CN109929773B - Bifidobacterium capable of being used for selenium-rich culture and active protein and application thereof - Google Patents

Abstract

The invention discloses a novel bifidobacterium DD98, the 16SrDNA nucleotide sequence of which is shown as SEQ ID No: 1, and the bifidobacterium is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 16573. The bifidobacterium DD98 can tolerate higher selenium pressure, can be used for selenium-enriched culture, has good intestinal tract colonization capacity, and has an intestinal tract protection function on the water-soluble protein obtained by extraction. The bifidobacterium DD98 can relieve liver injury caused by high-fat diet and alcohol and has a protective effect on the liver.

Description

Bifidobacterium capable of being used for selenium-rich culture and active protein and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to bifidobacterium, active protein and application thereof, and further, the bifidobacterium can be prepared into selenium-rich probiotics through selenium-rich culture.

Background

Probiotics are a class of microorganisms that produce beneficial effects on the host, and are closely related to the health of the human body. The probiotics are generally planted in the intestinal tract and the reproductive system of a human body and play an important role in maintaining the micro-ecological balance in the human body. The existing research results show that the stabilization of human microbiota is very important for maintaining human health, and aging, hypertension, diabetes, intestinal diseases and the like are all related to the human microbiota. An imbalance in the human microbiota can directly or indirectly contribute to the development of these diseases. The bifidobacterium is a common physiological probiotic in human bodies, is planted in the gastrointestinal tracts of the human bodies and is very important for maintaining and regulating the intestinal flora in the human bodies. Many researches show that the live bacteria preparation prepared by using the probiotics such as lactobacillus, bifidobacterium and the like can replace antibiotics to play roles in protecting intestinal tracts and maintaining intestinal tract balance, so that the digestion diseases such as diarrhea, constipation and the like are prevented and treated, and the live bacteria preparation has a plurality of obvious probiotic effects such as blood sugar reduction, cholesterol reduction, liver protection, immunity enhancement, cancer resistance and the like. According to the latest (2004) berjie's manual of identification of bacteria, the bifidobacteria obtained on the basis of 16SrDNA sequencing technology were grouped into a total of 34 different species, including bifidobacterium adolescentis, bifidobacterium horn, bifidobacterium animalis, bifidobacterium asteroids, bifidobacterium bifidum, bifidobacterium bovis, bifidobacterium breve, bifidobacterium odonta, bifidobacterium infantis (i.e., bifidobacterium longum subsp. infantis.), bifidobacterium lactis (i.e., bifidobacterium animalis subsp. cremoris), bifidobacterium longum, bifidobacterium pseudocatenulatum, and bifidobacterium thermophilum and bifidobacterium acidophilum, among others. Patent CN200910136613.7 reports a bifidobacterium BBMN68 which has the capability of adhering to intestinal epithelial cells and has the function of regulating flora. Patent CN201110388295.0 reports a strain of bifidobacterium infantis IFA09 capable of alleviating symptoms of gastrointestinal dysfunction.

Selenium is one of the essential trace elements for human body, and can participate in the synthesis of various seleno-amino acids and various selenoproteins (enzymes). Selenium element and selenoprotein synthesized by selenium element have a plurality of biological activities, such as oxidation resistance, tumor resistance, human immunity enhancement and the like, so selenium is closely related to human health. The world health organization proposes that selenium is an essential trace element for promoting human life health, and if the selenium is reasonably taken in daily life, the selenium can effectively prevent various diseases. The selenium-enriched yeast is a selenium supplement product which is widely applied in the current market, and the yeast can be used as a conversion carrier to convert external inorganic selenium into organic selenium, so that the selenium-enriched yeast is more beneficial to the absorption of a human body. Besides, probiotics such as bifidobacterium and lactobacillus can also be used for selenium culture and transformation. The selenium-enriched probiotics may have superior biological activity compared to the selenium-enriched yeast due to the probiotic effect of the probiotics themselves.

Although bifidobacteria are GRAS (generally recognized as safe) probiotics, studies have shown that they are capable of performing multiple probiotic functions after ingestion by humans. However, there are few strains of bifidobacteria available in food at home and abroad, because after the bifidobacteria are orally taken into human bodies, the bifidobacteria can affect the colonization in human intestinal tracts, for example, the acidic environment of the stomach with low pH value and digestive enzymes in the stomach can reduce the proton power and destroy the osmotic pressure balance inside and outside the bifidobacteria cells; high-concentration bile salt and endogenous antibacterial peptide in the small intestine also have defense systems of intestinal inherent flora to organisms, glycocholate and bezoar cholate have cell membrane toxicity, and bifidobacterium with high bile salt hydrolase activity hydrolyzes the cholate into secondary cholate to reduce the cell membrane toxicity; the intestinal colonization by bifidobacteria is influenced by the ability of bifidobacteria to adhere to intestinal epithelial cells and by the availability of nutrients (some proteinics, prebiotics such as oligosaccharides, etc.) that are not metabolized by the host. Therefore, the bifidobacterium which can be colonized in the intestinal tract is obtained by separation, and the method has very important application value and practical significance.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel bifidobacterium DD98 which can be further used for selenium-rich culture and has good intestinal tract colonization ability and intestinal tract protection and liver injury protection functions. In addition, in order to avoid the problem that the bifidobacteria lose activity due to environmental sensitivity, the invention extracts the active ingredients of the bifidobacteria DD98 to obtain the water-soluble protein thereof, thereby expanding the application of the bifidobacteria DD 98.

