CN116396905A - Bifidobacterium breve HC2953, microbial inoculum and application thereof - Google Patents

Bifidobacterium breve HC2953, microbial inoculum and application thereof Download PDF

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CN116396905A
CN116396905A CN202310367943.7A CN202310367943A CN116396905A CN 116396905 A CN116396905 A CN 116396905A CN 202310367943 A CN202310367943 A CN 202310367943A CN 116396905 A CN116396905 A CN 116396905A
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bifidobacterium breve
mice
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fat
cholesterol
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周亚男
解云芸
王永平
周群
李俊
韩海滨
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Weikaihaisi Shandong Bioengineering Co ltd
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Abstract

The invention belongs to the technical field of biological bactericides, and particularly relates to bifidobacterium breve HC2953, a microbial inoculum and application thereof. The invention relates to bifidobacterium breveBifidobacterium breve) HC2953 can reduce Total Cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and glutamic pyruvic transaminase (ALT) content, increase high density lipoprotein cholesterol (HDL-C), increase bile acid (TBA) excretion in obesity body, and improve body lipidThe metabolic disorder state has extremely strong colonization and adhesion capacity, so that the technical effect of reducing fat can be achieved, and the product for reducing weight and treating obesity can be prepared.

Description

Bifidobacterium breve HC2953, microbial inoculum and application thereof
Technical Field
The invention belongs to the technical field of biological bactericides, and particularly relates to bifidobacterium breve HC2953, a microbial inoculum and application thereof.
Background
Bifidobacterium breveBifidobacterium breve) The bifidobacterium belongs to one of the dominant bacteria in the intestinal tracts of mammals, belongs to the home flora in microecology, has dynamic change trend with age, and has close relation with a plurality of physiological and pathological change phenomena in the body. The bifidobacteria can be planted on the intestinal membrane of a host to form a biological barrier, and has important physiological functions of antagonizing pathogenic bacteria, improving microecological balance, providing nutrition, improving immunity, resisting tumors, reducing cholesterol level and the like.
Lipids in the human body are divided into fat and lipid, and in general, the content of fat in the human body often varies with factors such as nutritional status and energy consumption. The body fat ratio refers to the proportion of the weight of fat in the human body in the total weight of the human body, and is also called as body fat percentage, which reflects the content of fat in the human body. The normal range of body fat rate of adult is 20% -25% female and 15% -18% male respectively, if body fat rate is too high, body weight exceeds 20% of normal value, obesity can be regarded as, and obesity can increase risk of suffering from various diseases.
Probiotics can affect appetite and energy expenditure by producing short chain fatty acid acetate, propionate and butyrate, inhibit dietary fat absorption, and increase stool-discharged fat mass. Intestinal microorganisms secrete bioactive compounds by fermentation, induce a variety of responses within the intestinal mucosa, and simultaneously affect cellular metabolism of liver and adipose tissue, thereby regulating the homeostasis of lipids and glucose. Intestinal flora can also regulate intestinal barrier and endocrine functions and influence nutrient transport and absorption, so that regulation of intestinal flora is an effective strategy for improving and managing obesity, but the existing probiotics with lipid-lowering efficacy are fewer, and the probiotics resource library is not rich enough.
Disclosure of Invention
The invention aims to provide a bifidobacterium breveBifidobacterium breve) HC2953, the bifidobacterium breve HC2953 has the lipid-lowering effect, and can enrich the probiotic strain resource library.
The invention provides a bifidobacterium breveBifidobacterium breve) HC2953, wherein the preservation number of the bifidobacterium breve HC2953 is CCTCC NO: m20222007.
The invention also provides a microbial inoculum containing the bifidobacterium breve HC 2953.
Preferably, the viable count of the bifidobacterium breve HC2953 in the microbial inoculum is 100-2000 hundred million CFU/g.
Preferably, the microbial inoculum comprises the bifidobacterium breve HC2953 bacterial cells and/or bifidobacterium breve HC2953 fermentation broth.
The invention also provides application of the bifidobacterium breve HC2953 or the microbial inoculum in preparation of products with the weight-losing function.
Preferably, the product comprises a product having lipid lowering function.
Preferably, the product with lipid-lowering function comprises a product satisfying one or more of the following:
i: lowering total cholesterol concentration in the body;
II: lowering triglyceride concentration in vivo;
III: lowering the concentration of low density lipoprotein cholesterol in the body;
IV: reducing the content of glutamic pyruvic transaminase in the body;
V: increasing the concentration of high density lipoprotein cholesterol in the body;
VI: increase bile acid excretion in vivo.
The invention also provides a product with the weight-losing function, which comprises the bifidobacterium breve HC2953 or the microbial inoculum and the auxiliary materials in the technical scheme.
Preferably, the viable count of the bifidobacterium breve HC2953 in the product is 100-2000 hundred million CFU/g.
Preferably, the product comprises a pharmaceutical and/or functional food.
Advantageous effects
The invention provides a bifidobacterium breveBifidobacterium breve) HC2953, and has been subjected to biological preservation, the bifidobacterium breve HC2953 can reduce Total Cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) concentration and glutamic pyruvic transaminase (ALT) content in vivo, improve high density lipoprotein cholesterol (HDL-C) concentration, increase the excretion of bile acid (TBA) in obesity body, improve the state of lipid metabolism disorder in vivo, and have extremely strong colonisation and adhesion capability, so that the technical effect of reducing fat can be achieved, and the bifidobacterium breve HC2953 can be used for preparing products for losing weight and treating obesity.
Preservation of organisms
Bifidobacterium breveBifidobacterium breve) HC2953 was preserved in China center for type culture Collection (CCTCC NO) at the 12 th month 23 of 2022, with the preservation address of China, the university of Wuhan, and the preservation number of CCTCC NO: m20222007.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a standard curve for determining cholesterol content by the phthalic aldehyde colorimetry in example 1;
FIG. 2 is a phylogenetic tree of HC2953 strain in example 1;
FIG. 3 is a colony chart of HC2953 strain in example 1;
FIG. 4 is a microscopic morphological image of HC2953 strain in example 1;
FIG. 5-1 shows the measurement results of the body weight of the mouse high fat model in application example 1;
FIG. 5-2 shows the results of measurement of total cholesterol and triglyceride content in the mouse high fat model of application example 1;
FIG. 6 is a graph showing the results of weight measurement of mice at week 4 of the test period in application example 1;
FIG. 7 shows the results of measurement of feed utilization in mice at week 4 of the test period in application example 1;
FIG. 8 shows the results of measurement of body fat percentage of mice at week 10 of the test period in application example 1;
FIG. 9 shows the results of Lee' S index and BMI index measurements of mice at week 10 of the test period in application example 1;
FIGS. 10 to 11 are the results of measurement of organ indexes of mice at week 10 of the test period in application example 1;
FIG. 12 is a graph showing the results of measurement of blood-related indicators of mice at week 10 of the test period in application example 1;
FIGS. 13-1 to 13-2 are results of measurement of liver tissue-related index of mice at week 10 of the test period in application example 1;
FIG. 14 shows the results of the detection of mouse fecal bile acid in application example 1.
