CN115747121A - Bifidobacterium lactis HSLA-009, microbial inoculum and preparation method and application thereof - Google Patents
Bifidobacterium lactis HSLA-009, microbial inoculum and preparation method and application thereof Download PDFInfo
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- CN115747121A CN115747121A CN202211588750.6A CN202211588750A CN115747121A CN 115747121 A CN115747121 A CN 115747121A CN 202211588750 A CN202211588750 A CN 202211588750A CN 115747121 A CN115747121 A CN 115747121A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biological inoculants, and particularly relates to bifidobacterium lactis HSLA-009, an inoculant and a preparation method and application thereof. Bifidobacterium lactis of the present invention: (I)Bifidobacterium lactis) HSLA-009, complete biological preservation. The bifidobacterium lactis HSLA-009 is separated from camel milk, and can repair intestinal tissue injury and promote intestinal peristalsis by increasing the water content of excrement of organisms, so as to improve intestinal flora and promote intestinal healthThe technical effect of preventing and treating constipation; meanwhile, the bifidobacterium lactis HSLA-009 has stronger colonization ability in intestinal tracts, is safe and reliable and has no toxic or side effect.
Description
Technical Field
The invention belongs to the technical field of biological inoculants, and particularly relates to bifidobacterium lactis HSLA-009, an inoculant and a preparation method and application thereof.
Background
With the continuous improvement of living standard of people, the increasingly abundant dietary choices make the intestinal health problems caused by bad diet and habit also not negligible, such as chronic functional constipation.
Constipation can cause long-term defecation of patients, and can increase the occurrence rate of intestinal obstruction and intestinal cancer, thus seriously threatening the physical health of the patients. The most effective means for treating constipation at present is drug therapy, and constipation treatment drugs can be divided into two types according to different drug action modes, wherein one type is intestinal motility drugs, and the other type is constipation relieving drugs. Wherein the intestinal prokinetic drugs, such as cisapride, mosapride, prucalopride, tegaserod, etc., are mainly combined with 5-HT4 receptors, so as to increase acetylcholine released by excitatory neurons, enhance coordinated movement of esophagus, stomach, duodenum and small intestine, and promote evacuation of digestive system, thereby treating or relieving constipation, but the drugs have larger side effects, such as headache, nausea, syncope or arrhythmia, etc. Laxative drugs such as rhubarb, senna leaf, magnesium sulfate, polyethylene glycol and the like have quick response in treating constipation, but also have the problems of abdominal distension, abdominal colic and the like, and the excessive use of the laxative drugs can cause unbalance of water and electrolytes in vivo, cause permanent damage to intestinal walls and the like. According to the analysis, the discomfort caused by constipation can be only temporarily solved by using the medicine for treating constipation, and the medicine is accompanied by strong side effects, so that the constipation problem of a patient cannot be fundamentally solved, and secondary damage can be caused to the structure and the function of the gastrointestinal tract, and therefore, a method for treating constipation with small side effect and good effect needs to be found.
In recent years, the field of probiotics is rapidly developed, and various beneficial functions of probiotics are more and more accepted by people, so that a new idea is provided for treating constipation which is a common disease. However, the existing probiotic bacteria library for improving constipation and regulating intestinal health needs to be abundant and complete.
Disclosure of Invention
The invention aims to provide bifidobacterium lactis HSLA-009, a microbial inoculum and a preparation method and application thereof, wherein the bifidobacterium lactis HSLA-009 has the effects of preventing and treating constipation and promoting intestinal health, and can enrich a probiotic bank for improving constipation and regulating intestinal health.
The invention provides Bifidobacterium lactis (Bifidobacterium lactis) HSLA-009, wherein the preservation number of the Bifidobacterium lactis HSLA-009 is CGMCC NO.25322.
The invention also provides a microbial inoculum containing the bifidobacterium lactis HSLA-009 in the technical scheme.
Preferably, the microbial inoculum comprises the bifidobacterium lactis HSLA-009 thallus and/or a fermentation broth of the bifidobacterium lactis HSLA-009.
Preferably, the viable count of the bifidobacterium lactis HSLA-009 in the microbial inoculum is 1.35 multiplied by 10 10 ～1.85×10 10 CFU/mL。
The invention also provides a preparation method of the microbial inoculum in the technical scheme, which comprises the following steps:
inoculating bifidobacterium lactis HSLA-009 to a fermentation medium for anaerobic fermentation, and obtaining a fermentation liquid which is the microbial inoculum;
the fermentation medium takes water as a solvent and comprises the following components: 50-90 g/L of glucose, 28-40 g/L of yeast powder, 4-6 g/L of sodium acetate, 2-4 g/L of ammonium citrate, 2-4 g/L of dipotassium phosphate, 0.1-0.5 g/L of magnesium sulfate, 0.01-0.1 g/L of manganese sulfate and 1-2 g/L of Tween-80.
Preferably, the temperature of the anaerobic fermentation is 35-40 ℃, the pH value is 5.5-6.5, and the rotating speed is 100-300 rpm.
The invention also provides application of the bifidobacterium lactis HSLA-009 in the technical scheme or the microbial inoculum in the technical scheme in preparing products for preventing and treating constipation and/or promoting intestinal health.
The invention also provides a product for preventing and treating constipation and/or promoting intestinal health, which comprises the microbial inoculum and auxiliary materials in the technical scheme.
Preferably, the viable count of the bifidobacterium lactis HSLA-009 in the product is 2.85 × 10 11 ～3.15×10 11 CFU/g。
Preferably, the product is freeze-dried powder formed by the microbial inoculum and auxiliary materials, and the auxiliary materials comprise skimmed milk powder; the mass ratio of the microbial inoculum to the auxiliary materials is 1.
Has the beneficial effects that:
the invention provides bifidobacterium lactis (Bifidobacterium lactis) HSLA-009, and biological preservation is completed. The bifidobacterium lactis HSLA-009 is separated from camel milk, and can repair intestinal tissue injury and promote intestinal peristalsis by increasing the water content of excrement of an organism, so that the technical effects of improving intestinal flora, promoting intestinal health and preventing and treating constipation are achieved; meanwhile, the bifidobacterium lactis HSLA-009 has stronger colonization ability in intestinal tracts, is safe and reliable and has no toxic or side effect. And when the bifidobacterium lactis HSLA-009 disclosed by the invention is used for treating constipation, the bifidobacterium lactis HSLA-009 has the advantages of wide application range, obvious curative effect, high cure rate, low recurrence rate and no adverse side effect.
