CN111440750B - Bifidobacterium animalis subsp lactis with functions of relieving lactose intolerance and reducing triglyceride and application thereof - Google Patents
Bifidobacterium animalis subsp lactis with functions of relieving lactose intolerance and reducing triglyceride and application thereof Download PDFInfo
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- CN111440750B CN111440750B CN202010466436.5A CN202010466436A CN111440750B CN 111440750 B CN111440750 B CN 111440750B CN 202010466436 A CN202010466436 A CN 202010466436A CN 111440750 B CN111440750 B CN 111440750B
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- bifidobacterium animalis
- animalis subsp
- lactis
- strain
- triglyceride
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-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/515—Animalis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a Bifidobacterium animalis subsp lactis strain with functions of relieving lactose intolerance and reducing triglyceride, which is characterized in that the Bifidobacterium animalis subsp lactis strain is named as Bifidobacterium animalis subsp lactis (HCS 04-002), and is preserved in China general microbiological culture Collection center (CGMCC) at 03, 25 and 2020, with the preservation number of CGMCC No. 19509. The bifidobacterium animalis subsp lactis HCS04-002 has the functions of relieving lactose intolerance and reducing triglyceride. And has excellent acid resistance and cholate resistance. When ingested orally into the human body, it is able to withstand the lower pH gastric environment and the high bile salt environment to reach the intestinal tract with a higher survival rate.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bifidobacterium animalis subsp lactis strain with functions of relieving lactose intolerance and reducing triglyceride and application thereof.
Background
Lactose is a disaccharide specific to female mammals and is only present in the milk. Lactose cannot be directly absorbed in human body, and needs to be decomposed into glucose and galactose under the action of lactase. Lactase, also known as beta-galactosidase, is a digestive enzyme located in human small intestine villi. The enzyme can catalyze and decompose lactose, and has galactoside transferring effect. The activity and secretion of lactase in human body are influenced by primary and multiple secondary factors, and after people lack of lactase, the people cannot digest lactose in dairy products, and then symptoms of indigestion such as flatulence, borborborygmus, diarrhea and the like occur, which is called lactose intolerance.
At present, the treatment methods for lactose intolerance mainly comprise dietary lactose elimination therapy, bifidobacterium preparation and lactase preparation therapy. The dietary lactose elimination therapy is to reduce the intake of lactose, and lactose intolerance is avoided by eating lactose-free milk powder or milk, yoghourt and other alternative diets; the bifidobacterium preparation can show the activity of lactase by utilizing bifidobacterium, has the function of hydrolyzing lactose, can repair epithelial cells of small intestinal mucosa, promote the secretion of lactase, relieve clinical symptoms such as abdominal distension, borborborygmus and the like, and is a bifidobacterium triple viable tablet; the lactase preparation therapy can effectively improve lactose malabsorption without changing lactose-containing diet, and ensures normal dietary nutrition intake, and the therapy is widely accepted, but the domestic lactase raw material production technology is not mature, the existing production capacity cannot meet the domestic market demand, most of the lactase preparation therapy needs to be imported, and the cost is high.
Triglyceride (TG) is a fat molecule formed from 3 molecules of long-chain fatty acids and glycerol, and is a lipid with the highest content in the human body, most tissues can supply energy by using triglyceride decomposition products, and tissues such as liver and fat can also synthesize triglyceride, and the triglyceride is stored in fat tissues and is a main source of energy in the body. However, the excessive triglyceride in the body can cause a plurality of diseases of the human body, such as hypertension, atherosclerosis, cardiovascular diseases and the like. At present, the main methods for preventing and treating the hyperlipidemia comprise reasonable diet, proper exercise, proper physical therapy and drug therapy, but the long-term drug therapy for the severe hyperlipidemia has obvious side effects, including sleep problems, diarrhea or constipation, memory or mental problems and the like. Taking safe and effective bacteria preparation food or medicine with triglyceride degrading effect can avoid or reduce the side effect of the existing medicine, but according to the existing research, the triglyceride degrading rate of the bacteria preparation is generally low, and the bacteria preparation is listed in table 1.