Description of the embodiments
The invention provides a bifidobacterium breveBifidobacterium breve) HC2953, wherein the preservation number of the bifidobacterium breve HC2953 is CCTCC NO: m20222007.
The bifidobacterium breve HC2953 is preferably obtained by screening and separating from the feces of healthy adults. The 16S rDNA of the bifidobacterium breve HC2953 is preferably shown as SEQ ID NO.1, and is specifically 5'-CATGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGATCCATCGGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCATCACACCGCATGGTGTGTTGGGAAAGCCTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGCACTTTGTGTTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGATCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGTGCGAGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGCGGGAGGGAGCCGTCTAAGGTGAGGCTCGTGATTGGGACTAAGTCG-3'. The strain has up to 99% homology with bifidobacterium breve ATCC15700 in NCBI database as shown in fig. 1; this strain was shown to have extremely high similarity to bifidobacterium breve, and therefore was designated as bifidobacterium breve HC2953.
The colony morphology of the bifidobacterium breve HC2953 on the MRS solid culture medium is as follows: regular round colonies with regular edges are provided with luster, are opaque and milky white in surface, are moist in whole, soft and fine in texture, are in a small short rod shape, are in a branched V shape, and are in a short stick shape with one thick end and one thin end.
The invention also provides a microbial inoculum containing the bifidobacterium breve HC2953 according to claim 1. The viable count of the bifidobacterium breve HC2953 in the microbial inoculum is preferably 100-2000 hundred million CFU/g, more preferably 200 hundred million CFU/g. The microbial inoculum of the present invention preferably includes the bifidobacterium breve HC2953 cell and/or the bifidobacterium breve HC2953 fermentation liquid, and more preferably includes the bifidobacterium breve HC2953 cell and the bifidobacterium breve HC2953 fermentation liquid.
The invention also provides a preparation method of the microbial inoculum, which comprises the following steps:
fermenting bifidobacterium breve HC2953 to obtain fermentation liquor;
and centrifuging the fermentation liquor, and taking bacterial mud for coating, freeze-drying and crushing to obtain the microbial inoculum.
Before the fermentation, the bifidobacterium breve HC2953 is preferably subjected to tempering, primary seed culture and secondary seed culture.
The present invention preferably carries out tempering on the bifidobacterium breve HC 2953. The temperature of the tempering is preferably normal temperature, and the time is preferably 0.5-1.5 h, more preferably 1h.
After the tempering, the invention preferably carries out primary seed culture on the bifidobacterium breve HC2953 after the tempering. The volume ratio of the first-stage seed tank for the first-stage seed culture to the seed culture medium is preferably 3-5: 2 to 4, more preferably 5:3. the inoculation amount of the bifidobacterium breve HC2953 after the temperature return is preferably 2% -5%, more preferably 3%. The temperature of the primary seed culture is preferably 35-37.5 ℃, more preferably 36.5 ℃; the time is preferably 16 to 20 hours, more preferably 18 hours. The seed culture medium of the invention preferably comprises the following components: 10.0g/L peptone, 5.0g/L yeast extract powder, 20.0g/L glucose, 2.0g/L dipotassium hydrogen phosphate, 2.0g/L triammonium citrate, 5.0g/L sodium acetate, 0.1g/L magnesium sulfate, 0.05g/L manganese sulfate, 1.0g/L Tween 80, 0.5g/L cysteine and 1.0g/L edible oil.
After the primary seed culture, the primary seed liquid obtained by the primary seed culture is preferably subjected to secondary seed culture. The volume ratio of the secondary seed tank for secondary seed culture to the seed culture medium is preferably 80-100: 55 to 75, more preferably 100:75. the inoculation amount of the primary seed liquid is preferably 3-7% of the volume of the seed culture medium, and more preferably 4%. The temperature of the secondary seed culture is preferably 35-37 ℃, more preferably 36.5 ℃; the time is preferably 10 to 12 hours, more preferably 10 hours. The seed culture medium is preferably the same as the seed culture medium used for primary seed culture, and will not be described in detail.
In the present invention, the inoculum size at the time of fermentation is preferably 8 to 12% by volume of the fermentation medium, more preferably 10%. The volume ratio of the fermentation tank to the fermentation medium is preferably 1000-1200: 750 to 1000, more preferably 1000:750. the fermentation temperature is preferably 35-37 ℃, more preferably 37 ℃; the time is preferably 8 to 11 hours, more preferably 10 hours. The fermentation medium according to the invention preferably comprises the following components: 20g/L of whey powder, 43g/L of glucose, 12g/L of yeast powder, 6g/L of peptone, 0.05g/L of manganese sulfate, 0.1g/L of magnesium sulfate, 1.0g/L of dipotassium hydrogen phosphate, 1.0g/L of tri-ammonium citrate, 1.0g/L of sodium acetate, 0.2g/L of cysteine and 1.0g/L of edible oil.
After the fermentation, the fermentation liquid is preferably centrifuged, bacterial sludge is taken for coating, freeze-drying and crushing, and the raw powder is obtained, namely the bacterial agent. The invention is not particularly limited to the centrifugation, and bacterial sludge can be obtained according to a conventional centrifugation mode in the field.
The coating liquid for coating preferably comprises the following components in parts by weight: 15% of skimmed milk powder, 8% of trehalose, 3% of glycerol, 1% of sodium ascorbate and 73% of distilled water. The mass ratio of the coating liquid to the bacterial sludge is preferably 1-2: 2 to 3, more preferably 1:2.
The coating method of the present invention is preferably a freeze-dried microencapsulated coating.
The freeze-drying is preferably vacuum freeze-drying, the specific parameters of the vacuum freeze-drying are not particularly limited, and the freeze-drying is carried out by preparing the freeze-drying powder by adopting the conventional freeze-drying parameters in the field.
The particle size of the raw powder is preferably 200-450 μm, more preferably 200 μm.
In order to obtain the microbial inoculum with different viable count contents, the invention preferably further comprises the steps of mixing the raw powder with auxiliary materials for blending and diluting to obtain the microbial inoculum with different viable count contents. The adjuvants of the invention are preferably, but not limited to, fructo-oligosaccharides or/and isomaltooligosaccharides. When the auxiliary materials are fructo-oligosaccharide and isomaltooligosaccharide, the mass ratio of the fructo-oligosaccharide to the isomaltooligosaccharide is preferably 0.5-1.5: 0.5 to 1.5, more preferably 1:1. the mass ratio of the raw powder to the auxiliary materials is not strictly defined, and the microbial inoculum prepared by mixing the raw powder serving as an active ingredient with the auxiliary materials belongs to the protection scope of the invention.
The microbial inoculum is preferably used in the form of a microbial suspension when in use, and the microbial suspension is preferably obtained by diluting the microbial inoculum with physiological saline. The invention has no special limitation on the viable bacteria concentration of the bifidobacterium breve HC2953 in the bacterial suspension, and the viable bacteria concentration after dilution according to the product property belongs to the protection scope of the invention.
The invention also provides application of the bifidobacterium breve HC2953 or the microbial inoculum of the technical scheme in preparation of products with the weight-losing function.