The specific technical scheme of the invention is as follows:

a Bifidobacterium, named Bifidobacterium DD98, has 16SrDNA nucleotide sequence shown in SEQ ID No: 1 is shown.

The results of the 16S rRNA sequencing and nucleotide sequence BLAST analysis of the invention show that the Bifidobacterium DD98 strain is not completely identical to the corresponding sequences of all other Bifidobacterium longum strains known so far, and has sequence difference with the standard strain Bifidobacterium longum JCM11342 (the 16S rRNA sequence thereof is shown in Genebank number: LC 306854.1). Thus, it was found that Bifidobacterium DD98 is a novel Bifidobacterium longum which has not been isolated before.

The bifidobacterium is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (No. 3 of Western No.1 of Beijing Kogyang district, Taiwan province, zip code 100101), the preservation time is 2018.10.11, the preservation number is CGMCC No.16573, and the classification name is as follows: bifidobacterium longum, survival: and (6) survival.

Further, the bifidobacterium is subjected to selenium-rich domestication culture to prepare the selenium-rich bifidobacterium.

The invention also aims to provide the freeze-dried preparation of the bifidobacterium, the bifidobacterium and the freeze-drying protective agent, wherein the freeze-drying protective agent comprises trehalose, skimmed milk powder and Vc-Na in a mass ratio of 10:20: 2.

Another object of the present invention is to provide an active protein extracted from the above Bifidobacterium DD 98.

The preparation method comprises the following steps:

carrying out anaerobic culture on bifidobacterium DD98 in an RCM culture medium at 37 ℃, centrifuging and washing with physiological saline to obtain thalli, carrying out water heavy suspension on the thalli, carrying out ultrasonic crushing, adding nuclease into cell crushing liquid for digestion, centrifuging, removing cell debris, precipitating protein by using acetone in supernate, volatilizing the acetone after centrifuging, and re-dissolving and precipitating to obtain the active protein of the bifidobacterium.

The bifidobacterium DD98 and the active protein extracted from the bifidobacterium can be used for preparing products for preventing or treating gastrointestinal diseases or liver diseases.

The Bifidobacterium strain is present in the form of viable cells or non-viable cells in the above product. Preferably greater than 10 per gram of product¹⁰An amount of cfu of bifidobacteria.

The bifidobacterium has similar activity with bifidobacterium longum, and particularly has excellent activity in treating gastrointestinal diseases and liver diseases, such as Crohn's disease, ulcerative colitis, irritable bowel syndrome, chronic enteritis, colitis after infection, diarrhea, high fat diet and liver injury caused by alcohol.

The invention has the advantages that:

the bifidobacterium DD98 can tolerate higher selenium pressure and can be used for selenium-rich culture. The bifidobacterium DD98 has good intestinal tract colonization ability, and the extracted water-soluble protein has the intestinal tract protection function. The Bifidobacterium DD98 can relieve liver injury caused by high fat diet and alcohol, and has protective effect on liver.

Drawings

FIG. 1 shows the result of electron scanning microscopy of Bifidobacterium DD 98. The cells are in the form of long rods or branched rods, generally about 10 μm long, and are typical bacterial forms of Bifidobacterium longum.

FIG. 2 is a morphological feature of Bifidobacterium DD98 colony. The colony has neat edge, soft texture and smooth surface, and is milky convex.

FIG. 3 shows the result of gram-staining optical microscope with Bifidobacterium DD 98. The staining was purple, indicating gram positive bacilli.

FIG. 4 is a graph showing the change in the number of bifidobacteria in the intestinal tract following administration of Bifidobacterium DD98 to mice.

FIG. 5 shows the change in the number of intestinal enterobacteria in the intestine of mice administered Bifidobacterium DD 98.

FIG. 6 shows that the bifidobacterium DD98 water-soluble protein enhances the activity of IEC6 cells.

FIG. 7 shows that the water-soluble protein of Bifidobacterium DD98 inhibits LPS-induced apoptosis of IEC6 cells.

FIG. 8 shows that the water-soluble protein of Bifidobacterium DD98 inhibits LPS-induced mitochondrial membrane potential collapse of IEC6 cells.

