CN113813293A - Bifidobacterium longum microcapsule preparation and application thereof - Google Patents

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a bifidobacterium longum microcapsule preparation and application thereof. The microcapsule preparation comprises bifidobacterium longum VHProbi Y08 (Bifidobacterium longumVHProbi Y08), the strain is screenedIs selected from infant feces, and the preservation number is CCTCC NO: m2019780 can effectively reduce the genotoxic effect of carcinogens on human cells. The microcapsule preparation has strong stress resistance and good stability, and can be widely applied to the fields of food, health-care products and the like.

Description

Bifidobacterium longum microcapsule preparation and application thereof

Technical Field

The invention relates to the technical field of functional microorganism screening and application, in particular to a bifidobacterium longum microcapsule preparation and application thereof.

Background

Bifidobacteria are the most abundant member of the infant intestinal microbiota, with the highest content in the large intestine and also found in the oral cavity. Bifidobacteria are obtained from the mother by vertical transmission and their persistence in the infant gut is associated with their persistent glycolysis of glycans in the infant gut. Such as bifidobacterium adolescentis and bifidobacterium catenulatum, which are more common in adults, and bifidobacterium bifidum, bifidobacterium breve and bifidobacterium longum, which are more common in the intestine of breast-fed infants.

Cancer is a major public health problem worldwide and has become one of the biggest threats to human health in the world today. The development of tumors is a very complex medical and biological problem and is influenced by a variety of factors. Besides genetic factors, environmental factors play an important role in the development of tumors. Epidemiological studies suggest that most tumors are associated with environmental factors, and the stimulation of the external environment leads to chromosome instability, resulting in susceptibility of the host to tumorigenesis. The proportion of malignant tumors caused by chemical factors is more than 90 percent of all environmental factors, and is far higher than other carcinogenic factors such as physical and biological factors.

Genotoxicity refers to the property of causing damage to human cellular DNA either directly or indirectly. Genotoxic substances can cause mutations in genetic material. Various genotoxic substances such as carcinogenic aromatic hydrocarbon, benzopyrene, carcinogenic aromatic amine, nitrous acid compound, aflatoxin, etc. exist in nature. These substances are widely present in the natural environment and increase the risk of cancer if the human body is exposed to the environment with genotoxic substances for a long time.

The carcinogenic capacity of genotoxic substances can be evaluated by in vivo animal experiments, and the method can directly and reliably characterize the genotoxic level, but is long in time consumption, high in cost and incapable of meeting the requirement of large-scale screening. The invention adopts the mode of co-culturing human living cells and probiotics to directly identify the effect of the probiotics or the metabolites thereof on reducing the genotoxicity of carcinogens to human cells. The microorganism can directly adsorb or metabolize genotoxic substances into nontoxic substances, or enhance the detoxification capability of human cells, so as to realize the prevention effect on carcinogens and other toxic substances on cell genes.

Disclosure of Invention

The invention aims to provide a bifidobacterium longum microcapsule preparation and application thereof. The microcapsule preparation comprises bifidobacterium longum VHProbi Y08 (Bifidobacterium longumVHProbi Y08), which is selected from infant feces and can effectively reduce the genotoxic effect of carcinogen on human cells. The microcapsule preparation has strong stress resistance and good stability, and can be widely applied to the fields of food, health-care products and the like.

The invention relates to a bifidobacterium longum microcapsule preparation which is prepared by mixing bifidobacterium longum and a protective wall material to prepare a spray mixed solution and then carrying out spray drying on the spray mixed solution.

Said Bifidobacterium longum is preferably Bifidobacterium longum VHProbi Y08 (Bifidobacterium longumVHProbi Y08), which has been deposited in the chinese type culture collection of the university of wuhan in 2019, 10, 8, with a deposition number of CCTCC NO: and M2019780.

The protective wall material is composed of gelatinized starch, skimmed milk powder, sucrose and calcium lactate.

The weight volume ratio (g/ml) of each component of the protective wall material in the spray mixed solution is respectively as follows: 6-10% of gelatinized starch, 10-15% of skim milk powder, 4-8% of sucrose and 20-30% of calcium lactate.

The bacterial concentration of the bifidobacterium longum in the spray mixed liquor is 10⁹-10¹⁰ CFU/ml。

More preferably, the weight-to-volume ratio (g/ml) of each component of the protective wall material in the spray mixture is: 10% of gelatinized starch, 15% of skim milk powder, 4% of sucrose and 30% of calcium lactate.

The spray drying is carried out by adopting a centrifugal spray dryer, wherein the air inlet temperature of a drying tower is 110-130 ℃, the atomization pressure is 0.3-0.5MPa, the feeding speed is 20-30L/h, and the air outlet temperature is 60-70 ℃.

The viable count of Bifidobacterium longum in the Bifidobacterium longum microcapsule preparation is at least 10⁹CFU/g。

The invention also relates to application of the microcapsule preparation in degrading cholesterol.

The invention also relates to the application of the microcapsule preparation in preventing cancer.

The bifidobacterium longum VHProbi Y08 provided by the invention has strong tolerance to artificial gastric juice and artificial intestinal juice, is sensitive to common antibiotics such as erythromycin and the like, and has good biological safety. The strain has strong oxidation resistance, the clearance rates of DPPH free radicals and HRS free radicals reach 23.8 percent and 74.5 percent respectively, the lipid peroxidation inhibition rate of supernatant fluid is 41.99 percent, and the lipid peroxidation inhibition rate of thalli is 43.17 percent. The strain can also effectively degrade cholesterol, and the degradation rate reaches 20%.

The bifidobacterium longum VHProbi Y08 has higher cell adhesion index and stronger adhesion to small intestine epithelial cells, and is easier to colonize and survive in the intestinal tract. The strain can selectively utilize three different types of prebiotics, namely inulin, FOS and GOS, and metabolize to generate proteins with different molecular weights. The proteins can play a role in resisting pathogens, regulating immunity, increasing mineral absorption, improving intestinal functions, influencing metabolism and satiety and the like in the intestinal tract, and have important potential probiotic effects.

The bifidobacterium longum VHProbi Y08 can effectively relieve the genotoxicity of carcinogens such as 4NQO, IQ and the like on human cells, has an effective tumor prevention mechanism, and has a certain value in the aspect of preventing cancers caused by genotoxic substances. Wherein, when HT-29 cells exist alone, the activity retention rate is only 25.1 percent under the toxic action of 4NQO gene; when the bifidobacterium longum VHProbi Y08 exists, the HT-29 cells are weakened by the toxicity of the 4NQO gene, and the activity retention rate of the HT-29 cells reaches 49.7 percent; when NCI-H460 cells exist alone, the activity retention rate is only 49.0 percent under the toxic action of the 4NQO gene; in the presence of Bifidobacterium longum VHProbi Y08, NCI-H460 cells are weakened by the toxicity of the 4NQO gene, and the activity retention rate reaches 88.7 percent, which are all obviously improved. In addition, after SOS reaction detection, the average clearance rate of the bifidobacterium longum VHProbi Y08 to IQ reaches more than 94%, and the genotoxic effect of IQ can be obviously reduced.

The bifidobacterium longum VHProbi Y08 provided by the invention is used as a probiotic strain of natural source, has no toxicity to organisms, can greatly reduce the genotoxicity of carcinogens, and has important application value.

In addition, the bifidobacterium longum microcapsule preparation provided by the invention has the advantages of simple preparation process, cheap protective wall material, low industrialization cost and strong gastric acid and choline resistance, and the survival rates of the bifidobacterium longum VHProbi Y08 are respectively up to 89.1% and 74.6% after the bifidobacterium longum is digested by artificial gastric juice and artificial intestinal juice. The microcapsule preparation has high stability, the survival rate of the bifidobacterium longum VHProbi Y08 is still higher than 50% after the microcapsule preparation is stored for 12 months at 37 ℃, the survival rate of the bifidobacterium longum VHProbi Y08 is still 71.8% after the microcapsule preparation is stored for 12 months at 20 ℃, the actual use effect of the bifidobacterium longum VHProbi Y08 on the aspects of degrading cholesterol and preventing cancers can be effectively improved, and the microcapsule preparation can be widely applied to the fields of food, health care products and the like.

Drawings

FIG. 1 is a Riboprinter fingerprint;

FIG. 2 is a RAPD fingerprint;

FIG. 3 is a rep-PCR fingerprint;

FIG. 4 is the growth rate of Bifidobacterium longum VHProbi Y08 in media supplemented with 3 prebiotics and glucose, respectively;

FIG. 5 is a MALDI-TOF mass spectrum of a metabolite of Bifidobacterium longum VHProbi Y08 in a medium supplemented with 3 prebiotics and glucose, respectively;

FIG. 6 shows the adhesion of cells of Bifidobacterium longum VHProbi Y08.