The products of the present invention preferably include products having lipid lowering functions, more preferably products that meet one or more of the following: i: lowering total cholesterol concentration in the body; II: lowering triglyceride concentration in vivo; III: lowering the concentration of low density lipoprotein cholesterol in the body; IV: reducing the content of glutamic pyruvic transaminase in the body; v: increasing the concentration of high density lipoprotein cholesterol in the body; VI: increase bile acid excretion in vivo. The body according to the invention preferably comprises serum or liver.
The bifidobacterium breve HC2953 can reduce the concentration of Total Cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and glutamic-pyruvic transaminase (ALT) in vivo, improve the concentration of high-density lipoprotein cholesterol (HDL-C), increase the excretion of bile acid (TBA) in obesity body, improve the state of lipid metabolism disorder in body, and have extremely strong colonization and adhesion capacity, so that the technical effect of reducing fat can be achieved, the bifidobacterium breve HC2953 can be used for preparing weight-reducing products, and has better treatment effect on obesity.
The invention also provides a product with the weight-losing function, which comprises the bifidobacterium breve HC2953 or the microbial inoculum and the auxiliary materials in the technical scheme. The viable count of the bifidobacterium breve HC2953 in the product is preferably (100-2000) multiplied by 10 8 CFU/g, more preferably 200X 10 8 CFU/g. The product according to the invention preferably comprises a pharmaceutical product and/or a functional food. When the product is a medicine, the auxiliary material is preferably a pharmaceutically acceptable auxiliary material; when the product is a functional food, the auxiliary material is preferably an auxiliary material acceptable in food preparation.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
In the examples of the present invention, the partial reagents used and the preparation methods thereof are as follows:
seed culture medium: taking water as a solvent, 10.0g/L of peptone, 5.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium hydrogen phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 1.0g/L of tween 80, 0.5g/L of cysteine and 1.0g/L of edible oil;
Fermentation medium: taking water as a solvent, 20g/L of whey powder, 43g/L of glucose, 12g/L of yeast powder, 6g/L of peptone, 0.05g/L of manganese sulfate, 0.1g/L of magnesium sulfate, 1.0g/L of dipotassium hydrogen phosphate, 1.0g/L of tri-ammonium citrate, 1.0g/L of sodium acetate, 0.2g/L of cysteine and 1.0g/L of edible oil;
MRS solid culture medium formula: taking water as a solvent, 10.0g/L of peptone, 5.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium hydrogen phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 1.0g/L of tween 80 and 15g/L of agar powder;
TPY agar medium: with water as solvent, hydrolyzed casein 10.0g/L, yeast powder 2.0g/L, phytone 5.0g/L, glucose 5.0g/L, dipotassium hydrogen phosphate 2.0g/L, magnesium chloride (6H) 2 O) 0.5g/L, zinc sulfate (7H) 2 O) 0.25g/L, 0.15g/L calcium chloride, trace ferric chloride, 0.5 g/L-cysteine, 1.0g/L Tween 80 and 20.0g/L agar;
m17 agar medium: taking water as a solvent, 5.0g/L of soybean peptone, 2.5g/L of casein peptone, 2.5g/L of yeast extract powder, 5.0g/L of lactose, 0.5g/L of sodium ascorbate, 19.5g/L of beta-sodium glycerophosphate, 0.25g/L of magnesium sulfate and 15.0g/L of agar;
MRS broth: 10g of peptone, 10g of beef powder, 5g of yeast powder, 20g of glucose, 0.1g of magnesium sulfate, 5g of sodium acetate, 2g of ammonium citrate, 2g of dipotassium hydrogen phosphate, 0.05g of manganese sulfate, 1mL of Tween 80, 1000mL of distilled water, pH 6.2+/-0.2 and sterilizing at 121 ℃ for 20min;
MRS triglyceride medium: according to Tween 80: lard = 1:1, heating and dissolving the mixture in proportion, performing ultrasonic emulsification at a temperature of the mixture for 15min to obtain triglyceride micelles, sterilizing the triglyceride micelles at 121 ℃ for 20min, and adding the triglyceride micelles into MRS broth containing 0.2% sodium thioglycolate in a volume ratio of 10% under a sterile condition for standby;
MRS glycerol medium: sterilizing appropriate amount of glycerol at 121deg.C for 20min, and adding into MRS broth containing 0.2% sodium thioglycolate at volume ratio of 2% under aseptic condition;
MRS cholesterol medium: adding 0.1g of cholesterol and 0.1g of sucrose ester into 1mL of Tween 80, uniformly mixing, adding 1mL of glacial acetic acid, heating for dissolving, preserving heat and performing ultrasonic emulsification for 15min to obtain cholesterol micelle, and sterilizing at 121 ℃ for 20 min. Under the aseptic condition, adding the prepared triglyceride micelle into MRS broth containing 0.2% sodium thioglycolate according to the proportion of 1% for standby;
gastric buffer (ph 3.0): accurately weigh 0.5144g KCl,0.1225g KH 2 PO 4 ，2.75085g NaCl，2.1002g NaHCO 3 ，0.0203g MgCl 2 ·(H 2 O) 6 ，0.0480g (NH 4 ) 2 CO 3 ，0.0083g CaCl 2 Constant volume to 1000mL;
simulating gastric juice: 3g of pepsin is dissolved in 1000mL of gastric buffer solution, pH is regulated to 3.0,0.22 mu m by using 1mol/L HCl, and filtration sterilization is carried out by using a filter membrane, and the preparation is carried out on the spot;
intestinal buffer: accurately weigh 0.5069g KCl,0.1089g KH 2 PO 4 ，2.2442g NaCl，7.1408g NaHCO 3 ，0.0067g MgCl 2 ·(H 2 O) 6 ，0.0333g CaCl 2 Constant volume to 1000mL;
Simulated intestinal fluid (0.1% bile salts): 1000mL of intestinal buffer, 1g of trypsin and 1g of bile salt were added for dissolution, and the pH of the simulated intestinal solution was adjusted to 8.0,0.22 μm by NaOH at 1mol/L for filtration sterilization with a filter membrane.
Example 1
Isolation and identification of bifidobacterium breve HC2953, the steps are as follows:
1. sampling: taking a stool sample of a healthy person, storing in a cold storage box, and numbering and recording.
2. And (3) increasing bacteria: and (3) taking a small amount of samples in the step (1), and respectively dissolving the samples in an MRS liquid culture medium and a TPY liquid culture medium, wherein the MRS liquid culture medium is a lactobacillus selective enrichment culture medium, and the TPY liquid culture medium is an enrichment culture of bifidobacteria. Wherein, the sample dissolved in MRS liquid culture medium is placed under aerobic condition at 37 ℃ for static culture for 24 hours, the sample dissolved in TPY liquid culture medium is placed under anaerobic condition at 37 ℃ for static culture for 24 hours, and the sample after bacterial enrichment is obtained. Aerobic, i.e. naturally placed in an air aerobic environment, anaerobic needs to be placed in an anaerobic bag with anaerobic conditions.
3. Separating: under the aseptic condition, 10 times gradient dilution is carried out on the sample after the enrichment in the step 2 by adopting aseptic normal saline, 100 mu L of each proper gradient is selected continuously according to the growth vigor of the culture medium and is respectively coated on a lactobacillus separation culture medium-MRS solid culture medium and a bifidobacterium identification culture medium-TPY agar culture medium, and the two culture mediums are respectively cultured under the aerobic and anaerobic conditions for 48 hours at 37 ℃. Selecting different bacterial colonies, repeatedly marking, separating and purifying, naming and preserving the bacterial colonies for later use; and (3) carrying out selective separation on a selective medium, and primarily screening to obtain the anaerobic bacillus HC2953.