Biological preservation information
Bifidobacterium lactis (HSLA-009) was deposited in China general microbiological culture Collection center (CGMCC) at 15.07.2022, with a collection address of No. 3 Xilu No.1 Beijing, the sunward area of Beijing, and a collection number of CGMCC No.25322.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 is a colony map of Bifidobacterium lactis HSLA-009 in example 1;
FIG. 2 is a microscopic strain morphology of Bifidobacterium lactis HSLA-009 in example 1;
FIG. 3 is a dendrogram of Bifidobacterium lactis HSLA-009 in example 1;
FIGS. 4 to 7 are tissue section views of small intestine of mice in blank group, model group, low dose group and high dose group in example 2, respectively;
FIG. 8 is a graph showing the effect of Bifidobacterium lactis HSLA-009 on the fecal pellet count in male mice in example 2;
FIG. 9 is a graph showing the effect of Bifidobacterium lactis HSLA-009 on the fecal pellet count in female mice in example 2;
FIG. 10 is a graph showing the effect of Bifidobacterium lactis HSLA-009 on the water content of feces from male mice in example 2;
FIG. 11 is a graph showing the effect of Bifidobacterium lactis HSLA-009 on the water content of feces from female mice in example 2.
Detailed Description
The invention provides bifidobacterium lactis (Bifidobacterium lactis) HSLA-009, wherein the preservation number of the bifidobacterium lactis HSLA-009 is CGMCC NO.25322.
The bifidobacterium lactis HSLA-009 disclosed by the invention is preferably obtained by screening and separating from camel milk. <xnotran> HSLA-009 16SrDNA SEQ ID NO.1 , 5'-CATGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGATCCCTGGCAGCTTGCTGTCGGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCAACCTGCCCTGTGCACCGGAATAGCTCCTGGAAACGGGTGGTAATACCGGATGCTCCGCTCCATCGCATGGTGGGGTGGGAAATGCTTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGTTGGCGGGGTGATGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGGAGGCCTTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGTTTCGGCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTGCCGGATCGCCGTGGAGACACGGTTTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCGCATGTTGCCAGCGGGTGATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGCGGTGCGACACGGTGACGTGGGGCGGATCGCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGTAGCACCCGAAGCCGGTGGCCCGACCCTTGTGGGGGGAGCCGTCTAAGGTGAGACTCGTGATT-3'. </xnotran> The strain has 99% similarity with Bifidobacterium lactis JCM10602 by BLAST comparison in the GenBank of NCBI database, and is identified as Bifidobacterium lactis (Bifidobacterium lactis), and is named as Bifidobacterium lactis HSLA-009.
The bifidobacterium lactis HSLA-009 can form small colonies on an MRS solid culture medium, and the colonies are milky white, smooth in surface and opaque and round. The gram stain is positive, the microscopic shape is a short rod, the two ends are elliptic, the top end can form a bifurcation and is distributed in a single, paired or cluster manner.
The invention also provides a microbial inoculum containing the bifidobacterium lactis HSLA-009 in the technical scheme.
In the invention, the microbial inoculum preferably comprises the bifidobacterium lactis HSLA-009 thallus and/or bifidobacterium lactis HSLA-009 fermentation liquor, and more preferably comprises the bifidobacterium lactis HSLA-009 fermentation liquor. The number of viable bacteria of Bifidobacterium lactis HSLA-009 in the microbial agent of the present invention is preferably 1.35 × 10 10 ～1.85×10 10 CFU/mL, more preferably 1.65X 10 10 CFU/mL。
The invention also provides a preparation method of the microbial inoculum according to the technical scheme, which comprises the following steps:
carrying out anaerobic fermentation on bifidobacterium lactis HSLA-009 to obtain a fermentation broth, wherein the fermentation broth comprises the microbial inoculum;
the fermentation medium takes water as a solvent and comprises the following components: 50-90 g/L of glucose, 28-40 g/L of yeast powder, 4-6 g/L of sodium acetate, 2-4 g/L of ammonium citrate, 2-4 g/L of dipotassium phosphate, 0.1-0.5 g/L of magnesium sulfate, 0.01-0.1 g/L of manganese sulfate and 1-2 g/L of Tween-80.
In the invention, the seed liquid of the bifidobacterium lactis HSLA-009 is preferably prepared before the anaerobic fermentation. The preparation method of the seed solution preferably comprises the following steps: activating and culturing the bifidobacterium lactis HSLA-009, and performing expanded culture on the obtained activated culture solution to obtain the seed solution.
Preferably, the activated culture solution is obtained by activated culture of bifidobacterium lactis HSLA-009. The culture medium for the activation culture is preferably MRS liquid culture medium. The source of the MRS liquid culture medium is not particularly limited, and the MRS liquid culture medium can be prepared by self or purchased from a market channel. The activated culture is preferably anaerobic static culture; the temperature of the activation culture is preferably 34-41 ℃, and more preferably 37 ℃; the time is preferably 20 to 29 hours, more preferably 24 hours.
After the activation culture solution is obtained, the present invention preferably performs scale-up culture on the activation culture solution to obtain the seed solution. The culture medium for the amplification culture is preferably an MRS liquid culture medium; the volume ratio of the activation culture solution to the medium for the expanded culture is preferably 1 to 3:50 to 150, more preferably 1. The time of the amplification culture is preferably 28-40 h, and more preferably 36h; other fermentation conditions are the same as those of the activation culture, and are not described in detail.
After the seed culture solution is obtained, the seed culture solution is preferably inoculated to a fermentation medium for anaerobic fermentation to obtain a fermentation liquid, namely the microbial inoculum. In the anaerobic fermentation process, stirring is preferably carried out; the rotation speed of the stirring is preferably 100 to 300rpm, more preferably 100rpm, 200rpm or 300rpm, and still more preferably 100rpm. The temperature of the anaerobic fermentation is preferably 35-40 ℃, and more preferably 37 ℃; the time is preferably 20 to 29 hours, and more preferably 24 hours; the pH is preferably 5.5 to 6.5, more preferably 6.5.
The fermentation medium for fermentation culture of the invention takes water as a solvent and comprises the following components: 50-90 g/L of glucose, 28-40 g/L of yeast powder, 4-6 g/L of sodium acetate, 2-4 g/L of ammonium citrate, 2-4 g/L of dipotassium phosphate, 0.1-0.5 g/L of magnesium sulfate, 0.01-0.1 g/L of manganese sulfate and 1-2 g/L of Tween-80.
The fermentation medium of the invention comprises 50-90 g/L glucose, preferably 60-80 g/L, and more preferably 60g/L.
The fermentation medium comprises 28-40 g/L yeast powder, preferably 30-38 g/L, and more preferably 35g/L.
The fermentation medium comprises 4-6 g/L of sodium acetate, and preferably 5g/L of sodium acetate.
The fermentation medium comprises 2-4 g/L ammonium citrate, preferably 2g/L.
The fermentation medium comprises 2-4 g/L dipotassium hydrogen phosphate, preferably 2g/L.
The fermentation medium comprises 0.1-0.5 g/L magnesium sulfate, preferably 0.1g/L.
The fermentation medium comprises 0.01-0.1 g/L manganese sulfate, preferably 0.05g/L manganese sulfate.
The fermentation medium comprises 1-2 g/L of Tween-80, preferably 1g/L.