Detailed Description

The screening method of the present invention is not limited to the examples, and any known method capable of achieving the screening purpose is possible, and the screening description of the examples is only illustrative of the present invention and is not limiting the scope of the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

The invention will be further illustrated with reference to specific examples.

EXAMPLE 1 isolation and screening of strains

1. Preliminary screening of bifidobacterium

Preparing a BSM (Bifidus Selective Medium agar) solid culture Medium: 1000mL of purified water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 801.0 mL of tween, 2.0g of diamine citrate, 20g of calcium carbonate, 0.58 g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate, 15g of agar, adjusting the pH value to 6.2-6.5, and autoclaving at 121 ℃ for 15 min. After the culture medium is cooled, 5mL of cysteine solution (100 mg/mL) and 5mL of mupirocin lithium salt solution (10mg/mL) are respectively weighed and added into the agar culture medium, and the BSM solid culture medium is obtained after shaking up.

Taking 1g of fresh feces of infants which come from 0-3 years old and have not used probiotic preparation within half a year, diluting with 0.85% physiological saline, placing into a sterile sample bag, beating with a homogenizer, and mixing; and (3) taking 100 mu L of the uniformly mixed solution, diluting in a gradient manner, coating the uniformly mixed solution on a BSM solid culture medium, putting the BSM solid culture medium into an anaerobic jar, culturing for 72 hours at 37 ℃, and performing microscopic examination on a single colony growing on a plate. According to microscopic examination results, the applicant screened 9 potential bifidobacteria strains which are named as NH-1, NH-2, … … and NH-9 respectively.

2. Bifidobacteria rescreening

Preparing 1L BSM liquid culture medium, autoclaving at 115 deg.C for 20min, cooling, adding 3.2g pig mucosa pepsin, shaking for dissolving, and placing in 37 deg.C water bath shaker for 1 hr to obtain acid-resistant culture medium.

Respectively inoculating the screened 9 strains of bifidobacterium NH-1, NH-2, … … and NH-9 into the acid-resistant culture medium according to the inoculation amount of 6 percent, standing and culturing for 72 hours at 37 ℃ under anaerobic condition, and taking the fermentation liquor for counting the bacterial quantity.

The results show that the Log values of the viable bacteria amount in the 9 strains of bifidobacterium fermentation liquor are respectively 8.19, 8.32, 7.76, 6.56, 6.87, 7.62, 5.85, 6.84 and 7.74 Log CFU/mL, the viable bacteria amount of the NH-2 strain after being sieved again by an acid-resistant culture medium is the most, and the Log value of the viable bacteria amount is as high as 8.32 Log CFU/mL. Thus, the NH-2 strain screened by the invention has the highest acid resistance.

Example 2 Strain identification

2.1 colony morphology identification

NH-2 strains are inoculated on a BSM solid culture medium, after anaerobic culture at 37 ℃ for 72 hours, NH-2 single colonies are seen to be cream, the surface is slightly smooth and moist, the edges are neat, and the diameter of the colonies is 1-1.5 mm. The lower part of the microscope is rod-shaped, and one end of the microscope is forked.

2.2 characterization of physiological and biochemical characteristics

The inoculation solution in this example was prepared as follows: taking a proper amount of fresh NH-2 bacterial liquid under the aseptic condition, centrifuging at 5000rpm/min for 5min, washing with PBS buffer for 2 times, and diluting by 50 times after the thalli are weighed by the PBS buffer with the same volume to be used as inoculation liquid.

1. Salinity tolerance test

Under sterile conditions, 190 μ L of BSM liquid medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% was added to 96-well plates, 3 replicates per salt concentration, followed by 10 μ L of inoculum solution, and wells without inoculum were used as controls. 50 μ L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37 deg.C, and observing whether the culture medium turns turbid.

The result shows that the NH-2 strain grows under 1% of salt concentration, does not grow under 2% -8% of salt concentration and has the maximum tolerant salt concentration of 1%.

2. Catalase assay

Taking fresh bacteria liquid, dripping one drop of the bacteria liquid on a clean glass slide, then dripping one drop of 3% hydrogen peroxide solution on the bacteria liquid, and observing that NH-2 strains do not generate bubbles and are negative reaction.

3. Carbon source metabolism assay

The basic medium formulation used in this example was as follows:

peptone 1.5 g; yeast extract 0.6 g; tween 800.1 g; 0.5mL of salt solution; 18mg of phenol red; 100mL of distilled water; pH 7.4. + -. 0.2. Salt solution composition: MgSO (MgSO)₄·7H₂O 11.5g，MnSO₄·4H₂O2.8 g and distilled water 100 mL.

A10 g/mL solution of the sugar, alcohol and carbohydrate glycoside was prepared and filtered through a 0.22 μm sterile filter. Under aseptic conditions, 20 μ L of sterilized carbohydrate solution, 4 replicates of each carbohydrate, was added to a 96-well plate, followed by 170 μ L of sterilized phenol red-containing basal medium, followed by 10 μ L of inoculum, and no-inoculum reaction wells served as controls. 50 μ L of liquid paraffin was added to each well to prevent evaporation of water during the culture. Anaerobic culture at 37 deg.C, and observing the color change of the culture medium with phenol red as indicator. Specific results are shown in table 1.

TABLE 1 results of carbon source metabolism by NH-2 strains

Arabinogalactan	L-arabinose	D-Cellobiose	D-fructose	alpha-L-trehalose	D-galactosamine
						+	+	-	+	-	-
D-galactose	Sodium gluconate	Inulin	D-lactose	D-maltose	D-mannitol
						+	-	+	+	+	-
D-mannose	D-melezitose	D-melibiose	D-raffinose	D-ribose	Salicin
						-	+	+	+	+	-
D-sorbitol	D-sucrose	D-trehalose	Xylan	D-woodCandy
						-	+	-	-	+

Note: a "+" positive reaction; "-" negative reaction.

2.3 molecular biological identification

2.3.116 s rDNA Gene sequence analysis

1. Genomic DNA extraction

Reference was made to the Tiangen bacterium genomic DNA extraction kit (catalog No.: DP 302).

2. 16s rDNA Gene amplification

1) The primer sequence is as follows:

27F：AGAGTTTGATCCTGGCTCA；

1492R：GGTTACCTTGTTACGACTT。

2) reaction System (50. mu.L)

TABLE 2.16 s rDNA PCR amplification System

Composition of matter	Reaction volume
		10×PCR buffer	5μL
dNTPs	4μL
		27F	2μL
1492R	2μL
		DNA	2.5μL
rTaq	0.5μL
		ddH2O	34μL

3) And (3) the electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.

4) Sequencing of PCR products

Obtaining a 16s rDNA sequence SEQ ID NO 1 of the NH-2 strain through sequencing, comparing the sequence in an NCBI database, and preliminarily determining that the NH-2 strain is bifidobacterium longum (C) (1)Bifidobacterium longum）。

2.3.2 Riboprinter fingerprint

And (3) dipping the purified single colony from an agar culture medium plate by using a bacteria taking rod, putting the single colony into a sample tube with a buffer solution, stirring the single colony in the buffer solution by using a handheld stirrer to enable the single colony to be suspended in the buffer solution, putting a sample rack into a heater to inactivate, putting the sample rack into a Riboprinter system, and obtaining a bacteria identification result after DNA preparation, membrane conversion, imaging detection and data processing of the sample. The identification result shows that the NH-2 strain is bifidobacterium longum (b)Bifidobacterium longum) The Riboprinter fingerprint spectrum result is shown in figure 1.

2.3.3 RAPD and rep-PCR fingerprinting

1. RAPD fingerprint identification

1) The primer sequence is as follows: m13 (5'-GAGGGTGGCGGTTCT-3');

2) RAPD reaction system

TABLE 3 RAPD reaction System

Reaction components	Volume of
		TaqDNA polymerase (5U/. mu.L)	0.2 μL
10 × Buffer (containing Mg)2+）	2 μL
		Primer (10 uM)	1 μL
dNTPs（2.5 mM）	0.8 μL
		DNA template
	2 μL
		Sterile double distilled water	14 μL
Total volume	20 μL

3) Electrophoresis

Preparing a 1.5% agarose gel plate, using DL2000 DNA Marker as a result contrast, performing electrophoresis at a constant voltage of 100V for 80min, and finally detecting an electrophoretogram by using a gel imaging system. The RAPD fingerprint of the NH-2 strain is shown in figure 2.