1) And (3) activating HC2953 separated and purified in the step (3), inoculating 2% of the activated HC2953 into MRS broth, culturing at 37 ℃ until the HC is stable, and centrifuging at 6000rpm for 8 min to collect thalli. The bacterial suspension was adjusted to about 10 by resuspension after washing twice with sterile PBS buffer 9 CFU/mL was ready for use.
2) And (2) inoculating the bacterial suspension obtained in the step (1) into an MRS triglyceride culture medium, an MRS glycerol culture medium and an MRS cholesterol culture medium respectively in an inoculum size of 2% of the volume of the culture medium, culturing for 24 hours at 37 ℃, taking basic MRS broth as a blank, and measuring the degradation amount (degradation capacity) of the strain on the triglyceride, the glycerol and the cholesterol once every 6 hours, wherein the measurement method of the degradation amount of the strain on the triglyceride, the glycerol and the cholesterol is shown as 2.1-2.3.
2.1 Triglyceride degradation Capacity determination
Measuring the content of triglyceride by using an acetylacetone chromogenic method:
(1) Reagent: (1) the extracting agent comprises the following components: n-heptane: isopropanol was used in a 2:3.5, proportional configuration; (2) 0.04mol/L sulfuric acid; (3) saponification agent: 2.5g of analytically pure potassium hydroxide are dissolved in 50mL of distilled water and the volume is fixed to 500mL with isopropanol; (4) oxidizing agent: 77.0g of anhydrous ammonium acetate is weighed and dissolved in distilled water, 60mL of glacial acetic acid and 1.0g of periodic acid are added, and the distilled water is fixed to 1000mL; (5) color-developing agent: 2mL of acetylacetone was fixed to 500mL with isopropanol; (6) standard solution: 1.76mmol/L (150 mg/dL) triglyceride;
(2) The test process comprises the following steps:
the fermentation broth of HC2953 was sampled at 0h, 6h, 12h, 18h, 24h, 4mL of the fermentation broth was centrifuged at 4000rpm for 5min, and the supernatant was taken for use, and then the operations were performed as in Table 1:
TABLE 1 Triglycerides degradation Capacity test procedure
Figure SMS_1
After the addition of the above reagents, the tubes were thoroughly mixed, cooled in a water bath at 65℃for 15min, zeroed with a blank tube and absorbance was measured at 420 nm.
(3) The degradation rate of the strain on triglyceride was calculated according to the formula:
(1) triglyceride concentration at each sampling point (mmol/L) =A Sampling tube / A Standard tube ×C Standard tube Wherein:
A sampling tube : measuring absorbance of sample tube
A Standard tube : absorbance of standard tube
C Standard tube : standard tube concentration (mmol/L)
(2) Triglyceride degradation rate (%) = (a-B)/a×100% formula:
a:0h triglyceride concentration (mmol/L), i.e., total triglyceride concentration in the medium (mmol/L)
B: triglyceride concentration (mmol/L) at each sampling point
a-B: reduced triglyceride concentration (mmol/L).
2.2 Glycerol degradation Capacity determination
The glycerol content is determined by the periodate oxidation method:
sampling fermentation liquor of HC2953 strain at 0h, 6h, 12h, 18h and 24h, taking 4mL fermentation liquor, centrifuging at 4000rpm for 5min, and taking supernatant for later use. Accurately sucking 2mL of supernatant, adding 10mL of distilled water and 2 drops of phenolphthalein, titrating with 0.05mol/L sodium hydroxide standard solution until the color changes, then adding 10mL of 0.1mol/L sodium periodate solution, reacting for 5min in a dark place, adding 5mL of 25% glycol solution, reacting for 5min in a dark place, titrating with the sodium hydroxide again until the color changes, ensuring the consistency of the color changes, and recording the dosage of the sodium hydroxide.
And calculating the degradation rate of the strain to glycerol according to a formula:
(1) glycerin concentration (g/L) =92.1×c at each sampling point NaOH ×V NaOH In the formula/n:
C NaOH : naOH concentration (mol/L)
V NaOH : naOH consumption volume (mL)
n: sampling volume (mL)
(2) Glycerol degradation rate (%) = (a-B)/a×100% formula:
a:0h glycerol concentration (g/L), i.e., the total glycerol concentration (g/L) in the medium
B: glycerin concentration (g/L) at each sampling point
a-B: reduced glycerol concentration (g/L).
2.3 determination of cholesterol degradation Capacity
The content of cholesterol is determined by a phthalaldehyde colorimetric method:
(1) Drawing a standard curve: using normal hexane as a solvent to prepare a cholesterol standard solution with the concentration of 1mg/mL, accurately sucking 0.1mL, 0.2mL, 0.3mL, 0.5mL, 0.7mL and 0.9mL of the cholesterol standard solution into a colorimetric tube, drying by water bath nitrogen at 60 ℃, adding 4mL of the freshly prepared 0.5mg/mL of phthalic dicarboxaldehyde solution, standing at room temperature for 10min, slowly adding 2mL of concentrated sulfuric acid along the wall, uniformly mixing, standing for 20min for color development, measuring absorbance at the wavelength of 560nm, and drawing a standard curve, as shown in figure 1.
(2) Measuring the cholesterol content of fermentation liquor: sampling fermentation liquid of 0h, 6h, 12h, 18h and 24h, centrifuging 4mL of fermentation liquid at 4000rpm for 5min, adding 3.0mL of absolute ethyl alcohol and 2.0mL of 50% potassium hydroxide into 1mL of supernatant, keeping the temperature at 60 ℃ for 10min in a water bath, cooling, adding 5mL of n-hexane and 1mL of distilled water, carrying out shake extraction for 2min, standing for delamination, taking 3mL of upper n-hexane, drying in a water bath nitrogen at 60 ℃ and then adding 4mL of an freshly prepared 0.5mg/mL of phthalaldehyde solution, standing at room temperature for 10min, slowly adding 2mL of concentrated sulfuric acid along the wall, uniformly mixing and standing for 20min for color development, and measuring absorbance at a wavelength of 560 nm.
(3) And calculating the degradation rate of the strain on cholesterol according to a formula:
cholesterol degradation rate (%) = (a-B)/a×100% formula:
a:0h cholesterol level (mg/mL), the total cholesterol level in the medium
B: cholesterol amount (mg/mL) at each sampling point
a-B: reduced cholesterol amount mg/mL).
The measurement result shows that: the degradation rate of cholesterol, triglyceride and glycerol of the blank CK was 0, while the degradation rate of HC2953 on cholesterol was 73.55%, the degradation rate of triglyceride was 68.35%, and the degradation rate of glycerol was 52.10%.
