in constipated feces. The total short-chain fatty acid content of fecal microbial metabolism is as high as 50.664 mg per gram, which is significantly higher than that of the control bacteria. Among them, the fecal microbial metabolism of acetic acid, butyric acid and hexanoic acid is more significant, and the acetic acid content is as high as 17.62484 mg / g, which has obvious advantages compared with the control group. It is beneficial to lower intestinal pH, improve the intestinal physical and chemical environment, and promote colon peristalsis.

[0024] Bifidobacterium pseudocatenulatum BP-18 can significantly increase the diversity of bacteria in constipated feces, increase the accumulation of beneficial bacteria such as Bifidobacterium and Lactobacillus, and reduce the abundance of pathogenic bacteria Shigella in feces, thereby helping to regulate intestinal flora homeostasis.

[0025] In summary, the Bifidobacterium pseudocatenulatum BP-18 provided by the present invention has good physiological properties, can be widely used in the production of food, health products and medicines, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the colony morphology of Bifidobacterium pseudocatenulatum BP-18;

[0027] Figure 2 This is the morphological diagram of Bifidobacterium pseudocatenulatum BP-18 strain;

[0028] Figure 3 This is a scanning electron microscope observation of Bifidobacterium pseudocatenulatum BP-18;

[0029] Figure 4 This is the phylogenetic tree of 16srDNA sequences of Bifidobacterium pseudocatenulatum BP-18;

[0030] Figure 5 This is the trend diagram of pH change over time during the fecal digestion process of each experimental group;

[0031] Figure 6 The changes in fecal bacteria density in each experimental group;

[0032] Figure 7 Dilution curve analysis diagram for each experimental group;

[0033] Figure 8 PCoA analysis diagram of community structure of each experimental group;

[0034] Figure 9 The bar graph shows the abundance of different microbial communities at the genus level in each experimental group;

[0035] Figure 10 This is a comparison chart of the content of short-chain fatty acids in each experimental group after digestion;

[0036] Figure 11 Heatmap analysis chart of the correlation environmental factors of each experimental group;

[0037] Figure 12 This is the RDA analysis chart of short-chain fatty acids in each experimental group. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions of the present invention in conjunction with specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall fall within the scope of protection of the present invention.

[0039] The liquid culture medium in the present embodiment is formulated as follows: 10 g tryptone, 10 g beef extract, 5 g yeast extract powder, 20 g glucose, 2 g anhydrous sodium acetate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate monohydrate, 2 g diammonium hydrogen citrate, 2.6 g dipotassium hydrogen phosphate trihydrate, 1 ml Tween, 0.5 g L-cysteine hydrochloride, and 1 L distilled water. The pH is adjusted to 6.7 ± 0.5. Sterilize at 121°C for 20 min.

[0040] Example 1: Isolation statistics of experimental strains screened from feces of healthy infants

[0041] The present invention isolates and screens a strain of bifidobacterium from fecal samples of healthy infants (the guardians of the fecal sample volunteers are informed and have not taken any antibiotics and no history of prebiotics or probiotics in the three months before the study).

[0042] The bifidobacteria were inoculated into modified MRS liquid medium for activation, cultured in an anaerobic incubator at 37°C for 24 hours, and subcultured for three generations to restore strain viability. After the strains grew well, Gram staining and microscopic examination were performed. The isolates were preserved, and genomic DNA was extracted for subsequent analysis.

[0043] The colony morphology of the bifidobacterium is as follows Figure 1 As shown in the figure, the colony surface is smooth, milky white and round. After Gram staining, the morphology of the strain under the microscope is as follows Figure 2 As shown, it is Gram-positive, short rod-shaped, and bifurcated; its morphology under scanning electron microscopy is as follows Figure 3 As shown, the strain is partially bifurcated, with the head and tail swollen and shaped like a mallet, which is consistent with the growth state of Bifidobacterium at different stages.