Table 1 lists the triglyceride degradation rates of the bacterial preparations published in the prior art
The bifidobacterium animalis subsp lactis provided by the invention has the functions of relieving lactose intolerance and reducing triglyceride, and provides more choices for treating the lactose intolerance people suffering from hyperlipidemia.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a bifidobacterium animalis subsp lactis strain which has strong acid resistance, strong bile salt resistance and strong gastrointestinal fluid resistance, and has the functions of relieving lactose intolerance and reducing triglyceride, and an application thereof, in particular to an application in food or pharmaceutical compositions.
The Bifidobacterium animalis subsp lactis strain with the functions of relieving lactose intolerance and reducing triglyceride is named as Bifidobacterium animalis subsp lactis (HCS 04-002), and is preserved in China general microbiological culture Collection center (CGMCC) at 03, 25 and 2020, with the preservation number of CGMCC No. 19509.
The application of the bifidobacterium animalis subsp lactis in preparing food or medicines for relieving lactose intolerance.
The bifidobacterium animalis subsp lactis is applied to preparing foods or medicines with the function of reducing triglyceride.
The bifidobacterium animalis subsp lactis can be applied to the preparation of food or medicines with the functions of relieving lactose intolerance and reducing triglyceride.
The Bifidobacterium animalis subsp. lactis HCS04-002 is separated from the excrement of healthy 2-month-old male infants which are produced in a natural way, is a Bifidobacterium animalis subsp. lactis strain with strong capability of resisting the reverse environment of the digestive tract, has the typical characteristics of lactobacillus, is gram-positive, has no spores, and is in a multi-form rod shape with cells arranged in pairs. The physical and chemical characteristics are as follows: the contact enzyme is negative, the oxidase is negative, and the cell morphology and the physicochemical experiment result of the bifidobacterium animalis subsp lactis CGMCC No.19509 are shown in Table 2.
TABLE 2 cell morphology and physicochemical experimental results of Bifidobacterium animalis lactococcus CGMCC No.19509
The 16S rDNA sequence of Bifidobacterium animalis lactococcus CGMCC No.19509 of the present invention was compared with the sequence similarity of the GenBank-submitted strain by referring to the International Information Center for Biotechnology Information (NCBI) to identify the strain HCS04-002 as Bifidobacterium animalis lactococcus (Lactis), and the results of the comparison are shown in Table 3.
TABLE 3 comparison of similarity between 16S rDNA sequence of Bifidobacterium animalis subspecies lactis CGMCC No.19509 and GenBank submitted strain sequence
Compared with the prior art, the invention has the following beneficial effects:
1. the Bifidobacterium animalis subsp.lactis HCS04-002 has the functions of relieving lactose intolerance and reducing triglyceride;
2. the Bifidobacterium animalis subsp.lactis HCS04-002 has excellent acid resistance and bile salt resistance. When taken orally into a human body, the composition can tolerate a gastric environment with a low pH value and a high bile salt environment and reach the intestinal tract with a high survival rate;
3. the Bifidobacterium animalis subsp.lactis HCS04-002 is separated from human feces, and has high safety when being applied;
4. the culture medium and the neutralizer adopted in the strain purification and fermentation processes are all food grade raw materials, so that the strain has high safety and can be used for food fermentation or freeze-drying bacterial powder and the like;
5. the enzyme activity of Bifidobacterium animalis subsp.lactis HCS04-002 with the function of relieving lactose intolerance can reach 0.784U/mL, and the TG degradation rate with the function of reducing triglyceride reaches 43.0%.
The present invention provides a composition of Bifidobacterium animalis subsp.lactis (HCS 04-002) strain or a culture solution thereof.
The present invention provides a food composition comprising Bifidobacterium animalis subsp (lactis) HCS04-002 strain or a culture thereof.
The present invention provides a pharmaceutical composition of Bifidobacterium animalis subsp.lactis (HCS 04-002) strain or a culture solution thereof.