3. Determination of the colonization ability of the Strain under simulated digestion conditions of gastrointestinal fluids
3.1 Gastric juice simulated digestion
Taking 1mL of enriched thalli, adding 9mL of simulated gastric juice, setting 3 parallel bacteria per strain, carrying out constant-temperature water bath at 37 ℃ at a rotating speed of 100r/min, respectively oscillating for 0h, 1h, 2h, 3h and 4h, respectively calculating the number of viable bacteria by a dilution coating flat plate method, and coating each sample for 3 times. The colonisation survival rate of the strain was calculated according to the formula:
gastric colonization survival = number of colonized viable bacteria/number of initial bacteria x 100%.
3.2 Intestinal juice simulated digestion
Taking 1mL of enriched thalli, adding 9mL of simulated intestinal juice, setting 3 parallel bacteria for each strain, carrying out constant-temperature water bath at 37 ℃ at the rotating speed of 100r/min, oscillating for 0h, 1h, 2h, 3h and 4h, calculating the number of viable bacteria by a dilution coating flat plate method, and coating each sample for 3 times. The colonisation survival rate of the strain was calculated according to the formula:
Intestinal fluid colonization survival = number of colonized viable bacteria/number of initial bacteria x 100%.
3.3 gastric juice-intestinal juice two-step method simulated digestion
Taking 1mL of enriched thalli, adding 9mL of simulated gastric juice, setting 3 parallel bacteria per strain, carrying out constant-temperature water bath at 37 ℃ at the rotating speed of 100r/min, taking out and rapidly centrifuging after shaking for 3 hours, adding 9mL of simulated intestinal juice into the bacteria, carrying out shaking culture for 2 hours under the same conditions, finally counting by a dilution coating flat plate method, and coating each sample for 3 times. The colonisation survival rate of the strain was calculated according to the formula:
intestinal fluid colonization survival = number of colonized viable bacteria/number of initial bacteria x 100%.
The results of the determination of the colonization ability of the strain under the simulated digestion condition of the gastrointestinal fluid are shown in figures 3-5.
Gastric acid and bile salt have a certain inhibition effect on bacteria, and probiotics must pass through gastric juice smoothly when entering human body, and meanwhile, the gastric acid and bile salt tolerance of the strain is an important index for measuring probiotics. The colonization and adhesion of probiotics in the gastrointestinal tract of a host can extend their residence time in the body, thereby affecting the health of the host by improving the local microbiota or its metabolites.
After simulated gastric juice is digested for 0h, 1h, 2h, 3h and 4h, the survival rate of HC2953 is 100%, 99.11%, 96.30%, 93.03% and 87.42%, respectively, and the survival rate of HC2953 is reduced to different degrees along with the time, and the bacterial strain of HC2953 has stronger colonisation capability along with the time in the simulated gastric juice;
After simulated intestinal juice is digested for 0h, 1h, 2h, 3h and 4h, the survival rate of HC2953 is 100%, 93.93%, 86.00%, 77.95% and 70.38%, respectively, and the survival rate of HC2953 is reduced to different degrees along with the time extension, and the colonization capacity of HC2953 strain is stronger along with the time change in the simulated intestinal juice;
in the experimental process of the simulated gastric fluid-intestinal fluid two-step method, after gastric fluid is digested for 3 hours and intestinal fluid is digested for 2 hours, the survival rate of HC2953 is 80.15%.
4. Determination of Strain adhesion Capacity
4.1 measurement of the ability of the Strain to adhere to mucin:
mucin model establishment: 10mg of mucin was weighed and 1mg/mL mucin solution was prepared in sterile PBS buffer and stored at-20 ℃. Taking 500 mu L of mucin solution, fixing the mucin solution on a 24-hole cell culture plate for 1h, culturing overnight at 4 ℃, adding an equal volume of mucin, continuously culturing at 37 ℃ for 2h to make up for blank sites, and washing the blank sites for 2 times by using sterile PBS buffer solution.
Adhesion ability measurement: after collecting thalli, 3 strains cultured overnight are respectively resuspended in sterile PBS, and the bacterial count is adjusted to 10 8 CFU/mL, coating count and record. After adding 500. Mu.L of bacterial suspension to the mucin model and incubating for 1h at 37 ℃, the model was washed 5 times with sterile citric acid buffer solution to remove unbound bacteria, and 1mL of 0.5% Tween 80 (v/v) was added to collect adherent bacteria. Finally, counting was performed by the dilution-coated plate method, each sample being coated with 3 replicates. Strain adhesion rate was calculated according to the formula:
Mucin adhesion rate = number of adherent bacteria/number of initial bacteria x 100%.
4.2 Measurement of the adhesion ability of the strain to Caco-2 cells:
0.5mL of Caco-2 cells were seeded into 24-well cell culture plates, wherein the Caco-2 cells were adjusted to 2X 10 5 cell/mL, culture to monolayer, PBS wash 2 times, add 500. Mu.L bacterial suspension (10 8 CFU/mL), for 2h at 37 ℃, washing 3 times with pbs to remove non-adhered bacteria, adding 150 μl of pancreatin cell digestive juice, adding 350 μl of MEM complete culture liquid after complete cell shedding to terminate digestion, and finally counting by a dilution coating plate method, wherein each sample is coated with 3 replicates. The strain adhesion rate was calculated according to the formula:
caco-2 cell adhesion ratio = number of adherent bacteria/number of initial bacteria x 100%.
The colonization and adhesion of probiotics in the gastrointestinal tract of a host can extend their residence time in the body, thereby affecting the health of the host by improving the local microbiota or its metabolites.
In the measurement of the ability of the strain to adhere to mucin, the adhesion rate of HC2953 to mucin was 47.20%. In the measurement of the adhesion capacity of the strain to Caco-2 cells, the adhesion rate of HC2953 was 57.24%.
The adhesion rate of HC2953 to mucin and the adhesion rate to Caco-2 cells are relatively good by combining the two results, so that the strain is selected as a target strain for the experiment, and subsequent strain identification and animal experiments are carried out.
5. 1) carrying out physiological and biochemical identification on the strain HC2953, wherein the identification results are shown in table 2:
and screening partial indexes to carry out physiological and biochemical identification on the strain according to the method in the Berger's bacteria identification manual and the common bacteria system identification manual.
TABLE 2 physiological and biochemical identification results of HC2953 Strain
Figure SMS_2
2) And selecting a single colony of the purified HC2953 strain, preparing bacterial suspension in sterile water, and performing colony PCR amplification on the strain 16S rDNA by using the bacterial suspension as a PCR template. The PCR primer was synthesized and sequenced by Shanghai Biotechnology Co., ltd using a 27F (5'-AGAGTTTGATCCTGGCTCAG-3') sequence shown in SEQ ID NO.2 and a 1492R (5'-GGTTACCTTGTTACGACTT-3') universal primer shown in SEQ ID NO.3, the sequenced sequence was shown in SEQ ID NO.1, the sequences were aligned in the national center for Biotechnology information (National Center for Biotechnology Information, NCBI) database for 16S rDNA sequences of the strain, and the phylogenetic tree was constructed using MEGA 5.0 software, and the results are shown in FIG. 2.
From fig. 2, it can be derived that: the homology of the HC2953 strain with bifidobacterium breve ATCC15700 in NCBI database is as high as 99%, indicating that the strain is bifidobacterium breve and is named HC2953.