[0044] The 16S rDNA sequencing result of the bifidobacterium, SEQ ID NO: 1, was uploaded to the NCBI database for BLAST comparison. The comparison results showed that the bifidobacterium was Bifidobacterium pseudocatenulatum. Further, a phylogenetic tree was constructed using MEGA 7.0 software, as shown in FIG. Figure 4 As shown, the species classification status of the bifidobacterium was determined to be Bifidobacterium pseudoseudocatenulatum, and it was named BP-18.

[0045] On November 10, 2023, the present invention deposited Bifidobacterium pseudocatenulatum BP-18 in the General Microbial Culture Collection Center of China Committee for Culture Collection of Microorganisms, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 28940.

[0046] Example 2: Analysis of the ability of Bifidobacterium pseudocatenulatum BP-18 to produce short-chain fatty acids

[0047] Studies have confirmed that short-chain fatty acids, as important metabolites of intestinal microorganisms, play an important role in inhibiting the growth of intestinal pathogens, maintaining the intestinal barrier, oxidative stress, inflammation and enhancing immunity. Therefore, they are also considered to be the key material basis for the intestinal flora to exert its regulatory role. Strains with high short-chain fatty acid production have therefore become the focus of research.

[0048] The experiment used GC-MS to determine the content of short-chain fatty acids in the fermentation products of Bifidobacterium pseudocatenulatum BP-18, and three parallel experiments were set up; at the same time, Bifidobacterium bifidum CICC6071 (purchased from China Industrial Microorganism Culture Collection Center) was used as a control.

[0049] Experimental Method: Take an appropriate amount of fermentation broth and centrifuge it at 10,000 rpm for 10 minutes at 4°C. Take 500 μL of the supernatant and mix it with equal proportions of ether. Vortex for 2 minutes to allow the supernatant and organic solvent to react fully. Place the vortexed mixture on a floating plate and sonicate at 400W for 3 minutes. Allow it to stand for 10 minutes and filter it through a 0.22 μm inorganic filter membrane before testing. Short-chain fatty acid content in different strains was determined using gas chromatography-mass spectrometry (GC-MS).

[0050] GC-MS analysis conditions: chromatographic column: HPFF-AP, injection port temperature: 250°C; detector temperature: 250°C; programmed temperature: initial temperature 80°C, increase the temperature to 140°C at 7.5°C / min and hold for 1 min, increase the temperature to 200°C at 15°C / min and hold for 3 min; split ratio: 10:1; injection volume: 1.0 μL.

[0051] Table 1 Analysis of short-chain fatty acid production (μg / mL)

[0052]

[0053]

[0054] The test results are shown in Table 1. Compared with the control bacteria, Bifidobacterium pseudocatenulatum BP-18 has a stronger ability to produce short-chain fatty acids, among which the production of acetic acid, propionic acid, and butyric acid is the highest, reaching 28.00±0.67μg / mL, 10.91±0.80μg / mL, and 14.54±0.15μg / mL, respectively; the total acid production reaches 61.77±2.87μg / mL, which is 120% higher than that of the control bacteria, achieving unexpected technical effects. The high production of short-chain fatty acids by the Bifidobacterium pseudocatenulatum BP-18 strain will help regulate intestinal balance and improve intestinal function.

[0055] Example 3: Calcium-dissolving circle rescreening experiment on acid production capacity of Bifidobacterium pseudocatenulatum BP-18

[0056] The calcium dissolution circle is also an experimental method to verify the acid-producing ability of a strain. The principle is that the acidic substance produced by the strain reacts with CaCO3 to form a dissolution circle. The acid-producing ability of the strain is judged based on the size of the dissolution circle.

[0057] In this example, the calcium dissolution circle method was used to verify the ability of Bifidobacterium pseudocatenulatum BP-18 to produce short-chain fatty acids, with Bifidobacterium bifidum CICC6071 as a control.

[0058] The experimental method involves the following steps: Using an anaerobic workbench, a Bifidobacterium culture solution is diluted with sterile saline in a gradient fashion, with triplicate dilutions performed for each dilution. 1 mL of the appropriate gradient is then transferred to a sterile Petri dish and poured with MRS-CaCO₃ medium. After the medium solidifies, the dish is incubated in an anaerobic incubator at 37°C for 48 hours. After completion of the incubation period, the strain's acid production (expressed as the diameter of the calcium-dissolving zone, in mm) is observed.