The food composition can be health food composition, children food composition, and fermented food composition.
The formulation of the food composition or the pharmaceutical composition is not limited, and for example, it may be formulated into powder, tablet, pill, capsule, granule, oral liquid, etc.
Drawings
FIG. 1 is a concentration standard curve of the amount of an ortho-nitrophenol (ONP) substance.
The Bifidobacterium animalis subsp lactis strain with the functions of relieving lactose intolerance and reducing triglyceride is named as Bifidobacterium animalis subsp lactis (HCS 04-002), is preserved in China general microbiological culture Collection center (CGMCC) at 25/03 of 2020, has the preservation number of CGMCC No.19509 and has the preservation address of: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
Detailed Description
Example 1 screening of Bifidobacterium animalis subsp.lactis (Bifidobacterium animalis) HCS04-002CGMCC No.19509 Strain
1. Preliminary screening
Diluting 500 μ L of infant feces sample in 4.5mL of TPY synthetic liquid culture medium, mixing, adding 500 μ L of diluent into 4.5mL of TPY synthetic liquid culture medium, diluting, and diluting to 10 times by 10 times dilution method-6Respectively take 10-2～10-6And (3) coating 50 mu L of diluent on a synthesized BBL solid medium plate, enabling two gradients to be parallel, and culturing the plate in an anaerobic environment at 39 ℃ for 24-48 h (placing the plate in an anaerobic bag).
2. Purification of
Selecting a single bacterial colony which has typical characteristics (observation shape, size, color, transparency and the like) of a target bacterial strain, is large in bacterial colony and strong in activity, performing streak purification culture on a synthesized BBL solid culture medium plate, performing anaerobic culture at 37 ℃ for 24-48 h and 39 ℃ for 24-48 h, and repeating the steps for 2-3 times until the characteristics of the bacterial colony in a streak plate are consistent;
the BBL culture medium: 70g BBL synthetic medium is added with 1000mL purified water, sterilized at 121 ℃ for 15 min. After the medium was cooled to 45 ℃ X-Gal (0.04g X-Gal dissolved in 2mL formamide added to 600mL medium) was added.
Example 2 identification of Bifidobacterium animalis subsp.lactis (Bifidobacterium animalis) HCS04-002CGMCC No.19509 Strain
The pure culture separated and purified in the example 1 is further determined to be a pure culture by streaking and smear microscopy, and then strain identification including gram staining test, contact enzyme test and 16S rDNA full sequence sequencing identification is carried out. The finally identified and separated strain is a Bifidobacterium animalis subsp, is named as Bifidobacterium animalis subsp (lactis) HCS04-002, is preserved in China general microbiological culture Collection center (CGMCC) at 25.03.2020, and has a preservation number of CGMCC No. 19509.
The Bifidobacterium animalis subsp.lactis HCS04-002 strain is gram positive, and the cells are in the form of multi-morphology rods and are arranged in pairs. The physical and chemical characteristics are as follows: catalase-negative and oxidase-negative, and can be D-ribose, D-glucose, maltose, lactose, melibiose, sucrose, or raffinose.
Example 3 test of resistance to reverse environmental of digestive tract of Bifidobacterium animalis subsp.lactis HCS04-002CGMCC No.19509 Strain
The ability of Bifidobacterium animalis subsp.lactis (HCS 04-002) to withstand the adverse environment of the digestive tract is a prerequisite for its ability to reach the gut with a high survival rate and to survive, colonize and function in the gut.
1. Acid resistance test
The bacterial liquid after three passages is respectively inoculated in a blank control culture medium, a basic MRS culture medium with pH3.0 and pH2.0 according to the inoculum concentration of 10 percent. Standing and culturing at 39 ℃, sampling for 17h, performing 10-fold serial dilution by using sterilized normal saline, respectively taking 1000 mu L of bacterial liquid with proper dilution, performing mixed bacteria counting operation, repeating for 2 times at each dilution, and counting after standing and culturing for 48-72 h at 39 ℃.