FIG. 9 shows that the water-soluble protein of Bifidobacterium DD98 can inhibit LPS-induced decrease in ZO-1 and Occludin mRNA levels and increase ZO-1 and Occludin mRNA levels.

FIG. 10 shows that Bifidobacterium DD98 can inhibit the elevation of serum ALT and AST in mice treated with high fat diet and alcohol.

FIG. 11 shows that Bifidobacterium DD98 can reduce serum TG and TC levels in mice co-treated with high fat diet and alcohol.

FIG. 12 shows that Bifidobacterium DD98 can reduce the serum free fatty acid level of mice treated with high-fat diet and alcohol.

FIG. 13 shows that Bifidobacterium DD98 can reduce MDA level in mice treated with high-fat diet and ethanol.

FIG. 14 shows that Bifidobacterium DD98 can increase serum SOD level in mice treated with high-fat diet and ethanol.

FIG. 15 shows that Bifidobacterium DD98 can reduce LDH levels in mice treated with high fat diet and alcohol.

FIG. 16 shows that Bifidobacterium DD98 can reduce serum TNF- α levels in mice treated with high fat diet and alcohol.

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1 Bifidobacterium DD98 acquisition and Strain isolation, selection

1. Sample collection and amplification culture

Taking 1g of fresh middle-section feces of a sampling object, adding 10ml of normal saline, fully and uniformly mixing, standing at room temperature for 5min, and collecting upper-layer suspension. Taking 1ml of the supernatant suspension, inoculating the supernatant suspension into 50ml of RCM medium, and carrying out anaerobic culture at 37 ℃ for 24h to obtain a culture solution.

2. Strain isolation

Taking the culture solution, diluting with physiological saline gradient 10^-1，10^-2，10^-3，10^-4，10^-5Multiplying to obtain bacterial suspension, sucking 0.1ml of bacterial suspension, coating the bacterial suspension on a BBL culture medium, and carrying out anaerobic culture at 37 ℃ for 24h to obtain a single bacterial colony.

3. Strain purification

Selecting a single colony, streaking and inoculating a colony culture on a BBL culture medium, and carrying out anaerobic culture at 37 ℃ for 24 hours; and then, continuously streaking and inoculating the grown single colony on a BBL culture medium, carrying out anaerobic culture at 37 ℃ for 24h, and continuously separating and purifying for three times. And (3) performing gram staining on the finally picked single colony, and performing microscopic examination on the single colony to obtain gram-positive bacteria, wherein the purified strain with a long rod or a forked top part is the pure culture obtained by separation.

4. Screening of dominant growth strains

Screening growth dominant strains: inoculating the bacterial liquid of the pure culture strain into a 96-deep-hole plate, measuring the OD600 of the bacterial culture liquid in each hole, and selecting 12 single bacterial colonies with the highest OD600 for re-screening. Re-screening: the 12 strains are re-inoculated into a deep-well plate containing a fresh culture medium, and are subjected to anaerobic culture at 37 ℃ for 14-16h, and the strain with the highest OD600 is selected as the growth dominant strain of the bifidobacterium. Inoculating the strain with the growth dominance into an eggplant bottle for amplification culture, and preserving the strain by using 20% of glycerol.

RCM culture medium formula: 10g of peptone, 3g of yeast powder, 5g of glucose, 3g of sodium acetate, 1g of soluble starch, 5g of sodium chloride, 0.5g of L-cysteine, 15g of agar, 1000mL of distilled water and pH 6.8.

BBL culture medium formula: 15g of peptone, 2g of yeast powder, 20g of glucose, 25g of soluble starch, 5g of sodium chloride, 0.5g of L-cysteine, 5g of tomato extract powder, 2g of liver extract powder, 20g of agar, tween-801 mL, 1000mL of distilled water and pH 7.0.

Example 2 selenium enrichment acclimatization study of Bifidobacterium DD98

Preparing sodium selenite solution, filtering with 0.22um, adding into culture medium, and packaging into 96 deep-well plate and dish. Preliminary design of selenium-enriched domestication gradient: 5, 10, 15, 20, 25, 50, 100, 200, 400, 600, 800. mu.g/ml.