[0059] Table 2 Calcium dissolution circle analysis

[0060]

[0061] From the data in Table 2, it can be seen that the acid production capacity of Bifidobacterium pseudocatenulatum BP-18 provided by the present invention is significantly higher than that of the control Bifidobacterium.

[0062] Example 4: Analysis of the probiotic properties of Bifidobacterium pseudocatenulatum BP-18

[0063] 1. Self-aggregation and hydrophobic properties

[0064] Self-aggregation has been shown to play a role in promoting the persistence and survival of intestinal bacteria in the host gastrointestinal tract. The high hydrophobicity of bacteria is conducive to the survival of strains in the intestine and the maintenance of intestinal homeostasis, thereby colonizing the intestine and exerting prebiotic functions.

[0065] The experimental method includes the following steps: In an anaerobic workbench, the overnight culture of Bifidobacterium pseudocatenulatum BP-18 strain was centrifuged at room temperature for 10 minutes at 4500r / min, the cells were collected, and the cells were washed twice with sterile PBS (pH=7.2) buffer. Subsequently, the obtained cells were adjusted to a concentration of A 600 To 1 (A0), incubate at 37°C for 24 hours. Carefully aspirate the upper bacterial suspension at 2, 4, 6, 12, and 24 hours and measure the OD600nm value (A1). Repeat the measurement three times for each sample. Use PBS buffer as the control group and calculate the agglutination rate according to the following formula. In addition, use Bifidobacterium bifidum CICC6071 as a control.

[0066] The calculation formula is as follows:

[0067] Self-aggregation rate (%) = (A0-A1) / A0.

[0068] The hydrophobicity test of the strain was carried out in the same manner as above, and the concentration of the bacterial suspension was adjusted to A. 600 To 1 (A0), take 1 ml of xylene and add it to 3 ml of bacterial suspension, incubate at room temperature for 10 minutes, vortex and shake for 2 minutes, incubate at room temperature for 30 minutes, separate the layers, carefully aspirate the aqueous phase, and use sterile PBS buffer as the control group to measure the A of each group after xylene treatment. 600 The hydrophobicity is calculated using the following formula:

[0069] The calculation formula is as follows:

[0070] Hydrophobicity (%) = (A0-A) / A0.

[0071] Table 3 Comparison of self-aggregation and hydrophobicity

[0072]

[0073] As shown in Table 3, compared with the control bacteria, Bifidobacterium pseudocatenulatum BP-18 has stronger self-aggregation ability and hydrophobic properties, with its hydrophobicity reaching 81%±0.08%. The self-aggregation ability is proportional to the culture time and can reach 85%±0.04% after 24 hours of culture, achieving unexpected technical results.

[0074] 2. Antibacterial activity

[0075] Bifidobacterium can resist the invasion of certain pathogens in the human intestine and is of great significance for maintaining body health and protecting intestinal homeostasis. In this experiment, the quality control strain Bifidobacterium bifidum CICC6071 was used as the control bacteria, and the Oxford cup method was used for antibacterial test. Sterile MRS culture medium was used as the control, and Salmonella, Staphylococcus aureus and Escherichia coli were selected as biological indicator bacteria.

[0076] The experimental method is as follows: 200 μL of activated pathogenic bacteria culture fluid was evenly spread onto MRS solid medium using a sterile spreading rod. A sterilized Oxford cup was placed on top, and 100 μL of cultured Bifidobacterium pseudocatenulatum BP-18 was added. The culture was incubated at 37°C for 24 hours. Each experiment was repeated three times, and the diameter of the inhibition zone was measured and the average of the three replicates was used. The results are shown in Table 4.

[0077] Table 4 Analysis of antibacterial ability of Bifidobacterium pseudocatenulatum BP-18

[0078]

[0079] As shown in Table 4, Bifidobacterium pseudocatenulatum BP-18 has a strong antibacterial ability and has a significant inhibitory effect on Escherichia coli, Salmonella and Staphylococcus aureus, with the maximum diameter of the inhibition zone reaching 19.7 mm.