The volume ratio of the seed liquid to the fermentation medium is preferably 1:20 to 40, more preferably 1. The number of viable bacteria in the fermentation liquid prepared by the fermentation medium and the fermentation conditions is obviously increased, and the number of the viable bacteria is about 15 times of that in the seed culture liquid.
After the fermentation liquor is obtained, the invention preferably further comprises the step of centrifuging the fermentation liquor to obtain the wet cells of the bifidobacterium lactis HSLA-009. The time for centrifugation is preferably 10-20 min, and more preferably 15min; the rotation speed is preferably 8000 to 15000rpm, more preferably 12000rpm.
The invention also provides application of the bifidobacterium lactis HSLA-009 in the technical scheme or the microbial inoculum in the technical scheme in preparing products for preventing and treating constipation and/or promoting intestinal health. The product of the invention is preferably a product for preventing and treating constipation and promoting intestinal health. The constipation-preventing products of the present invention preferably include products for preventing chronic functional constipation. The product of the invention comprises medicines, health products and food additives. The bifidobacterium lactis HSLA-009 can repair intestinal tissue injury and promote intestinal peristalsis by increasing the water content of excrement of organisms, so thatThe technical effects of improving intestinal flora, promoting intestinal health and improving constipation are achieved. The product of the invention is preferably administered in a dose of 1X 10 11 CFU/day, the administration period is preferably 3 to 7 days, more preferably 4 to 6 days.
The invention also provides a product for preventing and treating constipation and/or promoting intestinal health, which comprises the microbial inoculum and auxiliary materials in the technical scheme. In the product, the viable count of the bifidobacterium lactis HSLA-009 is 2.85 multiplied by 10 11 ～3.15×10 11 CFU/g, more preferably 3.01X 10 11 CFU/g. The product of the invention is preferably freeze-dried powder formed by the microbial inoculum and auxiliary materials. The mass ratio of the microbial inoculum to the auxiliary materials is preferably 0.5-1.7: 1.1 to 2.0, more preferably 1; the microbial inoculum is preferably mixed with auxiliary materials in the form of wet thalli. The auxiliary material of the invention preferably comprises skimmed milk powder, sucrose or maltodextrin, and more preferably skimmed milk powder.
The invention also provides a preparation method of the product, which comprises the following steps: after the microbial inoculum and the auxiliary materials are mixed, the water content is adjusted to 90%, and the mixture is dried to obtain freeze-dried powder, namely the product. The microbial inoculum is preferably mixed with auxiliary materials in the form of wet thalli, and the preparation method of the wet thalli is described in the technical scheme and is not described any more. The present invention preferably adjusts the water content with physiological saline. The present invention preferably employs vacuum freeze-drying for the drying process. The vacuum freeze-drying conditions are not particularly limited in the invention, and the conventional vacuum freeze-drying conditions in the field can be adopted.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The method for separating and identifying the bifidobacterium lactis HSLA-009 comprises the following steps of:
the latex of the Xinjiang bactrian camel in the Xinjiang erenganic basin area is selected as a sample for strain separation. The separation of the strains was carried out immediately after the samples were cryopreserved. The camel milk sample is firstly diluted by 10 times of serial gradient with sterile normal saline, then 1mL of liquid is respectively absorbed from each dilution (containing stock solution) and is placed in a sterile empty plate, then the TPY agar culture medium which is cooled to about 45 ℃ is operated according to the operation requirement of a double-layer pouring method, after the culture medium is cooled to be completely solidified, the culture medium is placed in a sealed culture tank of Mitsubishi MGC and is placed in a matched anaerobic air producing bag of Mitsubishi MGC, the sealed culture tank is placed in a constant temperature culture box, and the culture is carried out for 72 hours at 37 ℃.
1) Morphological characterization of strains
Selecting single bacterial colonies with different sizes and morphologies on the plate, wherein one bacterial strain is selected and obtained and is marked as HSLA-009, and after the bacterial colonies are subjected to anaerobic culture in an MRS culture medium, smaller bacterial colonies can be formed, and the bacterial colonies are milky white, smooth in surface and round in opacity, as shown in figure 1. The gram stain of the strain is positive, the microscopic shape under an optical microscope is in a short rod shape, two ends are in an oval shape, the top end can form a fork, and the fork is distributed in a single, paired or cluster manner, as shown in figure 2.
2) Physiological and biochemical identification of strains
Physiological and biochemical identification of the strain HSLA-009 was carried out according to Bergey's Manual of identification of bacteria. The main physiological and biochemical characteristics of the bacterium are shown in Table 1.
TABLE 1 physiological and biochemical identification results of the strain HSLA-009
* Note: in the table, "+" represents a positive reaction and "-" represents a negative reaction.
3) Molecular biological identification of strain HSLA-009
The purified sample of the strain HSLA-009 was sent to bio-engineering (shanghai) gmbh for 16SrDNA sequencing, and then the 16SrDNA sequencing results of the strain were BLAST-aligned in GenBank database. The sequencing result of the 16SrDNA of the strain is shown as SEQ ID NO. 1. The sequence was aligned with Bifidobacterium lactis JCM10602 strain in GenBank by BLAST and found to have a similarity of 99% (see FIG. 3), and was identified as Bifidobacterium lactis. The strain was named Bifidobacterium lactis HSLA-009.
Example 2
The functional verification of the bifidobacterium lactis HSLA-009 comprises the following steps:
1. experiment on gastric acid tolerance of bifidobacterium lactis HSLA-009
(1) Preparing artificial gastric juice:
adjusting pH of sterile MRS liquid culture medium to 2.0 with 10% hydrochloric acid, adding pepsin to make the final concentration of pepsin in MRS liquid culture medium be 10g/L, and after it is fully dissolved, filtering with 0.22 μm microporous filter membrane to remove bacteria, and reserving.
(2) Preparation of bifidobacterium lactis HSLA-009 bacterial suspension:
inoculating bifidobacterium lactis SL08-01 into a test tube containing 10mL of sterile MRS liquid culture medium, performing static culture at 37 ℃ under anaerobic conditions for 24h, sucking 1mL of the solution, inoculating the solution into a triangular flask containing 100mL of sterile MRS liquid culture medium, and performing static culture at 37 ℃ under anaerobic conditions for 36h to obtain fermentation liquor of bifidobacterium lactis HSLA-009 strain. And then 8mL of fermentation liquor is taken to be centrifuged for 10min at 6000rpm, the supernatant is discarded, the bottom layer bacterial sludge is washed twice by sterile physiological saline, and then 1mL of sterile MRS solution is added for resuspension for later use.
(3) Gastric acid tolerance assay for bifidobacterium lactis HSLA-009
And (3) mixing the bacterial suspension obtained in the step (2) with the artificial gastric juice prepared in the step (1) according to the volume ratio of 1.
Wherein the number of the initial bacteria of Bifidobacterium lactis HSLA-009 is 7.86 × 10 8 CFU/mL, viable count of 7.71X 10 after incubation for 3h with artificial gastric juice 8 The survival rate is 98.09%. Therefore, the bifidobacterium lactis HSLA-009 has stronger gastric acid tolerance.