Subpackaging predominant strain of Bifidobacterium with glycerol tube into 96 deep-well plate containing 5 μ g/ml sodium selenite culture medium, anaerobically culturing for 24 hr, collecting 200 μ l of each well of 96 deep-well plate, detecting OD600, and diluting 3 wells with highest OD value to 10⁵～10⁶The double coated onto 5. mu.g/ml sodium selenite plates. After the plates are coated, the plates are placed into an anaerobic incubator to be cultured for 18 hours, and after single colonies grow out, single colonies with large spots and light colors (which indicate that the colonies grow well on sodium selenite culture medium with corresponding concentration and are difficult to convert sodium selenite into red elemental selenium) are picked and cultured in a 96-deep-hole plate filled with 5 mu g/ml of sodium selenite culture medium. After anaerobic culture for 18 hours, taking culture solution from each well to a 96 enzyme label plate for OD600 detection, and diluting by 10 in 3 wells with the maximum OD value⁵～10⁶The double coated onto 10. mu.g/ml sodium selenite plates. After the plate is coated, putting the plate into an anaerobic incubator to be cultured for 18 hours, after a single colony grows out, picking the single colony with large spot and light color by using a toothpick into a 96 deep-hole plate filled with 10 mu g/ml sodium selenite culture medium, after the plate is cultured for 18 hours in an anaerobic way, taking 200 mu l of the single colony into a 96 enzyme-linked immunosorbent assay plate per hole to carry out OD600 detection, and diluting 10 holes with 3 holes with the maximum OD value⁵～10⁶The double coated on 15. mu.g/ml sodium selenite plate, and the circulation is repeated until the pressure of 800. mu.g/ml sodium selenite is reached. Finally obtaining single bacterial colony with strong selenium pressure tolerance and good growth conditionAnd marking to obtain pure culture, and identifying strains. Test results show that the bifidobacterium DD98 has good selenium pressure tolerance capability.

Example 3 bacteriological characteristics of Bifidobacterium DD98

1. Morphological characteristics of bacterial colony

The separated and purified DD98 is observed by scanning electron microscope, and the thallus is in the shape of long rod or branched rod, and the thallus is generally about 10um long, as shown in FIG. 1.

Morphological characteristics of colonies: neat edges, soft texture, smooth surface, milky convex as shown in fig. 2.

Gram stain optical microscope observation: the staining was purple, as shown in FIG. 3, indicating gram positive bacilli.

2. Biochemical characteristics

The acid production characteristics of DD98 by sugar fermentation were examined by Bergey's handbook and the results are as follows:

the results show that: bifidobacterium DD98 can utilize L-arabinose, D-galactose, D-fructose, D-glucose, D-lactose, sucrose, maltose, melibiose, raffinose, D-mannitol, D-xylose, acacia, trehalose, D-mannitol, D-sorbitol, amylopectin, starch, xylan, inulin, salicin, cellobiose.

3. Characteristics of genetics

Extracting bacterial genome DNA, adopting 16S universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQID No: 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID No: 3) of the bacteria to amplify to obtain a 16SrRNA gene sequence of the bacteria, wherein the 16SrDNA sequence of the bacteria is shown as SEQ ID No: CGGAGTCTACTTAGACGGCTCATCCCACAAGGGGTTAGGCCACCGGCTTCGGGTGCTGCCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGACGTTGCTGATTCGCGATTACTAGCGACTCCGCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTTCAGGGATCCGCTCCGCGTCGCCGCGTCGCATCCCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTAACCCCGGCGGTCCCCCGTGAGTTCCCGGCATAATCCGCTGGCAACACGGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAAGCCGTATCTCTACGACCGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTAGCTCCGACACGGAACCCGTGGAACGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAAACCGCCTACGAGCCCTTTACGCCCAATAATTCCCGGATAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCAACGGGTAAACTCACTCTCGCTTGCTCCCGATAAAAGAGGTTACAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCCACTGCTGCCTCCCGTAGAGTCTGGACGTATTCCTCAGTCCATGTGACATCGCCCCTCTTCAGTCGGCTACGTTCGAAGCCTACGGGTGGGCCGTAGCCGGCGTTCAAGCCTGGTATATAG shown at 1).

By using 16s rrna sequencing and nucleotide sequence BLAST analysis, the strain is not completely identical to the corresponding sequence of all other currently known bifidobacterium longum, but has a certain homology. The strain was further confirmed to belong to Bifidobacterium longum by sugar fermentation characteristic analysis as compared with Bergey' S Manual of identification of bacteria, but the comparison of the 16SrRNA sequence with that of a standard strain Bifidobacterium longum JCM11342 (the 16S rRNA sequence thereof is shown in Genebank No. LC306854.1) shows that the strain of the present invention differs in sequence from the standard strain. Therefore, it can be seen that the strain of the present invention is a novel Bifidobacterium longum which has not been isolated before.

Example 4 Bifidobacterium DD98 lyophilized preparation

Mixing the bifidobacterium DD98 thallus with a protective agent aqueous solution according to the mass ratio of 1:1 is mixed and then placed in a vacuum freeze dryer (LGJ-22D type freeze dryer Beijing four-ring scientific instrument factory Co., Ltd.), and the freeze-drying process is as follows: the first stage is as follows: 4h at 40 ℃ below zero (vacuumizing after the pre-starting is finished); and a second stage: 5 ℃,10 h; and a third stage: 15 ℃,10 h, 10Pa vacuum degree and 0.5cm thickness. And after the freeze drying is finished, taking out the penicillin bottle, accurately counting the bacterial powder in the penicillin bottle by a flat plate counting method, and calculating the freeze drying survival rate.