Example 2
The preparation method of the microbial inoculum containing the bifidobacterium breve HC2953 comprises the following steps:
the preserved bifidobacterium breve HC2953 is warmed for 1h at normal temperature, inoculated in a 3.5L seed tank containing a seed culture medium for 16-20h to prepare first-stage seeds, then transferred to a 100L seed tank containing the seed culture medium for 6-8h to obtain second-stage seeds, and then transferred to a 1000L fermentation tank for fermentation with the inoculation amount of 10% volume to obtain fermentation liquor.
Centrifuging the obtained fermentation liquor to obtain bacterial sludge, coating the bacterial sludge, performing vacuum freeze drying to obtain freeze-dried blocks, and crushing the freeze-dried blocks to obtain bifidobacterium breve raw powder;
fully mixing the bifidobacterium breve raw powder with the mixture of the fructo-oligosaccharide and the isomaltooligosaccharide, and blending and diluting to obtain the microbial inoculum with different viable cell count specifications, wherein the mass ratio of the fructo-oligosaccharide to the isomaltooligosaccharide is 1:1.
Mixing 1.0g of raw bifidobacterium breve powder with 39.0g of mixture of fructo-oligosaccharide and isomaltooligosaccharide, wherein the viable count of bifidobacterium breve HC2953 in the obtained bacterial powder is more than or equal to 100 hundred million CFU/g;
mixing 1.0g of bifidobacterium breve raw powder with 19.0g of mixture of fructo-oligosaccharide and isomaltooligosaccharide, wherein the viable count of bifidobacterium breve HC2953 in the obtained bacterial powder is more than or equal to 200 hundred million CFU/g;
Mixing 1.0g of bifidobacterium breve raw powder with 7.0g of mixture of fructo-oligosaccharide and isomaltooligosaccharide, wherein the viable count of bifidobacterium breve HC2953 in the obtained bacterial powder is more than or equal to 500 hundred million CFU/g;
mixing 1.0g of bifidobacterium breve raw powder with 3.0g of mixture of fructo-oligosaccharide and isomaltooligosaccharide, wherein the viable count of bifidobacterium breve HC2953 in the obtained bacterial powder is more than or equal to 1000 hundred million CFU/g;
1.0g of raw bifidobacterium breve powder is mixed with 1.0g of mixture of fructo-oligosaccharide and isomaltooligosaccharide, and the viable count of bifidobacterium breve HC2953 in the obtained bacterial powder is more than or equal to 2000 hundred million CFU/g.
Application example 1
Animal experiments prove that bifidobacterium breve HC2953 has lipid-lowering function
(1) Basal feed: the corn flour comprises, by mass, 25% of corn flour, 20% of bran, 18% of flour, 20% of bean cake, 8% of rice flour, 2% of fish meal, 2% of calcium powder, 2% of bone meal, 1.9% of yeast powder, 0.9% of salt, 0.1% of compound vitamin and 0.1% of trace elements, and is uniformly mixed and granulated, and sterilized before use.
(2) High-fat feed: the feed comprises, by mass, 12% of egg yolk powder, 12% of lard, 5% of soybean oil, 6% of sucrose, 0.2% of bile salt and 64.8% of basic feed, and is prepared by uniformly mixing, granulating and sterilizing before use.
(3) Grouping and feeding:
60 SPF-class mice with the age of 5 weeks are subjected to initial weight of 20+/-2 g, free drinking and feeding, padding replacement at regular time, abnormal mice are removed after 1 week of adaptive feeding, and the mice are divided into 4 groups by adopting a random group grouping method: blank group (NCD), high fat model group (HFD), high fat probiotic group A1 (HFD-A1) and high fat probiotic group A2 (HFD-A2), wherein HFD and HFD-A2 are cultured together before modeling is successful, and the modeling is divided into 2 groups randomly, namely HFD and HFD-A2, and the mixture is fed according to a table, and the feed and water can be obtained from Obtained by the method. The viable bacteria source is obtained by diluting the raw powder obtained in example 2 with physiological saline, and the viable bacteria number of Bifidobacterium breve HC2953 in the prepared bacterial liquid is 1×10 9 CFU/mL. The feeding patterns of the test groups are shown in Table 3.
TABLE 3 animal experiments verify the feeding regimen of bifidobacterium breve HC2953
Test group Feeding mode Gastric lavage/(1 mL (100 g/bw. D)) Early and late 8:40 two lavages per day
Blank control group (NCD) Basic feed + water 0.9% physiological saline Self-grouping post-timing lavage
High fat model set (HFD) High fat feed + water 0.9% physiological saline Self-grouping post-timing lavage
High fat probiotic group A1 (HFD-A1) High fat feed + water Viable bacteria (1×10) 9 CFU/mL） Self-grouping post-timing lavage
High fat probiotic group A2 (HFD-A2) High fat feed + water Viable bacteria (1×10) 9 CFU/mL） Timing gastric lavage after modeling success
During the test, the feed intake of the mice, the weight of the mice, and the collection of the feces of the mice were recorded at fixed times per week; at the 4 th week of the test period, blood is taken from the eye sockets of the mice after grouping, various indexes are measured, and whether the high-fat model is successfully established is checked; and on the 10 th week of the test period, the mice are fasted without water control for 12 hours, the final weight and body length of the mice are recorded, the mice are killed by neck removal after blood is taken from eyeballs, all organs and fat are rapidly taken out in the dissection process, the weighing record is carried out, and the mice are temporarily stored in liquid nitrogen at-80 ℃ and are reserved for subsequent sample measurement.
And (3) establishing a high-fat model:
the weights of four groups of mice at the 4 th week of the test period were recorded, as shown in fig. 5-1, and the orbital blood was collected, and the collected blood was left standing at room temperature for 1 hour, and centrifuged at 8000rpm for 10 minutes at 4 ℃ to separate serum for later use, and the Total Cholesterol (TC) and Triglyceride (TG) contents in the serum were measured by using a Total Cholesterol (TC) test kit and a Triglyceride (TG) test kit, which were purchased from the institute of bioengineering in tokyo, and the results were shown in fig. 5-2.
According to the analysis of the body weight of mice in the fourth week of fig. 5, the difference of the body weight among mice in each group is obvious, the body weight of the HFD group is increased by 20.15% compared with that of the NCD group, and the conclusion of successful establishment of the high-fat model can be obtained by combining the obvious difference of the serum TC and TG of the mice in each group.
Body weight and food utilization rate of mice:
on day 20 a week: 00 weigh the mice, record the feed consumption weight, calculate the mice feed utilization according to formula.
The formula: feed utilization (%) =final body weight gain (g)/final feed consumption (g) ×100%, and the results are shown in fig. 6 to 7.
From fig. 6, it can be derived that: the weight difference was more pronounced in the mice of each group after week 2 of the test period, and the weight of the blank group (NCD) was used as a control, and the weight of the high fat model group (HFD) was 20.15% higher in week 4, after which the weekly weight overrun was higher than in the previous week. HFD-A1 group was gavaged 1X 10 on the basis of normal high-fat diet starting from the first week of the test period 9 CFU/mL viable bacteria, whereas HFD-A2 group began gavage 1X 10 after confirming that the modeling was successful 9 The CFU/mL viable bacteria can be seen from the process of weekly weight change, and the HFD-A1 group from which the stomach is irrigated at the initial stage of the test has obvious weight reduction difference compared with the HFD-A2 group from which the stomach is irrigated after the modeling is successful. And the HFD-A1 and HFD-A2 have very obvious weight reduction difference compared with the HFD group. It can be concluded that the bifidobacterium breve HC2953 strain powder can significantly reduce the weight of the high-fat mice, and the earlier the application time, the better the effect.