2. Bile salt tolerance test of Bifidobacterium lactis HSLA-009
(1) Preparing a bile salt solution:
adding bovine bile powder into MRS liquid culture medium to make its mass concentration in MRS liquid culture medium be 0.5%, then adding trypsin into experimental group to make its final concentration be 1.0g/L (W/V), after all the components are fully dissolved, using 0.22 micrometer microporous filter membrane to filter and sterilize, standing still for stand-by.
(2) Preparation of bifidobacterium lactis HSLA-009 bacterial suspension:
the preparation process of the bacterial suspension is consistent with the preparation method in the gastric acid tolerance experiment in the step 1.
(3) Bile salt tolerance assay
Adding the bacterial suspension of the bifidobacterium lactis HSLA-009 prepared in the step (2) into the bile salt solution prepared in the step (1), mixing according to the volume ratio of 1.
Wherein the number of the initial bacteria of Bifidobacterium lactis HSLA-009 is 7.86 × 10 8 CFU/mL, viable count of 7.81X 10 after incubation for 6h with bile salt solution 8 The survival rate is 99.36%. It can be seen that Bifidobacterium lactis HSLA-009 has strong bile salt tolerance.
3. Evaluation of Effect of Bifidobacterium lactis HSLA-009 in relieving constipation
1) Establishment of functional constipation mouse model
The main mode for modeling functional constipation is drug modeling, and one of the common drugs for modeling is loperamide hydrochloride. It acts as a peripheral opioid receptor agonist and delays fecal evacuation time and intestinal transit by inhibiting intestinal water secretion and colonic motility, which is primarily manifested by functional constipation symptoms due to decreased stool numbers, weight and water content in the animal.
The Kunming mouse is selected as an experimental object to carry out drug modeling of functional constipation. Firstly, 80 first-generation Kunming mice with 6-8 weeks of basically same mental state and activity are selected, wherein 40 female mice and 40 male mice are respectively divided into 4 groups on average, namely 10 female mice and 10 male mice are 1 group. One group was randomly drawn as a blank group, and other groups of mice were modeled except for the blank group. The main mode of modeling is performed by means of stomach irrigation with loperamide hydrochloride. After each group of mice is bred adaptively for one week, the mice of the other three groups except the blank group are subjected to the intragastric administration treatment by using the filtered and sterilized loperamide hydrochloride solution with the concentration of 1mg/mL of 0.25mL at 8 o' clock every day, and the mice of the blank group are subjected to the intragastric administration treatment by using the same amount of sterile physiological saline at the same time.
The feces of each group of mice are collected once every 24h and placed respectively, and the number of the feces of each group of mice is counted and the water content of the feces is measured. The measurement of the moisture content of the feces was performed by the baking method. The mouse feces are put into an oven at 105 ℃ and dried to constant weight, and then the water content of the mouse feces is calculated according to the following formula:
stool water content (%) = (stool weight before drying-stool weight after drying)/stool weight before drying × 100%
And when the number of the excrement grains of the other three groups of mice is obviously reduced and the water content of the excrement is obviously reduced compared with that of the blank group, the successful modeling of the mouse functional constipation model is shown.
During the experiment, mice were fed ad libitum with water and the feeding process was completed as per the requirements of the feeding instructions except for the necessary treatments. The change of the stool grain number and the stool water content in the mouse modeling process is shown in table 2.
TABLE 2 Change in stool Properties of mice during modeling
Note: the modeling data in the above table are the average of three groups of mice
As can be seen from Table 2, as the treatment time was prolonged, the stool volume and the stool water content of the mice in the modeling group were gradually decreased, and the stabilization was achieved on the third day, which is significantly lower than that of the blank group, indicating that the modeling of the mouse functional constipation model was successful. In addition, in consideration of the effective time of the loperamide hydrochloride drug, the modeling and gastric perfusion treatment needs to be carried out every day during the experiment, and the gastric perfusion time point and the gastric perfusion mode are the same as the modeling process.
2) Evaluation of Effect of Bifidobacterium lactis HSLA-009 in relieving constipation
(1) Preparation of bacterial suspension of Bifidobacterium lactis HSLA-009
Thawing the bifidobacterium lactis HSLA-009 preserved at the temperature of-80 ℃ at room temperature, then inoculating the bifidobacterium lactis into a test tube containing 10mL of sterile MRS liquid culture medium, performing static culture at the temperature of 37 ℃ under anaerobic conditions for 24 hours, sucking 1mL of the culture solution, inoculating the culture solution into a triangular flask containing 100mL of sterile MRS liquid culture medium, and performing static culture at the temperature of 37 ℃ under anaerobic conditions for 36 hours to obtain the fermentation liquid of the bifidobacterium lactis HSLA-009. Then, centrifuging 40mL of fermentation liquor for 10min at 6000rpm, discarding the supernatant, washing the bottom layer bacterial sludge twice with sterile normal saline, and adding the washed bottom layer bacterial sludge and 10mL of sterile normal saline for resuspension to obtain high-concentration bacterial suspension; then 1mL of high-concentration bacterial suspension is diluted by 10 times by sterile physiological saline to prepare low-concentration bacterial suspension. And hermetically preserving the two bacterial suspensions at the low temperature of 4 ℃ for later use.
(2) Experimental design for mouse experiments
Randomly numbering the three groups of mouse constipation models obtained in the step 1) into a model group, a high-dose group and a low-dose group. Except for the loperamide hydrochloride which is infused at 8 pm every day, the high-concentration bacterial suspension prepared by the method is infused with 0.25mL at 10 pm in the high-dose group, the low-concentration bacterial suspension prepared by the method is infused with 0.25mL at 10 pm in the low-dose group, the sterile physiological saline is infused with 0.25mL at 10 pm in the model group, and the sterile physiological saline is infused with 0.25mL in the blank group of mice at 8 pm and 10 pm respectively.
Collecting the excrement of the male and female mice in each group of mice every 24h, respectively placing the excrement, counting the number of excrement grains of the male and female mice in each group of mice, and measuring the water content of the excrement grains. The measurement and calculation of the water content of the excrement are the same as the modeling process of a mouse constipation model.
When the fecal grain number and fecal moisture content of each group of mice tend to be stable in the experimental process, the intestinal mobility and the first grain defecation time of the mice begin to be detected, the small intestine tissues of each group of mice are sliced and observed, meanwhile, the feces of each group of mice are taken for specific flora analysis, and the duration time of the action effect of the bifidobacterium lactis HSLA-009 is tested, so as to explore and research the effect and mechanism of the strain HSLA-009 in relieving and treating constipation. The influence of Bifidobacterium lactis HSLA-009 on the number of fecal pellets (pellets/mouse) and the water content of fecal samples in male and female mice is shown in tables 3 to 6.