The screening of lyoprotectants is as follows:

(1) selection of skimmed milk powder

Taking 5 sterilized penicillin bottles, adding a protective agent aqueous solution and bacterial sludge into each bottle, and mixing the raw materials in a mass ratio of 1:1, uniformly mixing, wherein the number of the protective agent aqueous solutions of 1-5 is 0, 5, 10, 15 and 20 percent of skimmed milk powder respectively, and freeze-drying penicillin bottles in a freeze drying box. The results of skim milk powder screening are shown in table 1:

table 1:

numbering	Skimmed milk powder (%)	Freeze-drying survival (%)
			1	0	3.3
2	5	28.5
			3	10	35.7
4	15	35.3
			5	20	33.5

From the results, when the freeze-drying survival rate of the skimmed milk powder is over 10%, the freeze-drying survival rate is not increased any more, the freeze-drying survival rate of the 10% skimmed milk powder is 35.7%, and the freeze-drying survival rate is improved by 10.7 times compared with the freeze-drying survival rate of the bacterial sludge directly freeze-dried without the protective agent.

(2) Screening of carbohydrate protectors

Taking 8 sterilized penicillin bottles, adding a protective agent aqueous solution and bacterial sludge into each bottle, and mixing the raw materials in a mass ratio of 1:1, uniformly mixing, adding 10% of glucose, stachyose, sucrose, trehalose, lactose, mannitol, lactulose and purified water into 10% of defatted milk powder serving as a protective agent aqueous solution with the number of 1-8, and freeze-drying a penicillin bottle in a freeze-drying box.

The results of the carbohydrate protectant screening are shown in table 2:

table 2:

the results show that the trehalose has the best freeze-drying protection effect on the selenium-enriched bifidobacteria, and compared with pure 10% skimmed milk powder, the freeze-drying survival rate is improved by 12.4%. Therefore, the addition ratios of trehalose were selected to be 10%, 15%, 20%, and 25%, respectively, and the specific procedures were as above, and the results are shown in table 3:

table 3:

numbering	Trehalose proportion	Freeze-drying survival (%)
			1	10％	48.1
2	15％	56.3
			3	20％	67.5
4	25％	70.2

From the above results, it was found that the protective effect was best when the addition amount of trehalose was 25%, but trehalose at 25% was relatively difficult to disperse, the concentration was too high, and the fluidity was poor, and 20% was selected as the optimum addition ratio of trehalose in consideration of the operability in the freeze-drying oven.

(3) Screening for antioxidants

Taking 4 sterilized penicillin bottles, adding a protective agent aqueous solution and bacterial sludge into each bottle, and mixing the raw materials in a mass ratio of 1:1, uniformly mixing, adding 1% of Vc-Na, sodium glutamate, L-cysteine hydrochloride and purified water into 10% of skimmed milk powder and 20% of trehalose which are numbered 1-4, and freeze-drying a penicillin bottle in a freeze-drying box.

Antioxidant screening results are shown in table 4:

table 4:

numbering	Species of	Freeze-drying survival (%)
			1	Vc-Na	82.4
2	Glutamic acid sodium salt	72.3
			3	L-cysteine hydrochloride	47.5
4	Blank control	67.5

From the above results, the additional addition of Vc-Na can increase the freeze-drying survival rate of the selenium-enriched bifidobacterium, so the Vc-Na addition ratio was screened, and the results are shown in table 5:

table 5:

numbering	Ratio of Vc to Na	Freeze-drying survival (%)
			1	1％	82.4
2	2％	85.5
			3	3％	85.2
4	4％	85.5

The results show that the protective effect on the selenium-enriched bifidobacterium hardly varies when the Vc-Na addition ratio is between 2 and 4%, and therefore 2% of Vc-Na is selected as the optimal addition amount in consideration of the cost.

To summarize:

the optimal formula of the aqueous solution of the freeze-drying protective agent is obtained by screening the freeze-drying protective agent and comprises the following components in parts by weight: 10% of skimmed milk powder, 20% of trehalose, 2% of sodium ascorbate and 68% of purified water. The weight addition ratio of the protective agent to the bacterial sludge is 1: 1. the freeze-drying process comprises the following steps: the first stage is as follows: -40 ℃, 4h (evacuation after pre-freezing), second stage: 5 ℃,10 h; and a third stage: 15 ℃,10 h, 10Pa vacuum degree and 0.5cm thickness. The freeze-drying survival rate is above 85%, and the viable count is 5 × 10¹⁰cfu/g or more.