Acid resistance test data indexes:
viable count (denoted by N') measured for media at different pH conditions; viable count (in N) in the blank control0Expressed), the acid-resistant viable cell count logarithmic ratio calculation formula is as follows:
the acid-resistant survival rate (%) of the tested strain is lg cfu N'/lg cfu N0×100％；
TABLE 4 HCS04-002 acid-resistance test data sheet
As can be seen from Table 4, the survival rate of the HCS04-002 strain still reaches 60.1% after 17 hours under the condition of pH2.0, which shows that the bacterium can still maintain higher activity after the inhibition of gastric acid, thereby exerting the probiotic effect.
2. Bile salt resistance test
The bacterial liquid after three passages is respectively inoculated in a liquid culture medium containing no bovine bile salt (blank control), and containing the concentrations of 0.3%, 0.5%, 1.0% and 1.5% of bovine bile salt (sigma) according to the inoculum size of 10%, and is statically cultured at 39 ℃, sampled for 17h, and the viable count is determined.
The indexes of the bile salt resistance test data are as follows:
n for measuring viable count of blank control0The viable count measured under other conditions of the bile salt concentration is expressed by N', and the calculation formula of the viable count logarithmic ratio of the bile salt resistance is as follows:
the survival rate (%) of the tested strain in the bile salt resistance test is lgcfuN'/lgcfu N0×100％；
TABLE 5 HCS04-002 cholate resistance test data Table
As shown in Table 5, although the number of viable bacteria of the HCS04-002 strain gradually decreased with the increase of the cholate concentration, the survival rate was still as high as 72.6% under the condition of 1.5% cholate concentration treatment.
3. Simulated gastric fluid test
Shaking the bacterial liquid after three passages evenly, centrifuging 10mL of bacterial suspension (5000 Xg, 10min, 4 ℃) to obtain bacterial sludge, washing the bacterial sludge for 2 times by using PBS buffer solution, suspending the obtained bacterial sludge in 10mL of simulated gastric juice, digesting the bacterial sludge for 3 hours at 39 ℃, and sampling for 0 hour and 3 hours respectively to measure the number of viable bacteria.
The viable count of the test strain after the third generation of activation in the third generation culture medium is represented by N, and the viable count is counted after the test strain is digested in a simulated gastric juice culture medium for 3 hoursN is used for the number of viable bacteria#The survival rate of the tested strain simulated gastric juice test is shown as the following formula:
the survival rate (%) of the tested strain in simulated gastric fluid test is lgcfu N#/lgcfuN×100％；
TABLE 6 HCS04-002 Table of simulated gastric fluid test data
As can be seen from Table 6, the HCS04-002 strain has strong survival ability in simulated gastric juice, the survival rate is as high as 99.9% after 3h treatment, and the strain still keeps high activity for a long time in the stomach.
4. Simulated intestinal fluid test
Shaking the bacterial liquid after three passages evenly, centrifuging 10mL of bacterial suspension (5000 Xg, 10min, 5 ℃) to obtain bacterial sludge, washing the bacterial sludge for 2 times by using PBS buffer solution, suspending the obtained bacterial sludge in 10mL of artificial intestinal juice, culturing the bacterial sludge at 39 ℃, and sampling for 0h, 2h and 4h respectively to measure the number of viable bacteria.
The viable count of the tested strain after the third generation of activation in the third generation culture solution is represented by N, the viable count measured after 2h and 4h of culture in the simulated intestinal fluid culture solution is represented by N, and the survival rate calculation formula of the tested strain simulated intestinal fluid test is as follows:
the tested strain simulates intestinal juice test survival rate (%) ═ lgcfuN/lgcfuN multiplied by 100%;
TABLE 7 HCS04-002 Table of data for simulated intestinal fluid test
As can be seen from Table 7, the HCS04-002 strain has strong survival ability in simulated intestinal fluid, and the survival rate of the strain is as high as 99.8% when the simulated intestinal fluid is treated for 4 hours.