[0080] Example 5: Safety Analysis of Bifidobacterium pseudocatenulatum BP-18

[0081] The antibiotic tolerance of a strain is one of the experimental methods for testing the safety of probiotics. In this example, the sensitivity of Bifidobacterium pseudocatenulatum BP-18 to eight antibiotics was detected by the MIC method.

[0082] The experimental method is as follows: the strain of Bifidobacterium pseudocatenulatum BP-18 was inoculated into the liquid culture medium at a rate of 3%, and anaerobically cultured at 37°C for 16-18 hours. The concentration of viable bacteria was determined using a microplate reader at an absorbance of 600. The concentration of the bacterial solution was diluted with broth culture medium to obtain the OD value. 600 nm=1.0. Prepare the stock solution of antibiotics with distilled water or anhydrous ethanol, dilute each antibiotic to the maximum concentration required for the experiment with broth culture medium, and then dilute 10 gradients of 1024, 512, 256, 128, 64, 32, 16, 8, 4, and 2 with broth culture medium (unit: μg / ml). Set up blank controls with physiological saline and culture medium. Use 96-well plates to add 100 μl of different concentrations of antibiotics and culture medium of Bifidobacterium pseudocatenulatum BP-18, with 3 parallels for each antibiotic. After anaerobic culture at 37°C for 24 hours, observe the growth of the strain. The lowest concentration at which the strain does not grow is recorded as the MIC of the antibiotic. The results are shown in Table 5.

[0083] Table 5 Resistance of Bifidobacterium pseudocatenulatum BP-18 to 8 antibiotics

[0084]

[0085] From the data in Table 5, it can be seen that Bifidobacterium pseudocatenulatum BP-18 is sensitive to sulfadiazine (Sdz), gentamicin sulfate (GentSulf), chloramphenicol (Cap) and ceftriaxone sodium (Cas), has good biosafety, and meets the screening criteria for probiotics.

[0086] Example 6: Analysis of the tolerance of Bifidobacterium pseudocatenulatum BP-18 to gastrointestinal fluid

[0087] The prerequisite for probiotics such as Bifidobacterium to work is that the strains can successfully pass through the acidic environment of the gastrointestinal tract and reach the intestine in the form of live bacteria to work.

[0088] Experimental method: The strain of Bifidobacterium pseudomicrocystis BP-18 was cultured under anaerobic conditions at 37°C and inoculated with 1% of the inoculum in a liquid culture medium containing artificial simulated gastric juice and intestinal juice (bile salts) and adjusted to OD 600 =1, take bacterial liquid at different time periods to measure its OD value, the experiment was set up in triplicate, and Bifidobacterium CICC6071 was used as the positive control.

[0089] Table 6 Survival rate of Bifidobacterium pseudocatenulatum BP-18 in artificial digestive fluid (%)

[0090]

[0091]

[0092] The experimental results are shown in Table 6. The Bifidobacterium pseudocatenulatum BP-18 provided by the present invention can better resist the influence of saliva digestion; after being treated in artificial gastric juice for 2 hours, its survival rate is as high as 85%, which is significantly higher than that of the control bacteria; after being treated in an artificial intestinal fluid environment for 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours, its survival rate is significantly higher than that of the control bacteria; after being treated in artificial gastrointestinal fluid for 24 hours, the survival rate of Bifidobacterium pseudocatenulatum BP-18 is still 30%.

[0093] The above results show that the Bifidobacterium pseudocatenulatum BP-18 provided by the present invention can maintain good stability and survival ability in the digestive tract environment, achieves unexpected technical effects, and is conducive to its wide application.