2. rep-PCR fingerprint

1) rep-PCR primer

GTGGTGGTGGTGGTG。

2) rep-PCR reaction system

TABLE 4 reaction System for rep-PCR

Reaction components	Volume of
		rTaqDNA polymerase	0.2μL
10 XEx Taq DNA Buffer (containing Mg)2+）	2μL
		Primer (10 uM)	1 μL
dNTPs（2.5 mM）	2μL
		DNA template	2μL
Sterile double distilled water	12.8 μL

3) Electrophoresis

DL2000 DNA Marker was used as a result control. The voltage is 100V, and the electrophoresis time is 80min to detect the amplification result. The rep-PCR fingerprint of the NH-2 strain is shown in FIG. 3.

In summary, the results of colony morphology and physiological and biochemical characteristics of the NH-2 strain were uploaded to the website http:// www.tgw1916.net/bacterial _ log _ desktop. html, and compared with the results published in the document De Clerck E, et al, Systematic and applied microbiology, 2004, 27(1) 50. By integrating the identification results of molecular biology, the conclusion can be drawn that the NH-2 strain is a new Bifidobacterium longum strain which is named as Bifidobacterium longum VHProbi Y08 (Bifidobacterium longum VHProbi Y08）。

Example 3 tolerance test of Bifidobacterium longum VHProbi Y08 to Artificial gastric and intestinal juices

3.1 preparation of Artificial gastric juice

5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl were weighed, respectively, 1000mL of distilled water was added, pH3.0 was adjusted with dilute hydrochloric acid, and then sterilization was carried out at 115 ℃ for 20 min. Then 3.2g of pig mucosa pepsin is added before use, shaken up and dissolved, and placed in a water bath shaker at 37 ℃ for a water bath for 1h to simulate the temperature of a human body.

3.2 preparation of Artificial intestinal juice

Separately weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH₂PO₄6.8g and 3.0g of ox-gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is adjusted to 1000mL, the pH value is adjusted to 6.8 +/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then 1g pancreatin is added before use, shaken up and dissolved, and put into a water bath shaker at 37 ℃ for water bath for 1h to simulate the temperature of a human body.

3.2 test methods

2mL of fresh bacterial liquid is taken, centrifuged at 5000rpm/min for 5min to collect thalli, the thalli are washed for 3 times by using normal saline, and then 2mL of normal saline is used for resuspension to serve as inoculation liquid. Taking 1mL of inoculation liquid, adding the inoculation liquid into 24mL of artificial intestinal juice, placing the artificial intestinal juice in a water bath shaker (200 rpm/min) at 37 ℃ for 3h, sampling 1mL, and detecting the amount of live bacteria.

The viable bacteria counting method is used for measuring the bacterial quantity according to the national standard GB 4789.35-2016-food microorganism test lactobacillus test, and the viable bacteria quantity (Log CFU/mL) of the bacterial strain after being digested by artificial intestinal juice is shown in the table 5.

TABLE 5 viable cell count of Bifidobacterium longum VHProbi Y08 after digestion with artificial gastric and intestinal juices

Before digestion	After the artificial gastric juice is digested	After digestion of the artificial intestinal juice
			8.57+ 0.17	8.01±0.18	4.98+ 0.21

As can be seen from Table 5, the survival rates of the Bifidobacterium longum VHProbi Y08 screened by the method are still as high as 93.5% and 58.1% respectively after the digestion by artificial gastric juice and artificial intestinal juice. Thereby showing that the strain has strong tolerance to artificial gastric juice and artificial intestinal juice.

Example 4 hemolytic and antibiotic resistance experiments of Bifidobacterium longum VHProbi Y08

1. Hemolytic test

(1) Preparing an inoculation solution: the method comprises the steps of streaking and inoculating a cryopreserved Bifidobacterium longum VHProbi Y08 strain into a BSM solid culture medium, culturing at 37 ℃ for 24-48 h, subculturing for 1 time through the BSM liquid culture medium, inoculating Bifidobacterium longum VHProbi Y08 into a fresh BSM liquid culture medium in an inoculation amount of 5%, and performing shake culture at 40 ℃ for 24-48 h to obtain a fresh bacterial liquid serving as an inoculation liquid.

(2) Blood cell culture medium preparation: weighing the components of TBS basic culture medium, dissolving, autoclaving at 121 deg.C for 15min, adding 5% sterile defibered sheep blood when the culture medium is cooled to 50 deg.C, mixing, and pouring into flat plate.

(3) And (3) streak culture: and streaking the test strain on a prepared blood cell plate, culturing in an incubator at 37 ℃, and observing whether the test strain has hemolysis or not for 24-48 hours.

The results show that: bifidobacterium longum VHProbi Y08 was unable to grow and the blood cell plates were unchanged, indicating that Bifidobacterium longum VHProbi Y08 did not produce hemolysin and was unable to lyse blood cells.

2. Antibiotic resistance test

(1) Preparing antibiotics: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were all prepared into 2048 μ g/mL stock solutions, and stored at-20 deg.C for further use. When in use, the stock solution is serially diluted into a use solution by 2 times of gradient with BSM liquid culture medium, and the gradient dilution concentration is 1-1024 mu g/mL, and the total concentration is 11 gradients.

(2) Preparing an inoculation solution: taking a proper amount of fresh bacterial liquid (cultured for 24-48 h at 40 ℃), centrifuging for 5min at 5000rpm, washing once with sterile normal saline, and then diluting by 50 times after resuspending the bacteria with the same volume of normal saline to serve as inoculation liquid.

(3) Determination of minimum inhibitory concentration MIC value of antibiotic to Bifidobacterium longum VHProbi Y08 by broth dilution method

a. Adding BSM liquid culture medium without antibiotics into the 96-well plate in the 1 st row as negative control, sequentially adding 190 mu L of BSM liquid culture medium containing antibiotics with different concentrations into the 2 nd to 12 th rows, then respectively inoculating 10 mu L of the inoculation liquid, making 3 parallel wells, and taking 1 well without adding bacteria liquid as blank.

b. Cover by adding 50 μ L of paraffin oil to prevent evaporation of water.

c. Culturing 96-well plate at 40 deg.C for 48 hr, taking out, and determining OD₆₀₀Values, the MIC values of the antibiotics to the strains were counted using the 48h results, and the specific results are shown in Table 6.

TABLE 6 antibiotic MIC values of Bifidobacterium longum VHProbi Y08

MIC unit μ g/mL

From the results in table 6, it can be seen that bifidobacterium longum VHProbi Y08 provided by the present invention is sensitive to common antibiotics such as erythromycin, and has good biosafety.

Example 5 Bifidobacterium longum VHProbi Y08 assay for antioxidant function

1. Determination of DPPH and Hydroxyl Radical (HRS) eliminating capability of strain

1) Preparation of PBS bacterial suspension

Inoculating single colony with excellent growth state into 3mL of BSM liquid culture medium, culturing at 37 ℃ for 18-20h, taking the culture solution as inoculation solution, inoculating into 50mL of BSM liquid culture medium according to the inoculation amount of 2%, and standing and culturing for 18h to obtain a culture solution of the strain. Sucking 1mL of bacterial liquid, collecting the bacteria, washing the bacteria for 2 times by using 1mLPBS buffer solution, and then adding 2mLPBS solution to resuspend the bacteria for later use.

2) Determination of DPPH free radical scavenging ability of strain

Taking 1mL of PBS bacterial suspension of the strain to be detected, adding 1mL of 0.4 mM of ready-prepared DPPH free radical solution, uniformly mixing, then placing at room temperature for shading reaction for 30min, then measuring the absorbance A sample of the sample at the wavelength of 517nm, and measuring for 3 times of parallelism. The control group samples were blank-zeroed with equal volumes of PBS solution and DPPH-ethanol mixture, and equal volumes of PBS suspension and ethanol mixture. The clearance rate is calculated according to the following formula: clearance% = [1- (a)_{Sample (I)}-A_{Blank space}）/A_Control]×100%。

Using Lactobacillus paracasei (L.), (Lactobacillus paracasei) IMC-4 strain was used as a positive control, and the results are shown in Table 7.

TABLE 7 DPPH radical scavenging Rate

Bacterial strains	Lactobacillus paracasei IMC-4	Bifidobacterium longum VHProbi Y08
			Clearance rate%	18.35%	23.80%
Standard deviation of	1.40%	1.20%

As can be seen from the data in Table 7, the Bifidobacterium longum VHProbi Y08 provided by the invention can effectively eliminate DPPH free radicals, and the clearance rate reaches 23.8 percent and is obviously higher than that of Lactobacillus paracasei ((L.))L. paracasei) IMC-4 strain.

3) Determination of HRS-removing ability of Strain

mu.L of 5mM sodium salicylate-ethanol solution, 100. mu.L of 5mM ferrous sulfate, 500. mu.L deionized water and 200. mu.L of lactic acid bacteria PBS suspension were mixed, 100. mu.L of hydrogen peroxide solution (3 mM) was added, and absorbance of the sample was measured at a wavelength of 510nm after water bath at 37 ℃ for 15 min. The hydroxyl radical clearance was calculated according to the following formula.