In conclusion, the strain has strong acid and bile salt resistance, and can effectively resist the influence of gastrointestinal fluids, so that high activity can be maintained after the strain passes through the digestive tract.
Example 4 determination of enzymatic Activity of Bifidobacterium animalis subsp.lactis HCS04-002 lactose intolerance alleviating function
The o-nitrophenol beta-D-galactopyranoside (ONPG) is a colorless compound which is easy to dissolve in water and can be used as a beta-galactosidase substrate. The glycosidic bond is broken under the catalytic action of enzyme to generate ortho-nitrophenol (ONP) and beta-D-galactopyranose, the product ONP is a yellow substance, the solution color gradually deepens along with the decomposition of a substrate, and the product ONP has a maximum absorption peak at 420nm, so that the yield of ONP can be measured and calculated by measuring the absorbance of the product ONP.
1. Preparation of test strains
1) Recovering the frozen strains: taking a bifidobacterium animalis subspecies lactis freezing tube stored in a low-temperature refrigerator, immediately putting the tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the freezing tube are melted;
2) first-stage culture: taking out the strain cryopreserving tube preserved at-80 ℃, thawing by hand, mixing uniformly, taking 1-ring bacteria liquid, streaking on a bifidobacterium lactis solid culture medium flat plate, carrying out anaerobic culture, and carrying out constant-temperature standing culture in an incubator at 39 ℃ for 36-48 h;
3) secondary culture: inoculating the single colony after the first-stage culture to a liquid culture medium containing 5mL of bifidobacterium lactis-lac, sealing, and performing static culture at the constant temperature of an incubator at 39 ℃ for 16-20 h;
4) and (3) third-stage culture: inoculating the bacterial suspension after the secondary culture to a triangular flask filled with 100mL of Bifidobacterium lactis-lac liquid culture medium according to the inoculation amount of 5 percent, and carrying out anaerobic culture in an incubator at the temperature of 39 ℃ for static culture for 16-20 h.
The formulation of the bifidobacterium lactis solid culture medium comprises: 30g/L of yeast peptone, 20g/L of yeast powder, 25g/L of glucose, 5g/L of fructo-oligosaccharide, 5g/L of L-malic acid and the balance of purified water, and adjusting the pH value to 7.2; agar 2%, 115 deg.C, 30min sterilizing.
The formula of the bifidobacterium lactis-lac liquid culture medium is as follows: 30g/L of yeast peptone, 20g/L of yeast powder, 20g/L of lactose, 5g/L of L-malic acid and the balance of purified water, and adjusting the pH value to 7.2; sterilizing at 115 deg.C for 30 min.
2. Drawing of o-nitrophenol (ONP) concentration standard curve
Weighing 0.1390g of o-nitrophenol (ONP) in a small beaker, adding 10ml of 96% ethanol for dissolving, transferring the solution into a 1L volumetric flask, adding water to a constant volume, and shaking up;
transferring 2mL, 4mL, 6mL, 8mL, 10mL, 12mL and 14mL solutions from the solutions to different 100mL volumetric flasks by a pipette, adding 25mL sodium carbonate solution, shaking up with buffer solution to constant volume to prepare ONP of 0.02mmol/L, 0.04mmol/L, 0.06mmol/L, 0.08mmol/L, 0.10mmol/L, 0.12mmol/L and 0.14mmol/L, and using sterile water as blank;
the quantity concentration of the o-nitrophenol substance is taken as the abscissa, and the absorbance delta OD of the diluent is taken420And drawing a standard curve for the ordinate to obtain a regression equation.