From fig. 7, it can be derived that: the utilization ratio of HFD > HFD-A2 > HFD-A1 > NCD of the four groups of feeds. Wherein, the HFD, HFD-A2 and HFD-A1 groups are all fed with high-fat feed, and the calorie and fat content of the high-fat feed are far higher than those of the basic feed. The analysis data can show that the utilization rate of the HFD group to the feed is 1.97 times of that of the NCD group; the utilization rate of HFD-A2 to feed is 1.55 times of that of NCD group, which is reduced by 27.70% compared with HFD group; the utilization rate of HFD-A1 to feed is 1.34 times of that of NCD group, and is reduced by 47.77% compared with HFD group. The above results indicate that the probiotic bifidobacterium breve can inhibit the weight gain of the mice by reducing the feed utilization of the obese mice.
Body fat rate of mice:
the test cycle was 10 weeks, the mice were fasted without water control for 12 hours, and after the mice were sacrificed, inguinal subcutaneous fat, epididymal fat, perirenal fat were precisely and rapidly separated in the dissection process, weighed and recorded. The body fat rate of the mice was calculated according to the formula.
The formula: body fat ratio (%) = [ inguinal subcutaneous fat (g) +epididymal fat (g) +perirenal fat (g) ]/body weight (g) ×100%, and the result is shown in fig. 8.
From fig. 8, it can be derived that: the HFD group body fat rate was 174.31% higher than that of the NCD group, while the HFD-A1 and HFD-A2 group body fat rates were significantly increased over the NCD group, they were significantly reduced by 99.77% and 27.79% over the HFD group, respectively. Body fat rate is the proportion of abdominal fat to body weight, and is mainly reflected in fat content, so that whether normal, lean or obese state is judged. The results show that the probiotics bifidobacterium breve has remarkable effect on reducing the body fat rate of the obese mice, and the earlier the use time, the better the use time.
Mouse Lee' S index and BMI index determination:
the test cycle was 10 weeks, the mice were fasted without water inhibition for 12 hours, the mice were weighed, and the length from the nose end to the anus of the mice was measured and recorded as body length. Lee' S index and BMI index of the mice were calculated according to the formula.
The formula: lee' S index= [ body weight (g) ×10 3 ] 1/3 Body length (cm);
BMI index = body weight (kg)/body length (m) 2 The results are shown in FIG. 9.
The BMI index reflects an important index for determining the obesity degree of a mouse by the relation between the body weight and the body length of the mouse, and the Lee' S index is also an effective index in evaluating the obesity degree. From the analysis of Lee 'S index and BMI index in each group of FIG. 9, it can be seen that the Lee' S index and BMI index of HFD-A1 and HFD-A2 groups are reduced to different degrees compared with the HFD group data, which indicates that the obesity degree of mice is remarkably reduced after using the bifidobacterium breve HC2953 bacterial powder.
Organ index measurement:
at the 10 th week of the test period, the mice are fasted without water control for 12 hours, and after the mice are sacrificed, the organs of the mice are accurately and rapidly taken out in the dissecting process: heart, kidney, spleen, liver, thymus, weighed and data recorded. And calculating the index of each organ of the mice according to the formula.
Liver index = liver weight (g)/body weight (g) x 100%;
spleen index = spleen weight (g)/body weight (g) ×100%;
heart index = heart weight (g)/body weight (g) x 100%;
kidney index = kidney weight (g)/body weight (g) ×100%;
thymus index = thymus weight (g)/body weight (g) x 100%.
The results are shown in FIGS. 10 to 11.
Obesity may lead to a difference in organ index from the normal index and is also an important reference for detecting the health index of obese mice. From fig. 10 to 11, it can be seen that the index of the other organs of the obese mice in the HFD group is significantly higher than that of the normal mice in the NCD group except the heart, and the index of the organs of the HFD-A1 and HFD-A2 groups subjected to the stomach infusion by the bifidobacterium breve powder is significantly lower than that of the HFD group, and the organs of the obese mice in the HFD group are close to the NCD group, so that the effect of the HFD-A1 group is most remarkable. In conclusion, the probiotic bifidobacterium breve HC2953 bacterial powder can help obese mice to maintain normal liver index, spleen index, heart index, kidney index and thymus index, so that the health state of the mice can be maintained.
Blood collection and related index measurement:
the test cycle was 10 weeks, the mice were fasted without water for 12 hours, the mice were subjected to eyeball extraction to obtain blood, the blood samples were collected and left standing for 1 hour at room temperature, and centrifuged at 8000rpm for 10 minutes to separate serum for later use, and the contents of Total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (glutamate pyruvate transaminase/ALT) in serum were measured by a Total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and glutamate pyruvate transaminase (ALT) kit purchased from a south-established bioengineering institute and calculated according to the formula.
Arteriosclerosis Index (AI) = [ Total Cholesterol (TC) (mmol/L) -high density lipoprotein cholesterol (HDL-C) (mmol/L) ]/high density lipoprotein cholesterol (HDL-C) (mmol/L), the results are shown in fig. 12.
High-fat diets result in significant decreases in HDL levels and significant increases in TC, TG and LDL levels in mice, resulting in lipid metabolism disorders in the body.
As can be seen from fig. 12, in this experiment, the serum HDL-C of the group of mice fed basal diet was significantly higher than that of the group of mice fed HFD (p < 0.05), while TC, TG, LDL-C, ALT and AI index was significantly lower than that of the group of obese mice fed high fat diet (p < 0.05), indicating lipid imbalance in the serum of obese mice fed high fat diet. The HFD-A1 and HFD-A2 groups which are subjected to dry prognosis of the probiotics bifidobacterium breve have significantly reduced TC, TG, LDL-C, ALT and AI indexes compared with the HFD groups, HDL-C is significantly higher than that of an obese group, and the effect of the HFD-A1 group is most significantly towards the normal level. The result shows that the probiotic bifidobacterium breve can achieve the aim of maintaining the lipid metabolism balance in the serum of the mice by reducing TC, TG, LDL-C and ALT in the serum of the mice and improving the content of HDL-C.
Liver tissue related index determination:
on the 10 th week of the test period, the mice are fasted without water control for 12 hours, after being killed, the mice are dissected to obtain the livers of the mice, and then the livers of the mice are stored in liquid nitrogen, 0.5g of liver tissue is accurately weighed, and 10 mL chloroform is added: mixing the methanol (2:1, v/v) mixed solution with shaking, preserving the temperature at 37 ℃ for 30min, centrifuging at 8000rpm for 10min at 4 ℃, collecting a chloroform layer in a sterile tube, adding 6mL of physiological saline for two times for centrifugation, collecting a bottom chloroform layer, blow-drying by a nitrogen blower, and adding 0.8mL of isopropanol: and (3) uniformly vibrating the Triton-100 (9:1, v/v) mixed solution, adding 1.2mL of distilled water, and fully and uniformly mixing to obtain the liver tissue total lipid solution.