Clearance% = (a)_{Sample (I)}-A_{Control of}）/（A_{Blank space}-A_{Control of}) X 100% where A_{Control of}To replace the sample with deionized water, A_{Blank space}Replacement of sample and H for deionized Water₂O₂. Using Lactobacillus paracasei (L.), (L. paracasei) IMC-4 was a positive control and the results are shown in Table 8.

TABLE 8 HRS radical clearance

Bacterial strains	Lactobacillus paracasei IMC-4	Bifidobacterium longum VHProbi Y08
			Clearance rate%	69.94%	74.50%
Standard deviation of	0.60%	3.20%

As can be seen from the data in Table 8, the Bifidobacterium longum VHProbi Y08 provided by the invention can effectively remove HRS free radicals, and the removal rate reaches 74.5 percent and is obviously higher than that of Lactobacillus paracasei: (A) (B)L. paracasei) IMC-4 strain.

2. Experimental identification of bacterial strain for resisting lipid peroxidation

1) Culturing lactic acid bacteria and preparing fermentation supernatant, thallus and intracellular extract:

culturing lactobacillus in BSM liquid culture medium at 37 deg.C for 24 hr, transferring for 3 generations, centrifuging at 6000 rpm/min at 4 deg.C for 10min, and collecting supernatant as fermentation supernatant. The collected cells were centrifuged at 6000r/min for 10min in PBS buffer (pH 7.4) and washed 3 times. The cells were resuspended in PBS buffer to adjust the cell concentration to 1.0X 10⁹cells/mL to obtain a bacterial suspension.

2) Preparing a linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.

3) 0.5mL of PBS solution (pH 7.4) was added 1mL of an emulsion of linoleic acid, 1mL of LFeSO₄(1%), adding 0.5mL of sample, carrying out water bath at 37 ℃ for 1.5 h, adding 0.2mL of TCA (4%), 2mL of TBA (0.8%), carrying out water bath at 100 ℃ for 30min, rapidly cooling, centrifuging at 4000 rpm/min for 15min, collecting supernatant, and measuring the absorbance at 532 nm to obtain A; the control group uses 0.5mL of distilled water to replace the sample to obtain A₀. Inhibition rate/% (A)₀－A)/ A₀×100%

Note: a is the absorbance of the sample group; a. the₀Absorbance was taken as the control. By auxiliary trunkLactobacillus casei (L.) ML. paracasei) IMC-4 was a positive control and the results are shown in Table 9.

TABLE 9 inhibition of lipid peroxidation

As can be seen from the data in Table 9, the supernatant of Bifidobacterium longum VHProbi Y08 provided by the invention has an anti-lipid peroxidation inhibition rate of 41.99 percent, which is slightly lower than that of Lactobacillus paracasei IMC-4 strain; the inhibition rate of the thalli for resisting lipid peroxidation is 43.17 percent and is obviously higher than that of lactobacillus paracasei IMC-4 strain.

Example 6 Bifidobacterium longum VHProbi Y08 in vitro Cholesterol degradation experiment

1. Preparation of cholesterol micelle solution: accurately weighing 1g of cholesterol, dissolving the cholesterol in absolute ethyl alcohol, diluting to a constant volume of 100mL, and filtering and sterilizing by using a 0.22 mu m microporous filter membrane under an aseptic condition.

2. 10.0 g of peptone and 10.0 g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0 g of glucose, 801.0 mL of Tween, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt and 1000mL of distilled water are weighed, the pH value is adjusted to 7.3, sterilization is carried out at 115 ℃ for 30min, and then cholesterol solution is added to ensure that the final concentration of cholesterol is 0.1%.

Inoculating fresh bacterial liquid according to the inoculation amount of 0.1%, performing static culture at 37 ℃ for 48h, then taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10min, centrifuging for 5min at 3000 rpm, and taking supernatant for measuring the cholesterol content. Method for measuring cholesterol GB/T5009.128-2003 < determination of cholesterol in food >.

The results show that: the degradation rate of the bifidobacterium longum VHProbi Y08 on cholesterol reaches 20 percent.

Example 7 Bifidobacterium longum VHProbi Y08 metabolism assay with 3 different prebiotics and glucose

1. Growth rate test

Weighing 1.5g of peptone; yeast extract 0.6 g; tween 800.1 g; 0.5mL of salt solution; 18mg of phenol red; distilled water 100mL, adjusted to pH7.4After +/-0.2, autoclaving at 121 ℃ for 15 min. 10g/mL of Glucose (Glucose), Fructooligosaccharide (FOS), Galactooligosaccharide (GOS) and Inulin (Inulin) were prepared and filtered through a 0.22 μm sterile filter. Under aseptic conditions, 20. mu.L of sterilized sugar solutions, 4 replicates of each, were added to a 96-well plate, followed by 170. mu.L of sterilized basal medium, followed by 10. mu.L of inoculum, and no-inoculum reaction wells served as controls. 50 μ L of liquid paraffin was added to each well to prevent evaporation of water during the culture. Placing into enzyme labeling instrument, culturing at 37 deg.C in anaerobic chamber, and measuring absorbance OD₆₀₀. The results of the experiment are shown in FIG. 4.

Bifidobacterium longum VHProbi Y08 provided by the invention is OD in culture medium containing inulin₆₀₀Up to 0.6, and secondly in a medium containing glucose, FOS and GOS, thus indicating that bifidobacterium longum VHProbi Y08 first had a plateau in a medium containing inulin and secondly in a medium containing glucose, FOS and GOS. Therefore, bifidobacterium longum VHProbi Y08 grew at the fastest rate in this prebiotic medium containing inulin, whereby this prebiotic component of inulin could be added during later fermentation cultures.

2. MALDI-TOF-MS (matrix-assisted laser desorption ionization-time of flight-mass spectrometry) detection of whole protein expression of Bifidobacterium longum VHProbi Y08 in 3 different prebiotics and glucose culture media

Weighing 1.5g of peptone; yeast extract 0.6 g; tween 800.1 g; 0.5mL of salt solution; 18mg of phenol red; distilled water (100 mL), adjusting pH to 7.4 + -0.2, and autoclaving at 121 deg.C for 15 min. Glucose (Glucose), Fructooligosaccharide (FOS), Galactooligosaccharide (GOS) and Inulin (Inulin) were prepared in an amount of 10g/mL, respectively, and added to the medium by filtration through a 0.22 μm sterile filter.

Inoculating fresh bacterial liquid into a culture medium according to the inoculation amount of 0.1%, performing static culture at 37 ℃ for 48h, uniformly coating a small amount of fresh bacteria on a target plate in a film form, adding 1 mu L of matrix solution to cover a sample, drying in the air, and placing the sample target into a mass spectrometer for identification. Irradiating the cocrystallized film formed by the sample and the matrix with laser to ionize the protein in the sample, accelerating the ions to fly through the flight pipeline under the action of 10-20KV electric field, and detecting the molecular weight of the protein according to the difference of flight time of the protein reaching the detector. The obtained 4 spectra were opened using the Autof ms 1000 analysis software Autof Analyzer v1.0 and the 4 spectra were presented in one list simultaneously by "spectra list" and "list peak marking" with the results shown in fig. 5.

As can be seen from fig. 5: besides glucose, bifidobacterium longum VHProbi Y08 can metabolize and utilize three prebiotics, inulin, FOS and GOS, and the species and amount of proteins in the metabolic products are significantly different. Wherein, when the bifidobacterium longum VHProbi Y08 utilizes inulin as prebiotics, the concentration of various proteins in the metabolite of the bifidobacterium longum VHProbi Y08 is obviously higher than that of other three prebiotics; when the bifidobacterium longum VHProbi Y08 utilizes glucose as prebiotics, the molecular weight of the metabolite is 5_KDThe following small proteins are more in variety, and when GOS is used as prebiotics, the molecular weight of the metabolites is 6_KDThe large proteins are more in variety; when the bifidobacterium longum VHProbi Y08 uses inulin and FOS as prebiotics, the protein types in the metabolites are similar.

The protein with a molecular weight of about 2693D is present in high concentrations in metabolites with glucose, inulin and FOS as prebiotics, and in very low concentrations in metabolites with GOS as prebiotics; the protein with the molecular weight of about 3575D exists in the metabolite taking inulin and FOS as prebiotics, but hardly exists in the metabolite taking glucose and GOS as prebiotics; in addition, there are significant differences in the concentrations of proteins with molecular weights of about 5385D, 6995D and 9840D among the metabolites of different prebiotics, and it is presumed that the genes encoding these proteins are involved in metabolism.