TABLE 3 Effect of Bifidobacterium lactis HSLA-009 on stool grain number in Male mice (grain/grain)
TABLE 4 Effect of Bifidobacterium lactis HSLA-009 on stool grain number in female mice (grain/grain)
TABLE 5 Effect of Bifidobacterium lactis HSLA-009 on moisture content of feces from Male mice (grains/grains)
TABLE 6 influence of Bifidobacterium lactis HSLA-009 on water content of female mouse feces (grains/grains)
From tables 3 to 6, it can be seen that: starting from the first day after the gastric lavage treatment of the bacterial suspension of the bifidobacterium lactis HSLA-009, the constipation symptom of the constipation mouse model is improved, the number of the fecal particles of the low-dose group reaches the normal level and tends to be stable on the fifth day, and the number of the fecal particles of the high-dose group reaches the normal level and tends to be stable on the fourth day, and meanwhile, the indexes of the fecal moisture content reach the normal level one day ahead of the number of the fecal particles, and no obvious difference exists between females and males in the same group. The bifidobacterium lactis HSLA-009 can treat the functional constipation by increasing the water content in the mouse excrement and has obvious treatment effect, and the cure efficiency is increased along with the increase of the dosage.
4. Effect of Bifidobacterium lactis HSLA-009 on intestinal mobility and first granule defecation time of mice
From the results in step 3, it was found that the constipation symptom of the mice disappeared and stabilized seven days after the beginning of the treatment with bifidobacterium lactis HSLA-009, and thus the intestinal mobility and the first-granule defecation time were measured on the eighth day.
(1) Intestinal mobility test
Intestinal mobility experiments were performed 6 pm the eighth day after the mice began gavage of bifidobacterium lactis HSLA-009, which was two hours earlier than the gavage of loperamide hydrochloride. Respectively randomly extracting 6 mice from a blank group, a model group, a low-dose group and a high-dose group, wherein the number of females and males is 3 respectively, filling 0.25mL of activated carbon suspension into each mouse after selection, killing the mice by a neck breaking method after 30min, quickly opening the abdominal cavity of the mice, taking out all intestinal tracts in the mice, straightening under the condition of no tension, measuring the length from a pylorus to a ileocecal part, recording the length as the total length of the intestinal tracts of the mice, measuring the distance from the front edge of activated carbon powder to the pylorus, recording the migration distance of the carbon powder in the intestinal tracts, and calculating the intestinal migration rate according to the following formula:
intestinal mobility (%) = migration distance of carbon powder in small intestine/full length of mouse intestinal tract × 100%;
the results of the intestinal mobility experiments are shown in table 7.
(2) Determination of black stool discharging time of first mouse particle
The first-grain defecation experiment of the mice is carried out 9 o' clock in the eighth night when the mice begin to gaze the bifidobacterium lactis HSLA-009, and the gazing operation is carried out for one hour later than the loperamide hydrochloride. 0.25mL of ink was gavaged into all of the remaining mice in the blank group, model group, low dose group and high dose group, and the time (in minutes) for each mouse to expel the first black stool was recorded from the gavage ink, and the average time required for the first black stool to be expelled in each group was taken as the group first black stool expelling time. The experimental results are shown in Table 7.
TABLE 7 Effect of the strain HSLA-009 on intestinal mobility and first-pellet defecation time in mice
From table 7, it can be seen that the intestinal mobility of the mice in the model group is significantly lower than that of the blank group, and the first-particle defecation time is significantly longer than that of the blank group, which indicates that the constipation model of the mice has stronger stability, i.e. the constipation symptoms of the mice are consistent and continuous; meanwhile, the results of the two experiments of the mice in the low-dose group and the high-dose group are not significantly different from those of the blank group, and in addition, the results of the two experiments are not significantly different between the male and female mice, which shows that the bifidobacterium lactis HSLA-009 can treat functional constipation by increasing intestinal peristalsis and has obvious treatment effect.
5. Effect of the strain HSLA-009 on the morphology of Constipation mouse small intestine tissue
The effect of bifidobacterium lactis HSLA-009 on constipation mouse intestinal histomorphology was performed at 6 pm on the tenth day after the mice began gavage of strain HSLA-009 to completely eliminate the effect of ink on the mice. 6 mice were randomly drawn from each of the blank, model, low dose and high dose groups, 3 of which were female and male each, and dissected. After the mice were sacrificed, the mice were dissected to take the small intestine portion from the pylorus, down to the ileocaecal region. Carefully cleaning out residual excrement in the small intestine, shearing the small intestine with the length of about 0.5cm, fixing the small intestine in tissue fixing liquid to be used as a sample of a tissue section, freezing the residual small intestine by liquid nitrogen, and storing the residual small intestine in a refrigerator at the temperature of 80 ℃ below zero to be used as a subsequent experiment sample.
The small intestine tissue sections of the mice in each group were observed under an optical microscope, and the results showed that: the small intestinal villi of the blank group of mice are regularly and uniformly arranged, the phenomenon of breakage or shrinkage does not exist, the intestinal villi structure is complete, the mucosal epithelium is complete and continuous, and goblet cells are abundant (figure 4); the mouse in the model group has severe rupture and shrinkage of intestinal villi and incomplete goblet cells (FIG. 5); although the villi of the small intestine of the high-dose group and the low-dose group also appeared to be shrunk and broken to some extent, the villi of the small intestine was significantly more intact than that of the model group mice and was closer to the characteristics of the intestinal tissue section of the blank group, and the villi integrity of the small intestine of the high-dose group and the low-dose group was not much different. Similarly, no significant difference was observed between female mice, and the small intestine tissue sections of the low dose group and the high dose group were shown in FIGS. 6 to 7, respectively.
The integrity of the villi of the small intestine is closely related to the peristalsis capability of the intestinal tract and the sensitivity of the intestinal tract to the stimulation of the feces, and the experiments show that the bifidobacterium lactis HSLA-009 can restore the sense of defecation and the normal peristalsis function of the villi of the small intestine in a way of repairing the tissue structure of the intestinal tract, thereby treating constipation and maintaining the health of the intestinal tract in this way.
6. Effect of Bifidobacterium lactis HSLA-009 on specific intestinal flora of constipation-treated mice
All the excrements of the male and female mice in each group of mice from 10 pm on the tenth day to 7 pm on the eleventh day after the beginning of gavage of the strain HSLA-009 are collected respectively, the excrements are sent into a laboratory after being refrigerated and immediately subjected to lactic acid bacteria counting work, and the influence of the strain HSLA-009 on the intestinal flora of the mice is researched by measuring the change of the content of the lactic acid bacteria in the excrements of the mice. The content of the lactobacillus in the mouse excrement is detected according to a method in GB4789.35-2016 food safety national standard food microbial inspection lactobacillus inspection. The results are shown in Table 8.
TABLE 8 Effect of the Strain HSLA-009 on Constipation mouse intestinal flora
Note: the data in Table 8 are the number of colonies contained per gram of feces (CFU/g).