3. Determination of beta-galactosidase Activity (ONPG method)
Centrifuging 15mL of fermentation liquor after three-stage culture at 10000r/min and 4 ℃ for 5 min. The bacterial sludge was collected and washed twice with equal volumes of 0.05mol/L phosphate buffer (pH 6.8). The cells were resuspended in 3mL of the above phosphate buffer and disrupted by sonication. The crushing conditions are as follows: the ultrasonic power is 200W, the working time/interval time is 1s/9s, 60 times and 4 ℃. Centrifuging at 4 deg.C at 10000r/min for 20min to obtain supernatant to obtain crude enzyme solution. The crude enzyme solution was diluted appropriately, 200. mu.L of the crude enzyme solution was then preheated in water bath at 30 ℃ for 5min, 1mL of ONPG solution preheated at 30 ℃ for 5min was added, shaken well, and then washed in water bath at 30 ℃ for 10min, and 400. mu.L of sodium carbonate solution was immediately added to terminate the reaction. The OD was measured at 420nm within 30 min.
Preparation of a blank: the order of addition of the ONPG substrate and sodium carbonate solution was reversed and the remaining steps were performed in the same manner as the sample treatment.
One unit of enzyme activity is defined as (U): the amount of enzyme required for the hydrolytic release of 1. mu. mol ONP per minute at 30 ℃.
The enzyme activity calculation formula is as follows:
in the formula: e is the enzyme activity in the fermentation liquor, U/mL; f is determinationDilution times of enzyme solutions; v is the total volume of the reaction system and is 1.6 mL; k is the slope of the standard curve, 3.1625; t is reaction time, 10 min; v1The volume of the enzyme solution was 0.2 mL.
Bifidobacterium animalis subsp.lactis HCS04-002 enzyme activity was 0.784U/mL.
Example 5 determination of enzyme Activity of lactose intolerance relieving functional Strain of control Strain
The same beta-galactosidase activity determination method (ONPG method) is adopted, and the existing part of commercial strains are selected as a reference to perform enzyme activity determination. The results of the measurements are shown in the following table. According to the results in Table 8, the enzyme activity of Bifidobacterium animalis subsp lactis BB-12 of Hansen of Danish family was the highest and was 0.731U/mL; secondly, Lactobacillus reuteri LR92 of Italian Saccharaceae, and the enzyme activity is 0.499U/mL. According to the results of the measurement in example 2, the enzyme activity of Bifidobacterium animalis subsp.lactis HCS04-002 was 0.784U/mL, which is higher than that of all the control strains.
TABLE 8 List of control Strain lactose intolerance relief test data
Example 6 Bifidobacterium animalis subsp.lactis HCS04-002 triglyceride lowering in vitro assay
1. Preparation of test strains
(1) First-stage culture: taking out the strain cryopreserved tube at-80 ℃, thawing by hand, mixing uniformly, taking 1-ring bacteria liquid, streaking on a bifidobacterium lactis solid culture medium flat plate, and performing static culture at 39 ℃ for 36-48 h;
(2) secondary culture: selecting single colony to 10mL of milk bifidobacterium culture medium, and culturing for 20h at 39 ℃;
(3) and (3) third-stage culture: inoculating 10mL of the bacterial liquid into 200mL of a culture medium of the bifidobacterium lactis, and culturing for 17h at 39 ℃;
(4) four-stage culture: the third-stage culture broth was inoculated in 100mL of a triglyceride medium at an inoculum size of 3% and cultured at 39 ℃ for 24 hours. Meanwhile, the triglyceride culture medium without inoculated bacteria liquid is cultured for 24 hours at 39 ℃.
Preparation of triglyceride medium formulation: mixing 2% polyvinyl alcohol aqueous solution and vegetable oil according to a volume ratio of 3: 1, mixing, treating with ultrasonic wave (controlling parameters, ultrasonic treatment for 5s each time, interval time for 5s, and ultrasonic treatment for 70 times total), and mixing to obtain vegetable oil emulsion as triglyceride source. Adding the prepared vegetable oil emulsion into Bifidobacterium lactis culture medium at a ratio of 5%, adjusting pH to 7.2 + -0.2, sterilizing at 115 deg.C for 30min, making into triglyceride culture medium, and cooling in refrigerator at 4 deg.C for use.