The above results illustrate that: three different types of prebiotics, namely inulin, FOS and GOS, can be selectively utilized by the bifidobacterium longum VHProbi Y08, and can promote the proliferation of the bifidobacterium longum VHProbi Y08. This combination of probiotics and prebiotics may improve the health of the host and the combination of colonic bacteria in the intestine. In addition, compared with glucose metabolism, bifidobacterium longum VHProbi Y08 is metabolized in a culture medium added with three different prebiotics of inulin, FOS and GOS to generate proteins with different molecular weights, and the proteins can play a role in resisting pathogens, regulating immunity, increasing mineral absorption, improving intestinal functions, influencing metabolism and satiety and the like in the intestinal tract, and have important potential probiotic effects.

Example 8 Bifidobacterium longum VHProbi Y08 cell adhesion assay

Caco-2 cells were cultured in RPMI 1640 (10% fetal bovine serum, 1% antibiotics) medium, and the medium was changed every two days until the cell density reached 80-90%. The cells were digested with 0.25% trypsin solution and adjusted to a cell concentration of 1X 10⁵one/mL, and inoculated in a six-well plate, into which 18X 18mm sterile coverslips were previously placed, and placed at 37 ℃ in 95% air/5% CO₂Culturing in an incubator until the cells grow into a compact monolayer. After washing the cells twice with PBS buffer (pH 7.2), 1mL of antibiotic-free RPMI medium and 1mL of the bacterial suspension cultured and resuspended in PBS (adjusted to 10 total bacteria count) were added to each well⁸CFU/mL), mixed well and incubated in a 5% carbon dioxide incubator with three replicates per strain. After culturing for 2h, the plates were removed, the cells were washed with PBS buffer pH7.2 until non-adherent cells were removed, and gram-stained on cell slides after 30min of fixation with anhydrous methanol. Randomly selecting 20 visual fields under a high power microscope for observation, and counting the number of lactic acid bacteria adhered to 100 Caco-2 cells. The adhesion index is shown in Table 10, and the adhesion results of Bifidobacterium longum VHProbi Y08 cells are shown in FIG. 6.

TABLE 10 Bifidobacterium longum VHProbi Y08 adhesion index to HT29 cells

Bacterial strains	Adhesion index CFU/cell	Adhesion index CFU/100 cell
			Bifidobacterium longum VHProbiY08	2.025	202.5

From the results in table 10, it can be seen that the cell adhesion index of bifidobacterium longum VHProbi Y08 provided by the invention is higher, which indicates that the strain has stronger adhesion to small intestine epithelial cells and is easier to colonize and survive in the intestinal tract, and this lays a foundation for exerting probiotic properties.

Example 9 Bifidobacterium longum VHProbi Y08 experiments to reduce the genotoxic effect of carcinogens in intestinal cells

Resuscitated HT-29 cells were treated with CO at 37 ℃ in basal Medium (BEM) supplemented with 10% calf serum and penicillin and streptomycin₂More than 90% of the adherent culture is incubated in the incubator. The cells were then washed 2 times with PBS buffer, digested with Trypsin-EDTA (0.05% Trypsin and 0.53mM EDTA), added to BEM medium and harvested by centrifugation.

Bifidobacterium longum was inoculated in 200mL of BSM liquid medium at an inoculum size of 3%, and anaerobically cultured at 37 ℃ for 12 hours until the suspension became turbid. Centrifuging culture solution at 6000r/min for 10min to collect thallus, washing thallus with physiological saline for 2 times, and resuspending in 50mL physiological saline to obtain bacterial suspension with concentration of 10⁹CFU/mL is ready for use.

4NQO is a nitroaromatic toxic substance, is a strong chemical mutagen and carcinogen, can form an adduct with a DNA chain to induce the formation of a tumor, and is prepared into 4 mu g/mL 4NQO working solution for later use. The 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) is a water-soluble yellow dye, can be converted into insoluble purple formazan under the action of mitochondrial succinate dehydrogenase in living cells, and the living cells do not have the function, can dissolve the purple formazan by using dimethyl sulfoxide (DMSO), has maximum absorbance at 570nm, and can indirectly react the number of the living cells. In a certain cell range, the formation amount of MTT converted into purple formazan is in direct proportion to the number of living cells.

Thus, inAdding HT-29 cells into deep-well plate containing BEM culture medium, and adjusting cell number to 10⁴Culturing for 16-18h overnight. 1mL of the bacterial suspension is added into the deep-well plate, and then 1mL of 4NQO working solution is added. After 12h of incubation, the supernatant was aspirated and then 2mL of BEM medium was added and 1mL of MTT was added. The mixture was incubated at 37 ℃ for 4h, after the supernatant was aspirated, 200uL of DMSO was added and the absorbance was measured at 570nm, and the cytotoxic effect of Bifidobacterium longum VHProbi Y08 on HT-29 cells by 4NQO inhibition, i.e., the cell viability retention rate, was calculated.

Wherein A is₅₇₀Adding 4NQO and Bifidobacterium longum VHProbi Y08, and reacting to obtain culture medium absorbance₅₇₀Absorbance of blank medium, C₅₇₀The absorbance of the medium was 4NQO added thereto.

The experimental result shows that the activity retention rate of HT-29 cells is only 25.1% under the toxic action of the 4NQO gene when the cells exist alone; when the bifidobacterium longum VHProbi Y08 exists, the toxicity of the HT-29 cell by the 4NQO gene is weakened, the activity retention rate of the HT-29 cell reaches 49.7 percent, and the activity retention rate is obviously improved. Therefore, the bifidobacterium longum VHProbi Y08 provided by the invention can obviously inhibit the genotoxic effect of 4NQO on HT-29 cells.

Example 10 Bifidobacterium longum VHProbi Y08 experiments to reduce the genotoxic effect of carcinogens in lung cells

Resuscitated NCI-H460 cells were CO-incubated at 37 ℃ in basal Medium (BEM) supplemented with 10% calf serum and penicillin and streptomycin₂The incubator incubates over 90% of the adherent. The cells were then washed 2 times with PBS buffer, digested with Trypsin-EDTA (0.05% Trypsin and 0.53mM EDTA), added to BEM medium and harvested by centrifugation.

Bifidobacterium longum was inoculated in 200mL of BSM liquid medium at an inoculum size of 3%, and anaerobically cultured at 37 ℃ for 12 hours until the suspension became turbid. Centrifuging the culture solution at 6000r/min for 10min to collect thallus, washing thallus with normal saline for 2 times, and resuspending in 50mL normal saline to obtain bacterial suspensionIts concentration is 10⁹CFU/mL is ready for use.

Adding NCI-H460 cells into a deep-well plate containing BEM medium, and adjusting the number of cells to 10⁴Culturing for 16-18h overnight. 1mL of the bacterial suspension is added into the deep-well plate, and then 1mL of 4NQO working solution is added. After 12h of incubation, the supernatant was aspirated and then 2mL of BEM medium was added and 1mL of MTT was added. The mixture was incubated at 37 ℃ for 4 hours, after the supernatant was aspirated, 200uL of DMSO was added and then the absorbance was measured at 570nm, and the cytotoxic effect of Bifidobacterium longum VHProbi Y08 on NCI-H460 cells by 4NQO inhibition, i.e., the cell viability retention rate, was calculated.

Wherein A is₅₇₀Adding 4NQO and Bifidobacterium longum VHProbi Y08, and reacting to obtain culture medium absorbance₅₇₀Absorbance of blank medium, C₅₇₀The absorbance of the medium was 4NQO added thereto.

The experimental result shows that when NCI-H460 cells exist alone, the activity retention rate is only 49.0 percent under the toxic action of the 4NQO gene; in the presence of bifidobacterium longum VHProbi Y08, the toxicity of the NCI-H460 cell on the 4NQO gene is weakened, the activity retention rate reaches 88.7 percent, and the activity retention rate is obviously improved. Therefore, the bifidobacterium longum VHProbi Y08 provided by the invention can obviously inhibit the genotoxic effect of 4NQO on NCI-H460 cells.