As can be seen from table 8, after the lavage with bifidobacterium lactis HSLA-009, the numbers of lactobacillus and bifidobacterium in both the low dose group and the high dose group were close to the blank group level, while the number of lactic acid bacteria in the model group was significantly lower than those in the other three groups, and although the experiment could not directly prove the relationship between the number of intestinal flora and constipation, the increase in the number of lactic acid bacteria in the mouse intestinal tract promoted the balance between intestinal health and intestinal flora, indirectly exerted a positive effect on the treatment of constipation. Also, there were no significant differences between male and female mice.
7. Effect of Bifidobacterium lactis HSLA-009 on duration of therapeutic action in constipated mice
The experiment is developed in a blank group, a low-dose group and a high-dose group, after the experiment is carried out for two weeks, the drug and probiotic intragastric administration treatment at 8 pm and 10 pm every day is stopped, then, 0.25mL of loperamide hydrochloride solution with the concentration of 1mg/mL is intragastric administered to three groups of mice every two days, namely, the fifteenth day, the eighteenth day and the twentieth day at 10 pm, after filtration and sterilization, is respectively collected, the feces of the mice on the seventeenth day, the twentieth day and the twenty-third day in each group of male and female mice are respectively collected, the number of feces of each group is recorded, and the water content of the feces is measured, and the experiment result is shown in figures 8-11.
From fig. 8 to 11, it can be seen that: after the blank group is perfused with the loperamide hydrochloride solution, the stool grain number and the stool water content of the mice in the blank group are obviously reduced, and the constipation symptom can be considered to appear, while in the low-dose group and the high-dose group, the stool grain number and the stool water content of the mice in the group are reduced after the stomach is perfused with the loperamide hydrochloride solution, but the stool grain number and the stool water content are basically maintained at normal levels, and are obviously different compared with the obvious reduction amplitude of the blank group, in addition, the reduction amplitude of the high-dose group is slightly lower than that of the low-dose group, but the difference is not large, and no obvious difference exists between the male and female mice. This indicates that, even after the use of bifidobacterium lactis HSLA-009 is stopped, it still has a certain therapeutic and prophylactic effect on constipation, and may also play a role in reducing the recurrence rate in actual use.
From the experiments in steps 1 to 7 in example 2, it can be concluded that: the bifidobacterium lactis HSLA-009 can prevent and treat constipation in both female mice and male mice by increasing the water content of excrement of the mice, repairing intestinal tissue injury, promoting intestinal peristalsis, recovering beneficial flora in intestinal tracts and the like, and experiments prove that the strain can also effectively prevent the recurrence of the constipation. In addition, adverse reactions such as diarrhea and the like do not occur in all groups of mice in the whole experimental process, except for a model group, the mental states of all groups of mice are good, and symptoms such as inappetence, hypomotility and the like do not occur, which indicates that the way of treating constipation by using bifidobacterium lactis HSLA-009 is not caused by causing diarrhea, and also indicates that adverse reactions do not occur when using bifidobacterium lactis HSLA-009 to treat constipation in mice, and indicates that bifidobacterium lactis HSLA-009 has great application potential in the aspects of treating constipation, promoting intestinal health and the like.
Example 3
Preparation of 1 Bifidobacterium HSLA-009 seed liquid
Taking out the bifidobacterium lactis HSLA-009 from a refrigerator at the temperature of-80 ℃, melting in a water bath at the temperature of 37 ℃, inoculating into a test tube containing 10mL of sterile MRS liquid culture medium, performing static culture at the temperature of 37 ℃ for 24 hours under anaerobic conditions, sucking 1mL of the liquid culture medium, inoculating into a triangular flask containing 100mL of sterile MRS liquid culture medium, performing static culture at the temperature of 37 ℃ for 36 hours under anaerobic conditions, and using the liquid culture medium as bifidobacterium lactis HSLA-009 seed liquid for later use.
(2) Using an MRS liquid culture medium as a fermentation culture medium, wherein the formula of the MRS liquid culture medium comprises the following components of using water as a solvent, 20g/L of glucose, 10g/L of peptone, 10g/L of beef powder, 5g/L of yeast powder, 5g/L of sodium acetate, 2g/L of ammonium citrate, 2g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 1mL/L of tween-80;
and (3) filling 100mL of fermentation medium into a 250mL triangular flask, sterilizing by using high-pressure steam at 121 ℃ for 20min, cooling to room temperature, inoculating 1mL of prepared seed liquid, and performing anaerobic standing culture at 37 ℃ for 24h to obtain the fermentation liquid of the bifidobacterium lactis HSLA-009. Then measuring the number of viable bacteria in the fermentation culture solution, and carrying out the detection method according to GB4789.35-2016 food safety national standard food microorganism inspection lactobacillus inspection.
Example 4
A bifidobacterium lactis HSLA-009 fermentation broth was prepared by the method in example 3, except that glucose in the MRS liquid medium was replaced with an equal amount of sucrose.
Example 5
A bifidobacterium lactis HSLA-009 fermentation broth was prepared by the method in example 3, except that glucose in MRS broth was replaced with an equal amount of whey powder.
Example 6
A fermentation broth of Bifidobacterium lactis HSLA-009 was prepared by the method in example 3, except that peptone 10g/L, beef powder 10g/L, yeast powder 5g/L in MRS liquid medium was replaced with peptone 25 g/L.
Example 7
A fermentation broth of Bifidobacterium lactis HSLA-009 was prepared by the method in example 3, except that peptone 10g/L, beef powder 10g/L, yeast powder 5g/L in MRS liquid medium was replaced with beef powder 25 g/L.
Example 8
A fermentation broth of Bifidobacterium lactis HSLA-009 was prepared by the method in example 3, except that peptone 10g/L, beef powder 10g/L, yeast powder 5g/L in MRS liquid medium were replaced with yeast powder 25 g/L.
The results of measuring the viable cell count of the bifidobacterium lactis HSLA-009 fermentation broth prepared in examples 3 to 8 are shown in table 9.
TABLE 9 measurement results of viable cell count in Bifidobacterium lactis HSLA-009 fermentation broth in examples 3 to 8
From table 9 it can be derived: when all nitrogen sources in the MRS liquid culture medium are replaced by yeast powder, the number of bacteria is higher than that of the composite nitrogen sources in the MRS liquid culture medium, so that 25g/L of yeast powder is selected as the nitrogen sources in the fermentation culture medium. Whey powder is not suitable for serving as a carbon source used for fermentation of the strain compared with sucrose and glucose, the sucrose and the glucose are not greatly different, and the glucose is selected as the carbon source in a fermentation medium in view of production cost.
Examples 9 to 17
The fermentation broth of bifidobacterium lactis HSLA-009 prepared by the method of example 8 was different from the method in that the amounts of glucose (carbon source) and yeast powder (nitrogen source) were adjusted, and the amounts of glucose and yeast powder and the number of viable cells after completion of fermentation in examples 9 to 17 are shown in table 10.