2. Triglyceride content determination test by single reagent method (GPO-PAP method)
10mL of the triglyceride culture medium of the bacteria liquid after the four-stage culture and the non-inoculated bacteria liquid are respectively centrifuged for 10min at 4 ℃ and 4000r/min by a refrigerated centrifuge, 2.5 mu L of sterile distilled water, a calibrator and a sample are respectively added into a blank hole, a calibration hole and a sample hole by a 96-well plate according to the specification of a triglyceride kit (Nanjing Bioreagent company A110-1-1), 250 mu L of working solution is added into each hole and is mixed evenly, incubation is carried out for 10min at 37 ℃, three groups are parallel, and the OD value at 510nm is measured by an enzyme labeling instrument.
3. Test result calculation method
The TG degradation rate of lactic acid bacteria was calculated by the following calculation formula.
Total TG content-Triglyceride (TG) content in triglyceride medium without inoculated bacterial solution;
residual TG content-Triglyceride (TG) content in the triglyceride medium of the inoculated broth after the fourth stage culture.
The degradation rate of Bifidobacterium animalis subsp (lactis) HCS04-002 TG is 43.0%.
Example 7 triglyceride reduction in vitro test with control strains
In the test, partial existing commercial strains are selected for comparison, and the degradation rate of triglyceride is determined by adopting the same GPO-PAP method. The test results are shown in the following table, the highest triglyceride degradation rate of the control strains is Bifidobacterium animalis subsp lactis BB-12 strain of Hansen of Danish family, and the TG degradation rate is 39.3%; secondly, Lactobacillus plantarum vegestar 2.0 of Hansen of Danish family, whose TG degradation rate was 35.7%. According to the results of the measurement in example 6, the TG degradation rate of Bifidobacterium animalis subsp.lactis HCS04-002 was 43.0%, which was higher than that of all the control strains.
TABLE 9 triglyceride degradation rates of control strains
Example 8 culture method of Bifidobacterium animalis subsp.lactis HCS04-002
1. Recovering the frozen strains: taking a strain freezing tube stored in a low-temperature refrigerator, immediately putting the strain freezing tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the freezing tube are melted;
2. first-stage culture: inoculating recovered 1mL of frozen tube strains into a test tube filled with 10mL of basal medium, sealing the opening of the test tube, performing static culture at constant temperature in an incubator at 39 ℃ for 16-20h, and storing in a refrigerator at 4 ℃ for later use;
3. secondary culture: inoculating the bacterial suspension obtained by activating the strain into 80mL of a basic culture medium according to the inoculation amount of 3%, filling the basic culture medium with 80% of liquid, sealing the basic culture medium with tinfoil, standing and culturing the basic culture medium for 16-20h at 39 ℃, and measuring the pH value and the OD value of the bacterial suspension;
the basic culture medium comprises the following components in percentage by weight: 35g/L of yeast peptone, 20g/L of yeast powder, 25g/L of glucose, 5g/L of lactose, 5g/L of L-malic acid and the balance of purified water, wherein the pH value is 7.20.
4. And (3) third-stage culture: inoculating the bacterial suspension subjected to secondary culture into 300mL of optimized culture medium according to the inoculation amount of 3%, filling the medium with 80% of liquid, sealing with tinfoil, standing and culturing at 39 ℃ for 16-20h, and determining the pH, yield and viable count of the bacterial suspension.
The formula of the optimized culture medium is as follows: 30g/L of yeast peptone, 20g/L of yeast powder, 15g/L of glucose, 5g/L of fructo-oligosaccharide, 10g/L of lactose, 5g/L of L-malic acid, 0.5g/L of calcium chloride and the balance of purified water, wherein the pH value is 7.20.