Example 11 SOS response assay for Bifidobacterium longum VHProbi Y08 Effect on reducing carcinogen genotoxicity

The SOS color reaction has the advantages of simple operation, high sensitivity and strong specificity in the aspect of evaluating the genotoxicity removing effect of the lactic acid bacteria. The principle is that the substance with genotoxic effect can make the indicator strain Escherichia coli PQ37 start the transcription, translation and expression of galactosidase gene, and judge whether the DNA is damaged or not and the damage degree according to the color reaction. Coli PQ37 replaces the normal lacZ gene with a sfIA lacZ fusion gene, so that the activity of beta-galactosidase is strictly affected by the expression of the sfIA gene. In addition, the uvrA mutation causes the thallus to lose the capability of shearing repair, so that the indicator bacteria are more sensitive to DNA damage reagents. The mutant Rfa causes the thallus to lack lipopolysaccharide, so that the carcinogen to be detected can more easily permeate into cells. Therefore, the SOS response activity of the indicator bacteria can be detected according to the expression level of the induction beta-galactosidase and the expression level of the alkaline phosphatase, and the genotoxicity of the sample can be detected according to the size of the SOS induction factors (IFsos). 2-amino-3-methylimidazol [ 4,5-f ] quinoline (IQ for short) is a heterocyclic aromatic compound, and IQ is easily generated in roasting and high-temperature cooking of fish and meat. In 2017, the world health organization listed IQ as a 2A carcinogen.

1. Reagent preparation

M63 medium: agar 15g, KH₂PO₄ 13.6g，(NH₄ )₂SO₄ 2g，FeSO₄·7H₂O 0.5mg，MgSO₄·7H₂O0.2 g/L distilled water, adjusted to pH7 with KOH.

No metabolically active STA media in SOS spot assay: lactose (20%)2mL, glucose (20%)0.5mL, tryptophan (1%)2mL, threonine (1%)2mL, histidine (1%)2mL, uracil (1%)2mL, thiamine (1%)2mL, ampicillin (10mg/mL)2mL, X-gal (20mg/mL DMSO)2mL filter sterilized were added to 1L M63 medium.

Buffer and reagent for SOS color reaction: b, buffer: na (Na)₂HPO₃ 16.1g，NaH₂PO₃· H₂O 5.5g， KCl 0.75g，MgSO₄· 7H₂0.25g of O, 1g of SDS, 2.7mL of beta-mercaptoethanol (beta-mercaptoethanol), preparing 1L of solution by using distilled water, and adjusting the pH to 7; p buffer: hydroxymethyl aminomethane tris (hydroxyymethyl) aminomethane (C)₄H₁₁NO₃): 121g of SDS, 1g of distilled water is used for preparing 1L of solution, and the pH value is adjusted to 8.8 by hydrochloric acid; ONPG (4 mg/mL): 400mg of ONPG was added to 100mL of pH7 buffer; pH7 buffer: 61mL of 0.1mol/L Na₂HPO₃·7H₂O，39mL 0.1mol/L NaH₂PO₃·H₂O; PNPP solution (4 mg/mL): 400mg PNPP was added to 100mL P buffer;

other reagents used for the SOS reaction: 1mol/L NaCO₃，2.5mol/L HCl，2moL/L Tris solution.

Standard procedure for the reaction

1mL of the indicator bacterium Escherichia coli PQ37 was added to 9mL of fresh La medium to evaluate the metabolic activity. 0.6 mL of the suspension was put into an EP tube, and the suspension was added with a test compound (IQ) and cultured with shaking at 37 ℃ for 2 hours. 0.3mL of each tube was added to another EP tube, centrifuged, washed, and resuspended in 0.3mL of the corresponding buffer. One series (X series) is used for beta-galactosidase reaction, the other series (Y series) is used for alkaline phosphatase reaction, and the two reactions can be simultaneously carried out.

Beta-galactosidase reaction: adding 2.7mL of B buffer into the X-series tube, and preserving the heat for 5-10 min at 37 ℃. Adding 0.6 mL of 4mg/mL ONPG (beta-galactosidase substrate: beta-D-galactoside) solution into each tube, reacting for 10-90 min, adding 2mL of 1mol/L Na₂CO₃The reaction was terminated. The reaction time A can be adjusted according to specific conditions₄₂₀The time of (a) is 0.1 to 0.4. Reading A with negative control of non-indicator bacteria as blank₄₂₀The value of (c).

Alkaline phosphatase reaction: adding 2.7mL of P buffer into the Y-series tube, and preserving the heat for 5-10 min at 37 ℃. Each tube is added with 0.6 mL of 4mg/mL PNPP (alkaline phosphatase substrate: p-nitrophenyl phosphate disodium salt) solution, the reaction is carried out for 10-90 min, and 1mL of 2.5mol/L HCl is added to stop the reaction, so that the color disappears. After standing for 5min, 1mL of 2mol/L Tris (hydroxyymethyl) aminomethane was added to recover the color, which remained stable for several hours. The reaction time A can be adjusted according to specific conditions₄₂₀ The time of (a) is 0.1 to 0.4. Reading A with sterile negative control as blank₄₂₀The value of (c).

The results express: enzyme unit =1000 × a₄₂₀T is calculated. In the formula, A₄₂₀Reading at 420 nm; t is the incubation time of the substrate (ONPG or PNPP).

Induction of the sfi a gene by carcinogens when protein synthesis is inhibited can be reflected by the ratio R of β -galactosidase to alkaline phosphatase.

R = β -galactosidase activity unit/alkaline phosphatase activity unit = (a)₄₂₀B×tP）/（A₄₂₀P×tB）。

Wherein A is₄₂₀B. tB and A₄₂₀P, tP represent beta-galactosidase activity and alkaline phosphatase activity at time t A₄₂₀The value of (c).

The parameter of induction index (Ic) is introduced, the induction index being the ratio of the R value R (c) at a concentration of the genetic carcinogen c to the R value R (0) at a concentration of 0.

Bifidobacterium longum VHProbi Y08 was inoculated in 200mL BSM broth at 3% inoculum size and anaerobically cultured at 37 ℃ for 24h until the broth became turbid. Centrifuging culture solution at 6000r/min for 10min to collect thallus, washing thallus with physiological saline for 2 times, and resuspending in 50mL physiological saline to obtain bacterial suspension with concentration of 10⁹CFU/mL is ready for use.

Adding 1mL of IQ solution (100mg/L) into 4mL of bacterial liquid to be detected by using a 1mL syringe, shaking uniformly, horizontally placing in an air shaking incubator at 37 ℃, and carrying out shaking culture at 150rpm/min for 3 h. The co-culture solution was centrifuged at 12000r/min for 3min, and the supernatant was carefully removed. SOS color reaction to determine the supernatant residual IQ gene toxicity effect. Meanwhile, physiological saline is used as a negative control, and IQ solution without adding bacteria liquid is used as a positive control.

Residual ratio of genotoxicity (%) = (IF)_X-1）/（IF_IQ -1）×100

Genotoxic clearance (%) = (1-residual rate) × 100

IF_XIs an inducing factor of SOS response after IQ-Bifidobacterium longum VHProbi Y08 co-culture, IF_IQThe positive control is an induction factor for IQSOS reaction, and 1 is an induction factor for the negative control (physiological saline).

The experimental data were averaged over three replicates and each experiment was independently repeated three times. The enzyme activities of β -galactosidase and alkaline phosphatase in the negative control, positive control and bifidobacterium longum VHProbi Y08 SOS reactions are shown in table 11.

TABLE 11 Bifidobacterium longum VHProbi Y08 SOS Standard reaction Programming enzyme Activity

As can be seen from the data in Table 11, after the detection of SOS reaction, the mean clearance rate of Bifidobacterium longum VHProbi Y08 to IQ reaches more than 94%, and the genotoxic effect of IQ can be remarkably reduced.

In conclusion, the bifidobacterium longum VHProbi Y08 provided by the invention shows stronger adhesion capability in vitro cell model experiments, which lays a foundation for preventing the bifidobacterium longum VHProbi Y08 from being rapidly discharged out of the body due to contraction of the digestive tract and keeping a certain amount in the gastrointestinal tract after being taken, thereby playing a probiotic function. In addition, the invention proves that the bifidobacterium longum VHProbi Y08 can obviously reduce the genotoxicity of two carcinogens of 4NQO and IQ through cell experiments and SOS reaction. Therefore, the bifidobacterium longum VHProbi Y08 has the effect of reducing the genotoxicity of carcinogens in human cells, has an effective tumor prevention mechanism and has certain value in the aspect of preventing and treating cancers.

The bifidobacterium is one of the most abundant intestinal flora in the digestive tract of infants, plays an important role in the health of the infant organism, and is one of edible strains in China, so that the safety of the bifidobacterium longum is widely accepted. The bifidobacterium longum VHProbi Y08 meets the regulatory requirements, can be used as a food raw material source, and does not have side effects or excessive risks after long-term administration. Multiphase taxonomy identification proves that the bifidobacterium longum VHProbi Y08 is a new bifidobacterium longum and has the potential probiotic characteristic of removing toxicity of 4NQO and IQ genes. Due to the rapid development of industrialization in the last hundred years, various carcinogens exist in human living factors such as food raw materials, water sources, air and the like, for example, the roasted food contains high-content benzopyrene which is a strong carcinogen, and when the food containing the benzopyrene is frequently eaten, the carcinogens can be accumulated in the body, so that the risk of inducing gastric cancer and intestinal cancer is caused; nitroaromatic compounds and azo dyes, which are widely present in soil, are typical indirect carcinogens, and the like. These carcinogens are widely present in the human living environment and increase the risk of cancer in humans. The bifidobacterium longum VHProbi Y08 provided by the invention is used as a probiotic strain of natural source, has no toxicity to organisms, can greatly reduce the genotoxicity of carcinogens, and has important application value.