TABLE 10 measurement results of the composition ratio of carbon source and nitrogen source and the number of viable bacteria in examples 9 to 17
From table 10 it can be derived: in the case of different amounts of yeast powder, the number of fermentation bacteria increases with the increase of the amount of glucose, increases with the increase of the amount of yeast powder, and finally the maximum number of fermentation bacteria is obtained by combining 60g/L glucose +35g/L yeast powder, so that the combination is used as the optimal carbon source and nitrogen source of the strain HSLA-009, and the culture medium is used as the optimal culture medium of the Bifidobacterium lactis HSLA-009, namely: 60g/L glucose, 35g/L yeast powder, 5g/L sodium acetate, 2g/L ammonium citrate, 2g/L dipotassium phosphate, 0.1g/L magnesium sulfate, 0.05g/L manganese sulfate and 80g/L Tween-80.
Example 18
The preparation method of the bifidobacterium lactis HSLA-009 microbial inoculum comprises the following steps:
preparation of 1 Bifidobacterium lactis HSLA-009 seed liquid
Taking out the bifidobacterium lactis HSLA-009 from a refrigerator at the temperature of-80 ℃, melting in a water bath at the temperature of 37 ℃, inoculating into a test tube containing 10mL of sterile MRS liquid culture medium, performing static culture at the temperature of 37 ℃ for 24h under anaerobic conditions, sucking 1mL of the liquid culture medium, inoculating into a triangular flask containing 100mL of sterile MRS liquid culture medium, and performing static culture at the temperature of 37 ℃ for 36h under anaerobic conditions to obtain the bifidobacterium lactis HSLA-009 seed solution for later use.
(2) Preparation of bifidobacterium lactis HSLA-009 fermentation liquor
Inoculating 100mL of the Bifidobacterium lactis HSLA-009 seed solution obtained in the step (1) into a 5L full-automatic fermentation tank containing 3.5L of a sterilized (sterilized by autoclaving at 121 ℃ for 25min and cooling to 37 ℃) fermentation medium for fermentation, wherein the fermentation medium comprises the following components: 60g/L of glucose, 35g/L of yeast powder, 5g/L of sodium acetate, 2g/L of ammonium citrate, 2g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate and 80g/L of Tween-80. And (3) fermenting the fermentation medium at a constant temperature of 37 ℃ for 24h under the condition that the pH value of the fermentation liquor is 5.5, adjusting the pH value of the fermentation liquor by using a 4mol/L sodium hydroxide solution in the culture process, keeping the pH value of 5.5 to obtain the Bifidobacterium lactis HSLA-009 fermentation liquor, and detecting the number of viable bacteria in the fermentation liquor after the fermentation is finished according to GB4789.35-2016 food safety national standard food microbial test lactobacillus test.
Example 19
Bifidobacterium lactis HSLA-009 broth was prepared as in example 18, except that the fermentation pH was adjusted to 6.0.
Example 20
Bifidobacterium lactis HSLA-009 broth was prepared as in example 18, except that the fermentation pH was adjusted to 6.5.
The viable cell counts in the fermentation broths of Bifidobacterium lactis HSLA-009 in examples 18 to 20 are shown in Table 11.
TABLE 11 results of measuring viable cell counts in Bifidobacterium lactis HSLA-009 fermentation broth in examples 18 to 20
As shown in Table 11, the number of fermentation bacteria increased with the increase of the pH value, and the optimum pH for liquid fermentation was 6.5 in the strain HSLA-009.
Example 21
A Bifidobacterium lactis HSLA-009 broth was prepared by the method of example 20, except that stirring was carried out at 100rpm during the fermentation.
Example 22
A Bifidobacterium lactis HSLA-009 broth was prepared by the method of example 20 except that the stirring speed was 200rpm.
Example 23
Bifidobacterium lactis HSLA-009 broth was prepared as in example 20, except that stirring was also carried out during the fermentation at a speed of 300rpm.
The number of viable bacteria in the fermentation broth of Bifidobacterium lactis HSLA-009 in examples 21 to 23 is shown in Table 12.
TABLE 12 detection results of viable count in Bifidobacterium lactis HSLA-009 fermentation broth in examples 21 to 23
From table 12 it can be derived: the number of cells in the fermentation broth decreased with increasing stirring speed, and the shearing force on the cells increased with increasing stirring speed, so 100rpm was selected as the optimum speed for fermentation of bifidobacterium lactis HSLA-009.
In conclusion, it can be seen from examples 3 to 23 that the number of the final fermentation bacteria of Bifidobacterium lactis HSLA-009 reaches 1.65X 10 by using the fermentation conditions in example 21 10 The number of bacteria is about 15 times that of the initial culture medium.
Example 24
The preparation method of the freeze-dried powder of the bifidobacterium lactis HSLA-009 comprises the following steps:
the fermentation broth obtained in example 21 was centrifuged at 12000rpm for 15min, and the supernatant was discarded to obtain wet cells of strain HSLA-009, which were then mixed in the following weight ratio of 1:1, adding skimmed milk powder, adjusting water content to 90% with sterile normal saline, drying by conventional vacuum freeze drying process, pulverizing the obtained lyophilized block, and measuring bacterial count according to GB4789.35-2016 national food safety StandardMaterial test lactobacillus test. Detection shows that the number of bacteria in the obtained freeze-dried powder of Bifidobacterium lactis HSLA-009 is 3.01 × 10 11 CFU/g。
The experiment in example 2 was repeated using 100-fold dilution of the above-mentioned bacterial powder in sterile physiological saline as a high dose group and 1000-fold dilution as a low dose group. The results show that the reproducibility of the mouse experiment is good, which not only confirms the positive effect of the strain in treating constipation, but also shows that the excellent properties of the bifidobacterium lactis HSLA-009 do not disappear after the processes of fermentation, vacuum freeze drying and the like.
Example 25
Verification of practical application effect of bifidobacterium lactis HSLA-009 freeze-dried microbial inoculum
(1) Test object
International universal roman iv diagnostic criteria for functional constipation are: (1) more than 25% of defecation feels strenuous; (2) more than 25% of the excrement is dry fecal pellets or hard feces; (3) more than 25% of defecation has obstruction feeling and/or blockage feeling of anorectal; (4) more than 25% of defecation needs manipulation assistance (such as finger-assisted defecation and basin bottom support); (6) spontaneous Bowel Movements (SBM) were less than 3 times per week. Therefore, the experimental subjects selected in the experiment were studied by 2 or more groups of people who met the above diagnostic criteria.
The experimental subject in the experiment is collected in an on-line collection and on-line registration mode, a deposit system is adopted during collection to eliminate human interference factors, and after the examination is passed, the experimental subject signs an informed and cooperative agreement.
(2) Procedure of experiment
(1) Preparation of bifidobacterium lactis HSLA-009 microbial inoculum
First, the lyophilized powder prepared in example 24 was diluted, and the ratio of the lyophilized powder to the nonfat dry milk powder by weight was 1:5 and 1:1, respectively diluting, respectively mixing, and detecting by the method described in GB4789.35-2016 food safety national standard food microbiological inspection lactic acid bacteria inspection, to obtain the product with 5.0 × 10 bacteria count 10 CFU/g and the number of bacteria is 1.5X 10 11 CFU/g of two specifications of bacterial powder. Then separately combining the two specificationsSubpackaging the fungus powder into small packages of 2 g/package, and placing into a refrigeration house at-18 ℃ for refrigeration and standby.