[0094] Example 7: In vitro intervention of Bifidobacterium pseudocatenulatum BP-18 in the intestinal environment of constipation

[0095] Research has shown that in vitro fecal digestion, a method that utilizes fecal microorganisms to ferment food, can simulate the digestive process by inoculating fecal microorganisms, and the results can be used to evaluate candidate probiotics and prebiotics. To date, in vitro fecal digestion has been used to simulate the digestion of a variety of substances. This study used a constipated fecal digestion model to simulate the effects of Bifidobacteria on the intestinal microbiome and intestinal metabolites.

[0096] Experimental method: 6 g of feces were collected using a disposable sterile sampler and immediately transferred to an anaerobic operating chamber and mixed with sterile saline (containing 0.05% cysteine hydrochloride) and vortexed to obtain a (10% w / v) fecal homogenate. The fecal homogenate was centrifuged (3000 r, 4°C, 5 min) to obtain a supernatant. 1 mL of the obtained supernatant was mixed with 9 mL of a basal nutrient growth medium (pH 5.5) containing a normal carbon source content. The basal nutrient medium preparation (per liter) included: 2 g peptone, 20 g glucose, 2 g yeast extract, 0.1 g NaCl, 0.04 g K2HPO4, 0.04 g KH2PO4, 0.01 g MgSO4·7H2O, 0.01 g CaCl2·6H2O, 2 g NaHCO3; Tween-80 2 mL, L-cysteine 0.5 g, ox bile powder 0.5 g, vitamin K 110 mL, bile salts 0.5 g, and cysteine 0.5 g. Incubate at 37°C under anaerobes for up to 48 h. Use basal nutrient medium as a blank control and CICC6071 as a Bifidobacterium control. Immediately after the experiment, collect the culture medium and store it at -80°C until further analysis.

[0097] 1. Effect of Bifidobacterium pseudocatenulatum BP-18 on fecal bacteria density

[0098] During in vitro fecal digestion, OD 600 The changes in bacterial abundance were analyzed, the pH changes of digestive fluid from 0h to 48h of digestion were measured, and the trend of acid accumulation in feces was determined.

[0099] The experimental analysis results are as follows Figure 5 As shown in the figure, as the digestion time continued to increase, the pH of the digestive fluid in each group showed a continuous downward trend, and the acid production content in the BP-18 experimental group was significantly increased compared with the blank control group and the CICC6071 experimental group (p<0.05). Figure 6 As shown in the figure, the bacterial density of each experimental group gradually increased with the increase of digestion time, and the microbial richness continued to accumulate during the digestion period. 600 Showing a significant upward trend.

[0100] 2. Effects of Bifidobacterium pseudocatenulatum BP-18 on fecal microorganisms

[0101] In order to study the effect of Bifidobacterium pseudocatenulatum BP-18 on the microorganisms in constipation feces, the fecal microbial structure of the HC healthy control group, BP-18 experimental group, constipation group, and CICC6071 experimental group was analyzed to preliminarily determine the effect of Bifidobacterium pseudocatenulatum BP-18 on the intestinal flora structure and provide data support for animal experimental research.

[0102] 2.1 Microbial diversity analysis

[0103] like Figure 7 As shown in the figure, from the Rarefaction curve reflecting the species abundance and sequencing depth of the sample and the coverage index reflecting the sequencing coverage, it can be seen that the number of OTUs in each group of samples basically reaches saturation with the increase of sequencing depth, with a very high sequencing coverage, the samples are real and effective, and the current sequencing depth is sufficient to discover most species in each sample habitat. Furthermore, principal coordinate analysis was performed on the 16S rRNA data of feces to study the effect of Bifidobacterium pseudocatenulatum BP-18 on the flora in constipation fecal samples and explore the differences in community composition between samples in different groups. Figure 8 As shown in the figure, after intervention with Bifidobacterium, there were differences between samples and groups, indicating that intervention with Bifidobacterium and its metabolites significantly increased bacterial diversity, resulting in a certain degree of change in species composition.