The applicant has already transformed said bifidobacterium longum VHProbi Y08 (8/10/2019: (b))Bifidobacterium longumVHProbi Y08) is deposited in the chinese type culture collection of the university of wuhan, china with a collection number of CCTCC NO: and M2019780.

Example 12A Bifidobacterium longum microcapsule preparation and a method for preparing the same

1. Preparation of Bifidobacterium longum bacterial sludge

Under the aseptic condition, inoculating the bifidobacterium longum VHProbi Y08 seed liquid into a fermentation culture medium according to the volume ratio of 2-4%, wherein the fermentation culture medium comprises the following components in percentage by volume: 1-3 g/L of glucose, 5-20 g/L of soybean meal, 0.1-1.0 g/L of calcium carbonate, 10-30 g/L of corn starch, 0.5-1.0 g/L of dipotassium phosphate, 0.2-1.0 g/L of ammonium chloride, 0.01-0.7 g/L of magnesium sulfate and 0.01-0.7 g/L of manganese sulfate; culturing at 37 ℃ for 23-36 h, wherein the rotating speed is 180-200 rpm, and the ventilation rate is 1: 1.2 to 1.5, and the tank pressure is 0.05 to 0.08 MPa. After fermentation, centrifuging the bifidobacterium longum VHProbi Y08 fermentation liquor for 10min at 4 ℃ at 5000r/min, and collecting thalli to obtain bifidobacterium longum VHProbi Y08 bacterial sludge;

2. preparation of spray mixture

Weighing four protective wall materials of gelatinized starch, skim milk powder, sucrose and calcium lactate according to a proportion, adding water, stirring and mixing uniformly to obtain a protective wall material solution; sufficiently and uniformly mixing the bifidobacterium longum VHProbi Y08 bacterial mud and the protective wall material solution to obtain a spray mixed solution; the concentration of Bifidobacterium longum VHProbi Y08 in the spray mixture was 1.4X 10⁹CFU/ml, the weight volume ratio (g/ml) of each component of the protective wall material is respectively as follows: 6% of gelatinized starch, 10% of skim milk powder, 8% of sucrose and 20% of calcium lactate;

3. spray drying process

Spray drying with centrifugal spray dryer at inlet air temperature of 130 deg.C, atomization pressure of 0.4MPa, feeding speed of 20-30L/h, outlet air temperature of 70 deg.C, feeding dried granules into cyclone separator with hot air diameter of 50-90 μm, collecting, and weighing.

Example 13A Bifidobacterium longum microcapsule preparation and method for preparing the same

1. Preparation of Bifidobacterium longum bacterial sludge

Under the aseptic condition, inoculating the bifidobacterium longum VHProbi Y08 seed liquid into a fermentation culture medium according to the volume ratio of 2-4%, wherein the fermentation culture medium comprises the following components in percentage by volume: 1-3 g/L of glucose, 5-20 g/L of soybean meal, 0.1-1.0 g/L of calcium carbonate, 10-30 g/L of corn starch, 0.5-1.0 g/L of dipotassium phosphate, 0.2-1.0 g/L of ammonium chloride, 0.01-0.7 g/L of magnesium sulfate and 0.01-0.7 g/L of manganese sulfate; culturing at 37 ℃ for 23-36 h, wherein the rotating speed is 180-200 rpm, and the ventilation rate is 1: 1.2 to 1.5, and the tank pressure is 0.05 to 0.08 MPa. After fermentation, centrifuging the bifidobacterium longum VHProbi Y08 fermentation liquor for 8min at 4 ℃ and 6000r/min, and collecting thalli to obtain bifidobacterium longum VHProbi Y08 bacterial sludge;

2. preparation of spray mixture

Weighing four protective wall materials of gelatinized starch, skim milk powder, sucrose and calcium lactate according to a proportion, adding water, stirring and mixing uniformly to obtain a protective wall material solution; sufficiently and uniformly mixing the bifidobacterium longum VHProbi Y08 bacterial mud and the protective wall material solution to obtain a spray mixed solution; the concentration of Bifidobacterium longum VHProbi Y08 in the spray mixture was 2.3X 10⁹CFU/ml, the weight volume ratio (g/ml) of each component of the protective wall material is respectively as follows: 8% of gelatinized starch, 12.5% of skim milk powder, 6% of sucrose and 25% of calcium lactate;

3. spray drying process

Spray drying with centrifugal spray dryer at inlet air temperature of 120 deg.C, atomization pressure of 0.3MPa, feeding speed of 20-30L/h and outlet air temperature of 65 deg.C, feeding the dried granules into cyclone separator with hot air diameter of 50-90 μm, collecting, and weighing.

Example 14A Bifidobacterium longum microcapsule preparation and a method for preparing the same

1. Preparation of Bifidobacterium longum bacterial sludge

Under the aseptic condition, inoculating the bifidobacterium longum VHProbi Y08 seed liquid into a fermentation culture medium according to the volume ratio of 2-4%, wherein the fermentation culture medium comprises the following components in percentage by volume: 1-3 g/L of glucose, 5-20 g/L of soybean meal, 0.1-1.0 g/L of calcium carbonate, 10-30 g/L of corn starch, 0.5-1.0 g/L of dipotassium phosphate, 0.2-1.0 g/L of ammonium chloride, 0.01-0.7 g/L of magnesium sulfate and 0.01-0.7 g/L of manganese sulfate; culturing at 37 ℃ for 23-36 h, wherein the rotating speed is 180-200 rpm, and the ventilation rate is 1: 1.2 to 1.5, and the tank pressure is 0.05 to 0.08 MPa. After fermentation, centrifuging the bifidobacterium longum VHProbi Y08 fermentation liquor for 5min at the temperature of 4 ℃ and the speed of 8000r/min, and collecting thalli to obtain bifidobacterium longum VHProbi Y08 bacterial sludge;

2. preparation of spray mixture

Weighing four protective wall materials of gelatinized starch, skim milk powder, sucrose and calcium lactate according to a proportion, adding water, stirring and mixing uniformly to obtain a protective wall material solution; sufficiently and uniformly mixing the bifidobacterium longum VHProbi Y08 bacterial mud and the protective wall material solution to obtain a spray mixed solution; the concentration of Bifidobacterium longum VHProbi Y08 in the spray mixture was 5.1X 10⁹CFU/ml, the weight volume ratio (g/ml) of each component of the protective wall material is respectively as follows: 10% of gelatinized starch, 15% of skim milk powder, 4% of sucrose and 30% of calcium lactate;

3. spray drying process

And (2) carrying out spray drying treatment by adopting a centrifugal spray dryer, wherein the inlet air temperature of a drying tower is 110 ℃, the atomization pressure is 0.5MPa, the feeding speed is 20-30L/h, the outlet air temperature is 60 ℃, dried particles enter a cyclone separator along with hot air in the tower, the particle size is 50-90 mu m, and the particles are collected and weighed.

Example 15 Bifidobacterium longum microcapsule formulation Performance test

1. Strain survival detection

The results of taking 2g each of the bifidobacterium longum microcapsule preparations prepared in examples 12 to 14, after diluting the preparation by a physiological saline gradient, counting the number of viable bacteria in the preparation by a plate coating method, and calculating the amount of viable bacteria per unit in the microcapsule preparations are shown in table 12. TABLE 12 comparison of viable bacteria per unit in Bifidobacterium longum microcapsule formulations

Preparation of bifidobacterium longum microcapsuleAgent for treating cancer	Unit viable bacteria amount
		Example 12	2.86×109 CFU/g
Example 13	4.11×109 CFU/g
		Example 14	8.13×109 CFU/g

From the results in table 12, it can be seen that the amount of viable bacteria per unit in the bifidobacterium longum microcapsule preparation provided by the present invention is high, thereby indicating that the protective wall material and the spray drying preparation process selected by the present invention have very little damage to bifidobacterium longum VHProbi Y08 and significant effect.

2. Stress resistance detection of bifidobacterium longum microcapsule preparation

2.1 preparation of Artificial gastric juice

5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl were weighed, respectively, 1000mL of distilled water was added, pH3.0 was adjusted with dilute hydrochloric acid, and then sterilization was carried out at 115 ℃ for 20 min. Then 3.2g of pig mucosa pepsin is added before use, shaken up and dissolved, and placed in a water bath shaker at 37 ℃ for a water bath for 1h to simulate the temperature of a human body.

