CN115851508B - Streptococcus thermophilus JYST-26 for reducing oxalic acid and improving kidney stones, and product and application thereof - Google Patents
Streptococcus thermophilus JYST-26 for reducing oxalic acid and improving kidney stones, and product and application thereof Download PDFInfo
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- CN115851508B CN115851508B CN202211253422.0A CN202211253422A CN115851508B CN 115851508 B CN115851508 B CN 115851508B CN 202211253422 A CN202211253422 A CN 202211253422A CN 115851508 B CN115851508 B CN 115851508B
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of probiotics, in particular to streptococcus thermophilus JYST-26 for reducing oxalic acid and improving kidney stones, and a product and application thereof. Streptococcus thermophilus @Streptococcus thermophilus) JYST-26 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the year of 27 of 2019, the preservation address is North Xili No.1, 3 of the Korean area of Beijing city, the preservation number is CGMCC No.18045, and the 16S rRNA sequence of JYST-26 of streptococcus thermophilus is shown as SEQ ID No. 3. The streptococcus thermophilus JYST-26 has stronger intestinal tract field planting capability, can reduce the formation of calcium oxalate kidney stones and the resolving capability of the calcium oxalate kidney stones, can avoid uncomfortable symptoms caused by medication and operation on bodies, and has no side effect.
Description
Technical Field
The invention relates to the technical field of probiotics, in particular to streptococcus thermophilus JYST-26 for reducing oxalic acid and improving kidney stones, and a product and application thereof.
Background
Urolithiasis is one of the common diseases of the urinary system, and it can be classified into upper urinary tract stones (kidney and ureteral stones) and lower urinary tract stones (bladder and urethra stones). Calcium oxalate kidney stones are a common type of kidney stones, and have a relatively high stone density and a relatively hard texture. Often, because of the excessive oxalic acid content in the body, calcium in urine is combined with the oxalic acid to form calcium oxalate stones over time. At present, the treatment means of the calcium oxalate kidney stones mainly comprise western medicine treatment, traditional Chinese medicine treatment, acupuncture treatment, operation treatment and the like.
Western medicine treatment: patients with long-term urolithiasis often experience severe inflammation of the urethra or skin on the sides of the body due to bacterial infection, and even cause pyelonephritis, renal failure, and septicemia. Therefore, it is necessary to treat urinary stones with local and systemic antibiotic therapy, such as amoxicillin, compound sulfamethoxazole, or bark drugs, such as bark tandine. But antibiotics have great side effects.
And (3) treating traditional Chinese medicine: the traditional Chinese medicine treatment is mainly to clear heat, promote diuresis, treat stranguria and remove urinary calculus, and is assisted with qi-flowing, blood circulation promoting, hardness softening and stagnation eliminating. The eight-ingredient powder is used for a large number, and other formulas such as double-row stone soup, stone-dissolving soup, pig soup and stone-dissolving powder are also used. The traditional Chinese medicine can only assist, and can not quickly treat severe symptoms.
Acupuncture treatment: the corresponding acupuncture points can obviously dilate the renal pelvis, ureter and urethra smooth muscle, strengthen the kidney function, increase the kidney blood flow, promote the kidney secretion function and increase the urine volume. The two-way regulation of needling makes the ureter shrink and then relax, and can correspondingly promote the increase of the peristaltic motion of the kidney and the ureter and expand the ureteral cavity, thereby reducing the calculus and accelerating the discharge of the calculus. But still requires drug co-therapy.
Surgical treatment: the treatment effect of the operation is rapid, but the side effect is large, and partial patients cannot bear the operation process. And the recurrence rate of the operation is high, so that the problem can not be fundamentally solved.
Disclosure of Invention
Aiming at the technical problems of large side effect, high cost and the like of the existing treatment means of calcium oxalate kidney stones, the invention provides a streptococcus thermophilus JYST-26 for reducing oxalic acid and improving kidney stones, and a product and application thereof. The streptococcus thermophilus JYST-26 strain provided by the invention has the functions of reducing oxalic acid and improving kidney stones.