The Total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and glutamic-pyruvic transaminase (ALT) contents in the liver homogenates were determined by Total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and glutamic-pyruvic transaminase (ALT) kit, and the results are shown in FIGS. 13-1 and 13-2.
The liver is the center of lipid metabolism and is closely related to metabolic diseases. Under normal conditions, lipid metabolism in the liver remains in an equilibrium state, and obesity caused by long-term high-fat diet can lead to liver lipid metabolism disorder, leading to abnormally high levels of TC, TG in the liver.
As can be seen from fig. 13, liver TC, TG, LDL-C and ALT were significantly reduced (p < 0.05) in the HFD group obese mice, HDL-C was significantly increased (p < 0.05), HFD-A1 and HFD-A2 groups, TC, TG, LDL-C, ALT and HDL-C tended to be normal levels after the probiotic bifidobacterium breve stem prognosis, with the HFD-A1 group effect being most pronounced, compared to the NCD group. The result shows that the probiotics bifidobacterium breve can reduce TC, TG, LDL-C and ALT in the liver of the mouse, and the HDL-C content is improved to achieve the aim of maintaining the lipid balance state in the liver of the mouse.
Liver probiotics detection:
and (3) at the 10 th week of the test period, the mice are fasted and not forbidden for 12 hours, after the mice are killed, the mice are dissected to obtain livers, the livers are rapidly placed in a sterile flat plate, 1g of the livers are cut and added into a sterile PBS buffer solution for suspension and homogenization, 1mL of the solution is coated on an MRS solid flat plate, the temperature is constant and the aerobic and anaerobic culture is carried out at 37 ℃ for 48 h, and whether probiotics grow in the flat plate is detected.
Studies have shown that after a high-fat model mouse is fed with certain probiotics, strains grow in the liver, thereby leading to liver failure.
In this experiment, liver tissue of each group of mice was aseptically coated on MRS plates, and colony formation was not observed after aerobic and anaerobic culture of 48 h. This suggests that the probiotic does not undergo liver and intestine translocation in animals.
Mouse fecal bile acid detection:
the collected mouse manure is dried to constant weight, 1g of dry manure is weighed, 10mL of absolute ethyl alcohol is used for three times at 80 ℃ to obtain an extracting solution, the extracting solution is evaporated to dryness at 90 ℃ in a water bath, 10mL of petroleum ether is added for dissolution, and the supernatant is discarded. The resulting solution was dissolved in 10mL of distilled water by shaking thoroughly with ethanol containing 2% TritonX-100, and the Total Bile Acid (TBA) content of the feces was measured with a TBA kit, and the results are shown in FIG. 14.
Cholesterol is a precursor substance for synthesis of bile acids, and promotion of bile acid excretion in the intestinal tract is a major pathway for lowering cholesterol metabolism in the body.
From fig. 14, it can be derived that: the content of bile acid in the feces of HFD-A1 and HFD-A2 mice subjected to probiotic intervention is 1.92 times and 1.56 times of that of HFD group respectively. Therefore, the bifidobacterium longum HC2953 bacterial powder can effectively increase the excretion amount of the TBA of the mice, thereby reducing the cholesterol content in the bodies.
From the above embodiments it can be derived that: the bifidobacterium breve HC2953 disclosed by the invention can be used for efficiently degrading cholesterol, triglyceride and glycerol, and has extremely strong colonization capacity and adhesion capacity; meanwhile, HC2953 can achieve the purpose of controlling the weight of the mice by reducing the feed utilization rate of the high-fat obese mice; the visceral organ index of the mice is improved, so that the fat synthesis amount of the mice is reduced, and the fat-rich obese mice reach a body health and body fat balance state; HC2953 improves the lipid metabolism disorder state of high-fat mice by reducing the concentration of Total Cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and glutamic-pyruvic transaminase (ALT) in serum and liver of the fat mice, increasing the concentration of high-density lipoprotein cholesterol (HDL-C), increasing the excretion of bile acid (TBA) in the fat mice; thereby achieving the technical effects of reducing blood fat and losing weight.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. Bifidobacterium breve @ sBifidobacterium breve) HC2953, wherein the preservation number of the bifidobacterium breve HC2953 is CCTCC NO: m20222007.
2. A microbial preparation comprising the bifidobacterium breve HC2953 as claimed in claim 1.
3. The microbial agent according to claim 2, wherein the viable count of bifidobacterium breve HC2953 in the microbial agent is 100-2000 hundred million CFU/g.
4. A bacterial agent according to claim 1 or 2, wherein the bacterial agent comprises the bifidobacterium breve HC2953 bacterial cells and/or a bifidobacterium breve HC2953 fermentation broth.
5. The use of the bifidobacterium breve HC2953 as claimed in claim 1 or the microbial inoculum as claimed in any one of claims 2 to 4 in the preparation of products with weight-reducing function.
6. The use according to claim 5, wherein the product comprises a product with lipid-lowering function.
7. The use according to claim 6, wherein the product with lipid-lowering function comprises a product satisfying one or more of the following:
I: lowering total cholesterol concentration in the body;
II: lowering triglyceride concentration in vivo;
III: lowering the concentration of low density lipoprotein cholesterol in the body;
IV: reducing the content of glutamic pyruvic transaminase in the body;
v: increasing the concentration of high density lipoprotein cholesterol in the body;
VI: increase bile acid excretion in vivo.
8. A product with weight-reducing function, which is characterized by comprising the bifidobacterium breve HC2953 according to claim 1 or the microbial inoculum and auxiliary materials according to any one of claims 2-4.
9. The product according to claim 8, wherein the viable count of bifidobacterium breve HC2953 in the product is 100-2000 hundred million CFU/g.
10. Product according to claim 8 or 9, characterized in that the product comprises a pharmaceutical product and/or a functional food.
CN202310367943.7A 2023-04-08 2023-04-08 Bifidobacterium breve HC2953, microbial inoculum and application thereof Pending CN116396905A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040089987A (en) * 2003-04-15 2004-10-22 (주) 피엘바이오 Korean Isolate Bifidobacterium breve PL9504 with High cholesterol-decomposing Activity
CN101314763A (en) * 2007-06-01 2008-12-03 统一企业(中国)投资有限公司 Short bifidobacteria with functions of anti-gastrointestinal tract pathogen, oxidation resistance and blood pressure reduction
US20120076829A1 (en) * 2009-03-10 2012-03-29 Hero Espana S.A. Isolation, identification and characterisation of strains with probiotic activity, from faeces of infants fed exclusively with breast milk

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040089987A (en) * 2003-04-15 2004-10-22 (주) 피엘바이오 Korean Isolate Bifidobacterium breve PL9504 with High cholesterol-decomposing Activity
CN101314763A (en) * 2007-06-01 2008-12-03 统一企业(中国)投资有限公司 Short bifidobacteria with functions of anti-gastrointestinal tract pathogen, oxidation resistance and blood pressure reduction
US20120076829A1 (en) * 2009-03-10 2012-03-29 Hero Espana S.A. Isolation, identification and characterisation of strains with probiotic activity, from faeces of infants fed exclusively with breast milk

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