Example 5 study of Bifidobacterium DD98 on the function of regulating intestinal flora

SPF grade ICR mice, 6 to 8 weeks old, male, weigh 18-22 g. The breeding environment is 22 +/-2 ℃ at room temperature, the lamp/dark cycle is 12h, and the water and food are freely drunk and taken. The mice are randomly grouped into groups, 6 mice are fed adaptively for 5 days, the bifidobacterium group is administrated with 0.2ml of bifidobacterium with the concentration of 6 multiplied by 10^8cfu/ml every day, and the normal control group is administrated with 0.2ml of 0.9 percent sodium chloride solution every day. And after 30 days, observing the intestinal field planting and the capability of adjusting intestinal flora of the bifidobacterium DD98 by using the number of the bifidobacterium and the enterobacter in the mouse excrement as detection indexes, and after 30 days of administration, aseptically adopting 0.1g of the mouse excrement and diluting by 10 times, and carrying out plate counting. The bacterial colony morphology, gram staining microscopic examination, biochemical reaction and the like are used for identifying and counting the bacterial colonies, and the bacterial count in each g of wet stool is calculated. Statistical analysis is carried out by using SPSS 16.0 analysis software; graph pad.prism.v5.0 software plotted against the population change data. After 30 days of continuous administration of bifidobacterium gastrum in experimental animals, the change of the quantity of bifidobacterium in animal feces is shown in figure 4 (n is 6, X +/-SD, P is less than 0.05 and P is less than 0.01 compared with a control group), and the result shows that the quantity of the bifidobacterium in the feces of mice in the bifidobacterium group is obviously higher than that of the control group (P is less than 0.05), which indicates that the quantity of the bifidobacterium in intestinal tracts of the mice can be increased by orally taking bifidobacterium DD 98. The change of the intestinal bacillus number in animal feces after 30 days of continuous administration of bifidobacterium gastrum in experimental animals is shown in figure 5 (n is 6, X +/-SD, P is less than 0.05, and P is less than 0.01 compared with the control group), and the result shows that the intestinal bacillus number in the feces of mice in the bifidobacterium group is lower than that in the control group, which indicates that oral administration of bifidobacterium DD98 can reduce the survival of the intestinal bacillus harmful in the intestinal tract. The experiments show that the DD98 bifidobacterium can improve the intestinal flora, increase the number of probiotics and reduce the number of pathogenic bacteria.

Example 6 function of water-soluble protein of Bifidobacterium DD98 for protecting mouse intestinal epithelial cells

Bifidobacterium DD98 was anaerobically cultured in RCM medium at 37 ℃ for 24 hours, and centrifuged and washed 3 times with physiological saline to give DD98 cells. Resuspending the cells with ultrapure water, placing the cells in a cell disruption instrument for ultrasonic disruption, and adding nuclease into the cell disruption solution to digest the nucleic acid. The cell disruption solution was then centrifuged at high speed to remove cell debris. The supernatant was used to precipitate the protein using 4 volumes of acetone. Centrifuging, volatilizing acetone, and re-dissolving with ultrapure water to obtain water-soluble protein of Bifidobacterium DD 98.

Rat intestinal epithelial cells IEC6 cells were treated with 50. mu.g/ml LPS, 10,30,100mg/L DD98 water-soluble protein was added, and after 24 hours of incubation, cell viability was measured by MTT method, and apoptosis and mitochondrial membrane potential were measured by flow cytometry, respectively. After IEC6 cells are treated by LPS for 24 hours, the activity of IEC6 cells can be inhibited, IEC6 cells are induced to die, and mitochondrial membrane potential is collapsed. The water-soluble protein of bifidobacterium DD98 can inhibit the decrease of IEC6 cell activity caused by LPS (figure 6), and can inhibit the IEC6 cell apoptosis caused by LPS (figure 7) and mitochondrial collapse (figure 8).

Rat intestinal epithelial cells IEC6 cells were treated with 50. mu.g/ml LPS, while 10,30,100mg/L of a water-soluble protein of Bifidobacterium DD98 was added, and after 24 hours of incubation, the cells were collected. Total cellular RNA was extracted and the mRNA levels of ZO-1 and Occludin were determined. The stimulation of IEC6 cells by LPS resulted in a decrease in the mRNA levels of ZO-1 and Occludin, while water-soluble proteins were able to inhibit this effect of LPS, increasing the mRNA levels of ZO-1 and Occludin (FIG. 9).

The researches show that the water-soluble protein of the bifidobacterium DD98 can inhibit the activity reduction of IEC6 cells caused by Lipopolysaccharide (LPS), the apoptosis of IEC6 cells and the mitochondrial collapse caused by LPS, and the reduction of IEC6 cell tight junction caused by LPS, so that the water-soluble protein of the bifidobacterium DD98 can inhibit the intestinal epithelial cell injury induced by LPS, reduce the expression reduction of tight junction protein and has a protection effect on the intestinal epithelial cell injury.

Example 7 protective Effect of Bifidobacterium DD98 on high fat diet and alcohol-induced liver injury

1. Sample preparation

Bifidobacterium DD98 was anaerobically cultured in RCM medium at 37 ℃ for 24 hours, and centrifuged and washed 3 times with physiological saline to give DD98 cells.