2.2 preparation of Artificial intestinal juice

Separately weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH₂PO₄6.8g and 3.0g of ox-gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is adjusted to 1000mL, the pH value is adjusted to 6.8 +/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then 1g pancreatin is added before use, shaken up and dissolved, and put into a water bath shaker at 37 ℃ for water bath for 1h to simulate the temperature of a human body.

2.3 test methods

2g of the Bifidobacterium longum microcapsule preparation prepared in example 13 of the present invention was resuspended in 2mL of physiological saline as a seed stock solution. Adding 1mL of inoculation liquid into 9mL of artificial gastric juice which is warmed in advance in a warm water bath for 1h, placing the artificial gastric juice in a water bath shaking table at 37 ℃ and oscillating the artificial gastric juice at the rotating speed of 200rpm/min for 2h, sampling 1mL at 0h and 2h respectively, and detecting the amount of viable bacteria. Then 1mL of the artificial gastric juice digested for 2h is taken and added into 24mL of the artificial intestinal juice, and the artificial intestinal juice is placed in a water bath shaker (200 rpm/min) at 37 ℃ for 3h, and 1mL of the artificial gastric juice is sampled, and the amount of the live bacteria is detected. The viable bacteria counting method is used for measuring the bacterial quantity according to the national standard GB 4789.35-2016-food microorganism test lactobacillus test, and the viable bacteria quantity (Log CFU/mL) of the bacterial strain after being digested by artificial gastric juice and artificial intestinal juice is shown in a table 13.

TABLE 13 tolerance effects of Bifidobacterium longum microcapsule formulations on artificial gastric and intestinal juices

	Before digestion	After the artificial gastric juice is digested	After digestion of the artificial intestinal juice
				Log CFU/mL viable bacteria amount	9.47±0.12	8.44±0.21	7.06±0.19

From the results in table 13, it can be seen that the survival rates of bifidobacterium longum VHProbi Y08 after the bifidobacterium longum microcapsule preparation provided by the present invention is digested by artificial gastric juice and artificial intestinal juice are respectively as high as 89.1% and 74.6%, thereby demonstrating that the bifidobacterium longum microcapsule preparation provided by the present invention has strong gastric acid and choline resistance.

3 detection of stability of bifidobacterium longum microcapsule preparation

The bifidobacterium longum microcapsule preparation prepared in the embodiment 13 of the invention is respectively packaged in food grade aluminum foil bags and stored at 37 ℃ and 20 ℃ after vacuum sealing, the viable bacteria amount is measured every 2 months and lasts for 12 months, and the survival rate of the bifidobacterium longum VHProbi Y08 in the microcapsule preparation is calculated.

TABLE 14 survival rate of strains in Bifidobacterium longum microcapsule formulations at different storage temperatures

Temperature of	2 month	4 month	6 month	8 month	10 month	12 month
							37℃	93.1%	89.6%	80.5%	73.2%	67.1%	62.0%
20℃	95.5%	90.2%	87.9%	82.1%	77.4%	71.8%

As can be seen from table 14, the survival rate of bifidobacterium longum VHProbi Y08 in the bifidobacterium longum microcapsule formulation provided by the present invention is still higher than 50% after 12 months of storage at 37 ℃, and the survival rate of bifidobacterium longum VHProbi Y08 in the bifidobacterium longum microcapsule formulation provided by the present invention still reaches 71.8% after 12 months of storage at 20 ℃. Thus, the bifidobacterium longum microcapsule preparation provided by the invention has higher stability.

The bifidobacterium longum microcapsule preparation provided by the invention has the advantages of simple preparation process, cheap protective wall material and low industrialization cost, can effectively improve the stability of bifidobacterium longum VHProbi Y08 in the storage, processing and transportation processes, powerfully improves the actual using effect of bifidobacterium longum VHProbi Y08 in the aspects of degrading cholesterol and preventing cancers, and can be widely applied to the fields of foods, health-care products and the like.

Sequence listing

<110> Islands Ulva Biometrics Ltd

QINGDAO VLAND BIOTECH GROUP Co.,Ltd.

<120> bifidobacterium longum microcapsule preparation and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1380

<212> DNA

<213> Bifidobacterium longum (Bifidobacterium longum)

<400> 1

cccacaaggg gttaggccac cggcttcggg tgctgcccac tttcatgact tgacgggcgg 60

tgtgtacaag gcccgggaac gcattcaccg cgacgttgct gattcgcgat tactagcgac 120

tccgccttca cgcagtcgag ttgcagactg cgatccgaac tgagaccggt tttcagggat 180

ccgctccgcg tcgccgcgtc gcatcccgtt gtaccggcca ttgtagcatg cgtgaagccc 240

tggacgtaag gggcatgatg atctgacgtc atccccacct tcctccgagt taaccccggc 300

ggtcccccgt gagttcccgg cataatccgc tggcaacacg gggcgagggt tgcgctcgtt 360

gcgggactta acccaacatc tcacgacacg agctgacgac gaccatgcac cacctgtgaa 420

cccgccccga agggaagccg tatctctacg accgtcggga acatgtcaag cccaggtaag 480

gttcttcgcg ttgcatcgaa ttaatccgca tgctccgccg cttgtgcggg cccccgtcaa 540

tttctttgag ttttagcctt gcggccgtac tccccaggcg ggatgcttaa cgcgttagct 600

ccgacacgga acccgtggaa cgggccccac atccagcatc caccgtttac ggcgtggact 660

accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc agtaacggcc 720

cagagacctg ccttcgccat tggtgttctt cccgatatct acacattcca ccgttacacc 780

gggaattcca gtctccccta ccgcactcaa gcccgcccgt acccggcgcg gatccaccgt 840

taagcgatgg actttcacac cggacgcgac gaaccgccta cgagcccttt acgcccaata 900

attccggata acgcttgcac cctacgtatt accgcggctg ctggcacgta gttagccggt 960

gcttattcaa cgggtaaact cactctcgct tgctccccga taaaagaggt ttacaacccg 1020

aaggcctcca tccctcacgc ggcgtcgctg catcaggctt gcgcccattg tgcaatattc 1080

cccactgctg cctcccgtag gagtctgggc cgtatctcag tcccaatgtg gccggtcgcc 1140

ctctcaggcc ggctacccgt cgaagccacg gtgggccgtt accccgccgt caagctgata 1200

ggacgcgacc ccatcccata ccgcgaaagc tttcccagaa gaccatgcga tcaactggag 1260

catccggcat taccacccgt ttccaggagc tattccggtg tatggggcag gtcggtcacg 1320

cattactcac ccgttcgcca ctctcaccac caagcaagct tgatggatcc cgttcgactg 1380

Claims (10)

1. A Bifidobacterium longum microcapsule preparation is characterized in that the microcapsule preparation is prepared by mixing Bifidobacterium longum with a protective wall material to prepare a spray mixed solution and then carrying out spray drying on the spray mixed solution.

2. The microcapsule formulation according to claim 1, wherein the bifidobacterium longum is bifidobacterium longum VHProbi Y08 (c) (h b)Bifidobacterium longumVHProbi Y08) with a preservation number of CCTCC NO: and M2019780.

3. The microcapsule formulation according to claim 1 or 2, wherein the protective wall material is composed of gelatinized starch, skim milk powder, sucrose, calcium lactate.

4. The microcapsule formulation according to claim 3, wherein the weight-to-volume ratio (g/ml) of the components of the protective wall material in the spray mixture is: 6-10% of gelatinized starch, 10-15% of skim milk powder, 4-8% of sucrose and 20-30% of calcium lactate.

5. The microcapsule formulation according to claim 4, wherein the concentration of Bifidobacterium longum in the spray mixture is 10⁹-10¹⁰ CFU/ml。

6. The microcapsule formulation according to claim 4 or 5, wherein the weight-to-volume ratio (g/ml) of each component of the protective wall material in the spray mixture is: 10% of gelatinized starch, 15% of skim milk powder, 4% of sucrose and 30% of calcium lactate.

7. The microcapsule formulation according to claim 4 or 5, wherein the spray drying is carried out by a centrifugal spray dryer, wherein the inlet air temperature of the drying tower is 110-.

8. A microcapsule formulation according to any one of claims 1 to 7, wherein the viable count of Bifidobacterium longum in the microcapsule formulation is at least 10⁹CFU/g。

9. Use of a microcapsule formulation according to any one of claims 1 to 8 for degrading cholesterol.

10. Use of a microcapsule formulation according to any one of claims 1 to 8 for the prevention of cancer.

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