(2) Contents of the experiment
In this experiment, the number of bacteria was 5.0X 10 10 CFU/g 2 g/bag and the number of bacteria is 1.5X 10 11 CFU/g 2 g/bag of two bacterial powder specifications, and two administration modes of once every 24h and once every 12h are simultaneously designed, namely four administration modes of HSLA-009 bacterial agent in the experiment are provided, wherein the number of bacteria in one bag is 5.0 × 10 after the bacterial powder is taken every 24h 10 The experimental group of CFU/g is group A; the number of bacteria per 12 hr is 5.0 × 10 10 The experimental group of CFU/g is group B; the number of bacteria per 24 hr is 1.5 × 10 11 The experimental group of CFU/g is group C; the number of bacteria per 12 hr is 1.5 × 10 11 The experimental group of CFU/g is group D.
The experimental subject can freely select any one of the administration modes, the microbial inoculum is issued according to the requirements of the experimental subject, and once the microbial inoculum is selected, the microbial inoculum cannot be changed in the whole experimental period. The administration mode is the conventional probiotic administration mode, the probiotic can be taken with warm water, and the probiotic powder can also be directly taken, which is not limited in the experiment; the taking time point is not required, but the medicine needs to be taken according to the selected taking mode of the experimental subject in the experimental period. The experimental period was 3 weeks.
(3) Test evaluation criteria
The experimental indexes are mainly designed according to the general roman IV diagnostic standard of functional constipation, and the related indexes are shown in a table 13.
TABLE 13 test evaluation criteria
Note: the above feces classification was carried out according to the Bristol stool VII classification method.
In addition to the evaluation indexes in table 13, the experiment also simultaneously investigates and counts other indexes of the experimental subject in the experimental process, such as the occurrence of side effects of no abdominal pain, abdominal distension, diarrhea and the like, the time for the microbial inoculum to take effect and cure, the satisfaction degree (classified in 1-10 minutes) of the experimental subject on the constipation treatment effect of the lyophilized microbial inoculum, and the like. In addition, the experiment also carries out call return visit three months after the experiment is finished, and inquires the recurrence rate condition of the experiment.
(4) Results of the experiment
500 volunteers are recruited in the co-recruitment process, wherein 460 volunteers can complete related experiments and carefully fill out the records of the experiments according to the requirements, and the experimental statistics results are shown in tables 14-15.
TABLE 14 Effect of lyophilized powder of example 24 on constipation patients of different sexes and ages
TABLE 15 Effect of different amounts of lyophilized powder of example 24 on constipation sufferers
As can be seen from the analysis in tables 14 to 15, the total effective rate of the lyophilized powder in example 24 for constipation patients is 97.83%, and the total cure rate is 96.09%; obvious effect can be seen in 3-4 days on average, and cure can be realized in about 6 days on average; during the experiment, no adverse reaction is caused, the recurrence rate is 0 after 3 months, and the experimental subject is more satisfactory to the treatment effect. These data indicate that Bifidobacterium lactis HSLA-009 has good therapeutic and prophylactic effects on constipation, and can be used as a drug, health product, food additive, etc.
In addition, as can be seen from the analysis, the cure rate under the effect of the dose A can reach 96.82%, which is slightly higher than the total cure rate, but the difference is not great, and as can be seen from the analysis of Table 15, the dose group A has no significant difference between the effective time and the curative time compared with the other three dose groups, so that the recommended dose for treating constipation by using the freeze-dried powder of Bifidobacterium lactis HSLA-009 is 5.0 × 10 bacteria/day 10 CFU/g bifidobacterium lactis HSLA-009 lyophilized powder 2g.
The embodiment can be used for obtaining that the strain is separated from camel milk, a strain with strong adaptability is found through a strain tolerance experiment, and the strain is identified to be bifidobacterium lactis (Bifidobacterium lactis). Meanwhile, the high-activity powdery preparation is prepared by optimizing fermentation conditions and performing a vacuum freeze-drying process, and is convenient to process, store and transport. Mouse experiments and crowd practical application experiments prove that the bacterium has a very remarkable effect in the aspect of treating functional constipation, can play a better treatment role no matter sex or age when used for treating constipation, and has the characteristics of wide application range, remarkable curative effect, high cure rate, low recurrence rate, no adverse side effect and the like when used for treating constipation.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. A Bifidobacterium lactis (B)Bifidobacterium lactis) HSLA-009, and the preservation number of the Bifidobacterium lactis HSLA-009 is CGMCC NO.25322.
2. A microbial preparation comprising Bifidobacterium lactis HSLA-009 as claimed in claim 1.
3. The microbial inoculum according to claim 2, which comprises the bifidobacterium lactis HSLA-009 thallus and/or bifidobacterium lactis HSLA-009 fermentation broth.
4. The microbial agent according to claim 2 or 3, wherein the viable count of Bifidobacterium lactis HSLA-009 in the microbial agent is 1.35 x 10 10 ~1.85×10 10 CFU/mL。
5. The preparation method of the microbial agent according to any one of claims 2 to 4, wherein the preparation method comprises the following steps:
inoculating bifidobacterium lactis HSLA-009 into a fermentation culture medium for anaerobic fermentation, wherein the obtained fermentation liquor is the microbial inoculum;
the fermentation medium takes water as a solvent and comprises the following components: 50-90g/L of glucose, 28-40g/L of yeast powder, 4-6g/L of sodium acetate, 2-4g/L of ammonium citrate, 2-4g/L of dipotassium hydrogen phosphate, 0.1-0.5g/L of magnesium sulfate, 0.01-0.1g/L of manganese sulfate and 1-2g/L of Tween.
6. The method of claim 5, wherein the anaerobic fermentation temperature is from 35 to 40 ℃, the pH is from 5.5 to 6.5, and the rotation speed is from 100 to 300rpm.
7. Use of the bifidobacterium lactis HSLA-009 of claim 1, the microbial inoculum of any one of claims 2 to 4 or the microbial inoculum prepared by the preparation method of claim 5 or 6 in preparing products for preventing and treating constipation and/or promoting intestinal health.
8. A product for preventing and treating constipation and/or promoting intestinal health, which is characterized by comprising the microbial inoculum according to any one of claims 2 to 4 or the microbial inoculum prepared by the preparation method according to any one of claims 5 to 6 and an auxiliary material.
9. The product of claim 8, wherein the viable count of the bifidobacterium lactis HSLA-009 in the product is 2.85 x 10 11 ~3.15×10 11 CFU/g。
10. The product according to claim 8 or 9, wherein the product is a lyophilized powder formed by the microbial inoculum and an auxiliary material, and the auxiliary material comprises skimmed milk powder; the mass ratio of the microbial inoculum to the auxiliary materials is 1.
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