2. Grouping animals

32 SPF-grade C57/BL6 female mice were taken, fed normally for 3 days, and then divided into 5 groups according to weight average, namely a control group, a model group and a high, medium and low dose group of Bifidobacterium longum, wherein each group contains 6 mice. The high, medium and low dose components of Bifidobacterium longum are respectively gavaged with 25, 50 and 100mg/kg daily. Mice except the control group were treated with 15% ethanol and high-fat diet for 28 days.

The 15% ethanol is prepared by adding purified water into red star Erguotou wine, and blending by using an alcohol densimeter. The high fat diet formula is as follows: 10% lard, 2% cholesterol, 0.2% bile salt, 5% whole milk powder, 5% egg yolk powder and 77.8% basal feed.

3. Sample collection

Fasting is carried out for 12h before treatment, the mouse picks eyeballs and takes blood, the blood is kept still at normal temperature and is solidified, centrifugation is carried out for 15min at 3000 r/min, and serum is taken and stored at minus 80 ℃ for testing.

4. Biochemical index detection

The serum and liver homogenate of the mouse with the same sample amount are taken from each group, and the content determination of the serum ALT, AST, Triglyceride (TG), Total Cholesterol (TC), High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), SOD, MDA and LDH of the mouse is carried out according to the operation method of the kit.

5, detecting the content of TNF- α in mouse serum by ELISA method

Collecting mouse serum, measuring the level of serum TNF- α according to the operation of a kit specification, taking an ELISA measuring kit of TNF- α out of a refrigerator, balancing for 30 minutes at room temperature, taking out a required enzyme standard strip from a sealed bag, adding a specimen or a standard product with different concentrations into corresponding holes except for blank holes, respectively, adding a biotinylation antibody working solution (100 mu l/hole) except for the blank holes according to the standard curve with the following concentration of 2000, 1000, 500, 250, 62.5, 31.25 and 0pg/ml, sealing the reaction holes with a sealing plate gummed paper, incubating for 90 minutes at 37 ℃, washing the plate for 4 times with a concentrated washing solution diluted by double distilled water, adding a biotinylation antibody working solution (100 mu l/hole) except for the blank holes, sealing the reaction holes with a sealing plate gummed paper, washing the plate for 30 minutes at 37 ℃, adding a color developing agent (100 mu l/hole) after washing the plate for 4 times, adding a reaction working solution (100 mu l/hole) after 37 ℃, adding the reaction solution (100 mu l/hole) according to the blank, adding the enzyme binding working solution (100 mu l/hole), sealing the reaction holes, incubating for 30 minutes at 37 ℃, adding the standard solution, calculating the OD value of the standard product after adding the standard product, and uniformly mixing, and calculating the OD value of the standard curve, and calculating the OD value of the standard curve at a standard curve, wherein the horizontal coordinate value of the horizontal coordinate value.

6. Data processing

The experimental results are expressed as mean + -SEM. Statistical analysis was performed using one-way variance, and Student-Newman-Keuls was used to test differences between groups, with p <0.05 indicating significant differences between groups.

The bifidobacterium DD98 provided by the invention can improve the liver injury of mice caused by co-treatment of ethanol and High Fat (HFD) diet, protect liver cells, regulate blood fat disorder, and has certain effects of reducing oxidative stress and resisting inflammation, the bifidobacterium DD98 shows that the bifidobacterium DD98 can obviously reduce the elevation of serum transaminase ALT and AST caused by ethanol and high fat diet (figure 10), the bifidobacterium DD98 can obviously reduce the serum TG and TC level (figure 11), the high-dose DD98 can obviously reduce the serum FFAs level (figure 12), the high-dose DD98 has a reducing effect on the serum MDA level (figure 13), but the DD98 of each concentration has no obvious effect on the serum SOD level (figure 14), the DD98 also does not obviously influence the serum LDH level (figure 15), but the DD98 can reduce the serum TNF- α level (figure 16).

Example 8 preparation of fermented milk containing Bifidobacterium DD98 Strain

Bifidobacterium DD98 fermented milk is prepared by fermenting Bifidobacterium DD98 with milk and corn steep liquor dry powder as substrate. Inoculating Bifidobacterium DD98 into the fermentation tank at 3-5% inoculum size, controlling fermentation temperature at 36-38 deg.C, stirring speed at 250-350rpm, controlling pH at 6.4-6.6, and culturing for 16-20 h. The fermentation liquid obtained by canning is the bifidobacterium DD98 fermented milk. Fermentation medium: 80g/L of skimmed milk powder and 5g/L of corn steep liquor dry powder, and adjusting the pH value to 6.50 +/-0.1 by using a sodium hydroxide solution and dilute hydrochloric acid.