[0104] 2.2 Analysis of bacterial community composition

[0105] Constipation is a common gastrointestinal disease. Studies have shown that the main characteristics of constipation patients are the relative decrease in beneficial bacteria, the relative increase in potential pathogenic bacteria, and the decrease in species richness. After in vitro digestion experiments, the composition of fecal flora in each group is as follows Figure 9 As shown, the horizontal axis is the sample name, and the vertical axis is the proportion of the species in the sample. Columns of different colors represent different species, and the length of the column represents the proportion of the species. An analysis of the top 10 species in abundance showed that at the genus level, the abundance of Bifidobacterium and Lactobacillus in the feces of the BP-18 experimental group was significantly increased compared to the constipation group, proving that short-chain fatty acids have a certain promoting effect on the proliferation of beneficial bacterial genera. At the same time, in the constipation group, the abundance of the pathogenic bacterium Escherichia-Shigella was high. After BP-18 intervention, the abundance of the pathogenic bacterium Escherichia-Shigella decreased significantly, indicating that the intervention of Bifidobacterium pseudocatenulatum BP-18 affected the bacterial community structure in constipation feces. It may be that short-chain fatty acids have a strong inhibitory effect on potential pathogens, which will have a positive effect on the relief of constipation.

[0106] 3. Effects on the ability of fecal microorganisms to metabolize short-chain fatty acids

[0107] Among the metabolites produced by intestinal flora, short-chain fatty acids (SCFAs) have attracted considerable attention due to their pharmacological and physiological properties. Factors such as intestinal transit time, colonic pH, and the number and composition of intestinal flora influence the type and abundance of SCFAs produced. The experiments presented in this study demonstrate this, consistent with previous research.

[0108] The experimental method and GC-MS conditions were the same as in Example 2: Thaw the digested fecal supernatant on ice, take 500 μL of the supernatant and thoroughly mix it with 500 μL of ether, vortex, and sonicate. Carefully aspirate the upper organic phase and collect it with a 1 ml disposable sterile syringe. Filter the organic phase through a 0.22 μm microporous filter into a new sterile centrifuge tube and immediately store it in liquid nitrogen until analysis. Individual and total SCFAs in the fecal samples were quantified by gas chromatography-mass spectrometry (GC-MS).

[0109] Depend on Figure 10 It can be seen that the content of short-chain fatty acids in feces has been steadily increasing. Experimental analysis showed that after intervention with Bifidobacterium pseudocatenulatum BP-18, the total short-chain fatty acid content of fecal microbial metabolism reached 50.664 mg per gram, significantly higher than that of the CICC6071 control experimental bacteria and the blank group fecal fluid. Among them, fecal microbial metabolism of acetic acid, butyric acid, and hexanoic acid was particularly significant, with acetic acid content reaching 17.62484 mg / g, which was significantly higher than that of other control groups.

[0110] 4. Correlation Analysis

[0111] like Figure 11 As shown, the X-axis and Y-axis are environmental factors and species, that is, the relationship between the 10 bacterial genera with the highest correlation with short-chain fatty acids and acetic acid, propionic acid, butyric acid, valeric acid, and hexanoic acid produced by fecal digestive fluid. Escherichia-Shigella has a significant negative correlation with acetic acid (P≤0.001), propionic acid (P<0.05), and butyric acid (0.001<P≤0.01); Bifidobacterium has a significant positive correlation with acetic acid (P<0.05) and butyric acid (P<0.05). Figure 12As shown in the figure, the points of different colors represent sample groups under different short-chain fatty acid conditions. The red arrows represent the relationship between each acid and the sample. The length represents the degree of influence of each acid on the sample effect. The angle between the environmental factor arrows represents positive and negative correlation. According to the RDA analysis diagram of each experimental group, there is a correlation between the metabolism of acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid. The distance from the projection point to the arrow of the quantitative environmental factor represents the relative influence of the environmental factor on the sample community distribution. There are differences between the experimental groups. BP-18 is positioned as a key strain for the production of short-chain fatty acids, and its total short-chain fatty acid production capacity is outstanding. It proves that the ability of Bifidobacterium to produce short-chain fatty acids affects the sample effect, that is, the effect of alleviating constipation. Among them, the amount of acetic acid produced is a significant reason why BP-18 affects the community composition structure.