In a first aspect, the present invention provides a Streptococcus thermophilus strainStreptococcus thermophilus) JYST-26 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the year of 27 of 2019, the preservation address is North Xili No.1, 3 of the Korean area of Beijing city, the preservation number is CGMCC No.18045, and the 16S rRNA sequence of JYST-26 of streptococcus thermophilus is shown as SEQ ID No. 3.
In a second aspect, the invention provides an application of streptococcus thermophilus JYST-26 in preparing oxalic acid reducing and kidney stone improving products.
In a third aspect, the present invention provides a method for preparing a product for reducing oxalic acid and improving kidney stones from streptococcus thermophilus JYST-26, comprising the following steps:
(1) Streptococcus thermophilus JYST-26 was first activated on MRS plate medium;
(2) Inoculating the activated streptococcus thermophilus JYST-26 to an MRS liquid culture medium for culture to obtain bacterial liquid;
(3) Centrifugally collecting bacterial liquid, washing the bacterial liquid with sterile physiological saline, and then re-suspending the bacterial liquid in 15wt% reconstituted skim milk to obtain suspension;
(4) The concentration of the suspension is regulated to be 1.0-2.0X10 10 cfu/mL to obtain bacterial suspension, and freeze-drying the bacterial suspension to obtain the oxalic acid reducing and kidney stone improving product.
Further, the raw materials of the MRS plate culture medium in the step (1) comprise: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar and 1000mL of distilled water;
the preparation method comprises mixing the above materials, stirring at natural pH, sterilizing at 121deg.C under 0.1MPa for 20min, pouring sterilized culture medium into a dish, and cooling.
Further, the raw materials of the MRS liquid culture medium in the step (2) comprise: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water;
the preparation method comprises mixing the above materials, adjusting pH to 6.8, stirring, and sterilizing at 121deg.C under 0.1MPa for 20 min.
Further, the culture conditions in the step (2) were 37℃for 24 hours.
Further, glucose is also included in the oxalic acid reducing and kidney stone improving products.
Further, the bacterial quantity of the streptococcus thermophilus JYST-26 in the oxalic acid reducing and kidney stone improving product is 10 10 cfu/g。
The invention has the beneficial effects that:
the streptococcus thermophilus JYST-26 has stronger intestinal tract field planting capability, can reduce the formation of calcium oxalate kidney stones and the resolving capability of the calcium oxalate kidney stones, can avoid uncomfortable symptoms caused by medication and operation on bodies, and has no side effect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is the results of the adhesion test in example 3.
FIG. 2 shows the results of in vitro oxalic acid decomposition test in example 4.
FIG. 3 is a graph showing the effect of inhibiting calcium oxalate stone formation in example 5.
FIG. 4 is a graph showing the comparative effect of dissolution of stones in Drosophila Markov tube in example 6.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
The glucose used in the embodiments of the present invention was purchased from shan east-west kingdom company, inc.
The MRS plate culture medium used in the specific embodiment of the invention comprises the following raw materials: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar and 1000mL of distilled water;
the preparation method comprises mixing the above materials, stirring at natural pH, sterilizing at 121deg.C under 0.1MPa for 20min, pouring sterilized culture medium into a dish, and cooling.
The MRS liquid culture medium used in the specific embodiment of the invention comprises the following raw materials: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water;
the preparation method comprises mixing the above materials, adjusting pH to 6.8, stirring, and sterilizing at 121deg.C under 0.1MPa for 20 min.
The MRS-sodium oxalate culture medium used in the specific embodiment of the invention comprises the following raw materials: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 2.68g of sodium oxalate and 1000mL of distilled water;
the preparation method comprises mixing the above raw materials, stirring at natural pH, and sterilizing at 121deg.C under 0.1MPa for 20min to obtain MRS-oxalic acid culture medium with sodium oxalate content of 20 mmol/L.