The viable count of the bacterial suspension obtained by three-stage culture of Bifidobacterium animalis subsp (lactis) HCS04-002 is 2.7 × 109cfu/mL。
<110> Shenchen's (Shenyang) Children product Co., Ltd
<120> bifidobacterium animalis subsp lactis with functions of relieving lactose intolerance and reducing triglyceride and application thereof
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CGGCTTCGGGTGCTACCCAC TTTCATGACT TGACGGGCGG TGTGTACAAG GCCCGGGAAC 60
GCATTCACCGCGGCGTTGCT GATCCGCGAT TACTAGCGAC TCCGCCTTCA CGCAGTCGAG 120
TTGCAGACTG CGATCCGAAC TGAGACCGGT TTTCAGCGAT CCGCCCCACG TCACCGTGTC 180
GCACCGCGTT GTACCGGCCA TTGTAGCATG CGTGAAGCCC TGGACGTAAG GGGCATGATG 240
ATCTGACGTC ATCCCCACCT TCCTCCGAGT TGACCCCGGC GGTCCCACAT GAGTTCCCGG 300
CATCACCCGC TGGCAACATG CGGCGAGGGT TGCGCTCGTT GCGGGACTTA ACCCAACATC 360
TCACGACACG AGCTGACGAC GACCATGCAC CACCTGTGAA CCGGCCCCGA AGGGAAACCG 420
TGTCTCCACG GCGATCCGGC ACATGTCAAG CCCAGGTAAG GTTCTTCGCG TTGCATCGAA 480
TTAATCCGCA TGCTCCGCCG CTTGTGCGGG CCCCCGTCAA TTTCTTTGAG TTTTAGCCTT 540
GCGGCCGTAC TCCCCAGGCG GGATGCTTAA CGCGTTGGCT CCGACACGGG ACCCGTGGAA 600
AGGGCCCCAC ATCCAGCATC CACCGTTTAC GGCGTGGACT ACCAGGGTAT CTAATCCTGT 660
TCGCTCCCCA CGCTTTCGCT CCTCAGCGTC AGTGACGGCC CAGAGACCTG CCTTCGCCAT 720
TGGTGTTCTT CCCGATATCT ACACATTCCA CCGTTACACC GGGAATTCCA GTCTCCCCTA 780
CCGCACTCCA GCCCGCCCGT ACCCGGCGCA GATCCACCGT TAGGCGATGG ACTTTCACAC 840
CGGACGCGAC GAACCGCCTA CGAGCCCTTT ACGCCCAATA AATCCGGATA ACGCTCGCAC 900
CCTACGTATT ACCGCGGCTG CTGGCACGTA GTTAGCCGGT GCTTATTCGA ACAATCCACT 960
CAACACGGCC GAAACCGTGC CTTGCCCTTG AACAAAAGCG GTTTACAACC CGAAGGCCTC 1020
CATCCCGCAC GCGGCGTCGC TGCATCAGGC TTGCGCCCAT TGTGCAATAT TCCCCACTGC 1080
TGCCTCCCGT AGGAGTCTGG GCCGTATCTC AGTCCCAATG TGGCCGGTCA CCCTCTCAGG 1140
CCGGCTACCC GTCAACGCCT TGGTGGGCCA TCACCCCGCC AACAAGCTGA TAGGACGCGA 1200
CCCCATCCCA TGCCGCAAAA GCATTTCCCA CCCCACCATG CGATGGAGCG GAGCATCCGG 1260
TATTACCACC CGTTTCCAGG AGCTATTCCG GTGCACAGGG CAGGTTGGTC ACGCATTACT 1340
CACCCGTTCG CCACTCTCAC CCCGACAGCA AGCTGCCAGG 1380
Claims (4)
1. The Bifidobacterium animalis subsp lactis strain with the functions of relieving lactose intolerance and reducing triglyceride is named Bifidobacterium animalis subsp lactis (HCS 04-002), and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.19509 at 03, 25 and 2020.
2. Use of bifidobacterium animalis subsp lactis as claimed in claim 1 in the manufacture of a food or pharmaceutical product for the relief of lactose intolerance.
3. Use of bifidobacterium animalis subsp lactis as claimed in claim 1 for the preparation of a food or pharmaceutical product having a triglyceride lowering effect.
4. Use of bifidobacterium animalis subsp lactis as claimed in claim 1 for the preparation of a food or pharmaceutical product having both lactose intolerance alleviating and triglyceride reducing effects.
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