[0112] Example 8: Application of Bifidobacterium pseudocatenulatum BP-18 in yogurt preparation

[0113] Bifidobacterium pseudocatenulatum BP-18 can be used to prepare probiotic yogurt. The specific preparation process of yogurt is as follows:

[0114] 1. The raw material components and their weight proportions are:

[0115] 90 parts of fresh milk, 0.8 parts of whole milk powder, 2 parts of concentrated whey protein, 3 parts of xylitol, 2 parts of food additives (including sodium hydroxymethylcellulose, citric acid, sodium citrate, pectin, natural vitamins), 3 parts of inulin.

[0116] 2. Preparation Method

[0117] (1) Sterilization of raw materials: Mix the raw materials according to the proportion, stir for 20 minutes, mix evenly, sterilize and homogenize;

[0118] (2) Inoculation: Inoculate the activated bacterial solution of Bifidobacterium pseudocatenulatum BP-18 into the above raw materials at a volume ratio of 3-4%, and stir thoroughly;

[0119] (3) Fermentation: Ferment at 42°C for at least 6 hours. When the acidity is below 4.5, the fermentation can be stopped and the temperature can be quickly lowered to 10°C.

[0120] (4) Filling: Aseptic filling to obtain probiotic yogurt.

[0121] In summary, the Bifidobacterium pseudocatenulatum BP-18 provided by the present invention has excellent prebiotic properties, strong adhesion and antibacterial ability, can produce high amounts of short-chain fatty acids, and has good biosafety. This strain has strong survival ability in the gastrointestinal fluid digestion environment, can effectively improve the richness of the fecal flora in constipation patients, increase the abundance of dominant species at the genus level, and has a good ability to inhibit potential pathogens. This is related to the metabolism of short-chain fatty acids. Short-chain fatty acids continue to accumulate as the digestion time increases, optimize the intestinal physicochemical environment, and have a significant positive effect.

[0122] The Bifidobacterium pseudocatenulatum BP-18 provided by the present invention can be widely used in foods, health products, and medicines with the efficacy of preventing or alleviating constipation, and has broad market prospects.

Claims

1. A strain of Bifidobacterium pseudocatenulatum, characterized in that The deposit number of the Bifidobacterium pseudocatenulatum is CGMCC NO.28940.

2. Use of the Bifidobacterium pseudocatenulatum according to claim 1 in the preparation of food or health products.

3. Use of the Bifidobacterium pseudocatenulatum according to claim 1 in the preparation of a medicine for preventing or treating constipation.

4. A probiotic preparation, characterized in that The probiotic preparation comprises the Bifidobacterium pseudocatenulatum according to claim 1.

5. The probiotic preparation according to claim 4, wherein The probiotic preparation further comprises any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Tetragenococcus halophilus, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus acidilactici, and Leuconostoc lactis.

6. A probiotic yogurt, characterized in that: The probiotic yogurt is prepared by using the Bifidobacterium pseudocatenulatum described in claim 1 as a fermentation strain.

7. The probiotic yogurt according to claim 6, wherein The preparation method of the probiotic yogurt comprises the following steps: (1) Sterilization of raw materials: Mix the raw materials according to the proportion, stir for 20 minutes, mix evenly, sterilize and homogenize; (2) Inoculation: Inoculate the activated bacterial solution of Bifidobacterium pseudocatenulatum BP-18 into the above raw materials at a volume ratio of 3-4% and stir thoroughly; (3) Fermentation: Ferment at 42°C for at least 6 hours. When the acidity is below 4.5, stop the fermentation and quickly cool to 10°C. (4) Filling: Aseptic filling to obtain probiotic yogurt.

8. The probiotic yogurt according to claim 7, wherein The components of the raw materials in step (1) and their weight parts are: 90 parts of fresh milk, 0.8 parts of whole milk powder, 2 parts of concentrated whey protein, 3 parts of xylitol, 2 parts of food additives, and 3 parts of inulin.

9. The probiotic yogurt according to claim 8, wherein The food additives include any one or more of sodium hydroxymethyl cellulose, citric acid, sodium citrate, pectin, and natural vitamins.

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