Example 9 preparation of live bacterial preparation containing Bifidobacterium DD98 Strain

Mixing wet bifidobacterium DD98 with a freeze-drying protective agent (trehalose, skimmed milk powder and sodium ascorbate) according to a ratio of 1:1, and freeze-drying, wherein the freeze-drying parameters are as follows: the pressure is 10-50Pa, the temperature of the heating plate is-40-35 ℃, and the thickness of the sample is 8-12 mm; drying for 40-72 hr to obtain viable bacteria microecological preparation with viable bacteria count of 5 × 10¹⁰CFU/g。

Sequence listing

<110> Jiangsu De xi Biotech Co., Ltd

SHANGHAI INSTITUTE OF PHARMACEUTICAL INDUSTRY

<120> bifidobacterium capable of being used for selenium-enriched culture and active protein and application thereof

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>1227

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

cggagtctac ttagacggct catcccacaa ggggttaggc caccggcttc gggtgctgcc 60

cactttcatg acttgacggg cggtgtgtac aaggcccggg aacgcattca ccgcgacgtt 120

gctgattcgc gattactagc gactccgcct tcacgcagtc gagttgcaga ctgcgatccg 180

aactgagacc ggttttcagg gatccgctcc gcgtcgccgc gtcgcatccc gttgtaccgg 240

ccattgtagc atgcgtgaag ccctggacgt aaggggcatg atgatctgac gtcatcccca 300

ccttcctccg agttaacccc ggcggtcccc cgtgagttcc cggcataatc cgctggcaac 360

acggggcgag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 420

gacgaccatg caccacctgt gaacccgccc cgaagggaag ccgtatctct acgaccgtcg 480

ggaacatgtc aagcccaggt aaggttcttc gcgttgcatc gaattaatcc gcatgctccg 540

ccgcttgtgc gggcccccgt caatttcttt gagttttagc cttgcggccg tactccccag 600

gcgggatgct taacgcgtta gctccgacac ggaacccgtg gaacgggccc cacatccagc 660

atccaccgtt tacggcgtgg actaccaggg tatctaatcc tgttcgctcc ccacgctttc 720

gctcctcagc gtcagtaacg gcccagagac ctgccttcgc cattggtgtt cttcccgata 780

tctacacatt ccaccgttac accgggaatt ccagtctccc ctaccgcact caagcccgcc 840

cgtacccggc gcggatccac cgttaagcga tggactttca caccggacgc gacgaaaccg 900

cctacgagcc ctttacgccc aataattccc ggataacgct tgcaccctac gtattaccgc 960

ggctgctggc acgtagttag ccggtgctta ttcaacgggt aaactcactc tcgcttgctc 1020

ccgataaaag aggttacaac ccgaaggcct ccatccctca cgcggcgtcg ctgcatcagg 1080

cttgcgccca ttgtgcaata ttcccccact gctgcctccc gtagagtctg gacgtattcc 1140

tcagtccatg tgacatcgcc cctcttcagt cggctacgtt cgaagcctac gggtgggccg 1200

tagccggcgt tcaagcctgg tatatag 1227

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

agagtttgat cctggctcag 20

<210>3

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ggttaccttg ttacgactt 19

Claims (7)

1. A bifidobacterium, characterized in that the bifidobacterium has a nucleotide sequence of 16SrDNA as set forth in SEQ ID No: 1 is shown in the specification; the Bifidobacterium isBifidobacteriumIongumIs preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 16573.

2. The bifidobacterium of claim 1, wherein the bifidobacterium may be further subjected to selenium-enriched acclimation culture to produce selenium-enriched bifidobacterium.

3. A freeze-dried preparation of bifidobacterium, characterized by comprising the bifidobacterium as claimed in claim 1 or 2 and a freeze-drying protective agent, wherein the freeze-drying protective agent comprises trehalose, skimmed milk powder and Vc-Na in a mass ratio of 10:20: 2.

4. A bifidobacterium active protein is characterized by being prepared by the following method: carrying out anaerobic culture on the bifidobacterium of claim 1 or 2 in an RCM culture medium at 37 ℃, centrifuging and washing the bifidobacterium by using physiological saline to obtain thalli, carrying out water resuspension on the thalli, carrying out ultrasonic disruption, adding nuclease into a cell disruption solution for digestion, centrifuging, removing cell debris, precipitating protein by using acetone in a supernatant, volatilizing the acetone after centrifugation, and re-dissolving the precipitate to obtain the active protein of the bifidobacterium.

5. Use of a bifidobacterium as claimed in claim 1 or 2 or a lyophilised formulation of a bifidobacterium as claimed in claim 3 or a bifidogenic protein as claimed in claim 4 in the manufacture of a product for the prophylaxis or treatment of gastrointestinal or hepatic disorders.

6. Use according to claim 5, characterized in that the Bifidobacterium strain is present in viable cell form or in non-viable cell form.

7. The use according to claim 5, wherein the gastrointestinal and hepatic disorders comprise Crohn's disease, ulcerative colitis, alcoholic liver disease, fatty liver.

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