The PBS buffer used in the specific embodiment of the invention is prepared according to the following method: 0.27g of monopotassium phosphate, 1.42g of disodium phosphate, 8g of sodium chloride and 0.2g of potassium chloride, adding about 800mL of deionized water, fully stirring and dissolving, adding concentrated hydrochloric acid to adjust the pH value to 7.4, adding deionized water to fix the volume to 1L, and sterilizing at 121 ℃ for 20min to obtain the finished product.
The common standard food used in the specific implementation mode of the method is prepared according to the following method: mixing sucrose 40g, yeast 25g, corn flour 67g, soybean powder 10g, agar powder 10g, sodium benzoate 1g, maltose 40g with distilled water 1000mL, water-bathing at 33deg.C for 1h, boiling, adding propionic acid 7mL when the temperature is reduced to 60-70deg.C, mixing, and packaging.
The mouse food used in the specific implementation mode of the method is prepared by mixing the following raw materials: 19g of flour, 23g of corn flour, 6g of sorghum flour, 10g of bran, 15g of soybean meal, 2g of vegetable oil, 1g of cod liver oil, 10g of fish meal, 1g of bone meal, 1g of beer yeast, 1g of salt, 6.6g of starch, 3.4g of glycine, 0.5g of methionine and 0.5g of calcium carbonate.
Example 1
1. Screening of strains
1. Bacterial source:
and 7 months 2012, in the home of the inner Mongolian Dart flag herdsman, naturally fermented cheese.
2. Preparing a sample:
(1) Placing sterilized normal saline (0.85%) into a sterile triangular flask, adding 1g of bacteria source sample, and oscillating for later use;
(2) Diluting the solution obtained in the step (1) to obtain samples with different concentration gradients, namely 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 The reference numerals are 1#, 2#, 3#, 4#, 5#, 6#, 7#, respectively, for standby;
3. culturing:
coating the No.1, no. 2, no.3, no. 4, no. 5, no. 6 and No. 7 solutions in MRS plate culture medium by using a coater, and culturing for 48h under anaerobic condition at 37 ℃;
4. selecting bacterial colonies:
the colony features 1-2mm diameter, round colony, regular edge, white light color with raised middle, and 5 single colonies.
5. And (3) separating and purifying:
inoculating the single colony to a screening culture medium by streaking, culturing at 37deg.C under anaerobic condition for 48 hr, picking single colony, and preserving in glycerol tube at-70deg.C.
2. Identification of species
Single colony after separation and purification is sent to identification unit: biological engineering (Shanghai) Co., ltd
1. In the identification process, the primers used were as follows:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'；
1492R：5'-GGTTACCTTGTTACGACTT-3'。
2. the gene sequences identified were as follows:
CGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCGAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGC。
3. through identification, the bacterium is streptococcus thermophilusStreptococcus thermophilus) The classifying units are as follows: bacteria; firmics. Bacillli; lactobacillales; streptococcaceae; streptomyces sp.
4. The strain is named as streptococcus thermophilus JYST-26 and is sent to China general microbiological culture Collection center for preservation, and the preservation information is as follows;
the streptococcus thermophilus JYST-26 was deposited in China general microbiological culture Collection center, address: beijing, chaoyang area, north Chenxi way No.1, no.3, post code: 100101, accession number: CGMCC No.18045.
Example 2
According to the bacterial count of the streptococcus thermophilus JYST-26 in the product of 10 10 cfu/g, the JYST-26 bacterial powder of streptococcus thermophilus and glucose are compounded.
The streptococcus thermophilus JYST-26 bacterial powder is prepared by the following preparation method:
(1) Streptococcus thermophilus JYST-26 was first activated on MRS plate medium;
(2) Inoculating the activated streptococcus thermophilus JYST-26 to an MRS liquid culture medium according to an inoculum size of 1% (v/v) and culturing for 24 hours at 37 ℃ to obtain bacterial liquid;
(3) Centrifugally collecting bacterial liquid, washing the bacterial liquid with sterile physiological saline, and then re-suspending the bacterial liquid in 15wt% reconstituted skim milk to obtain suspension;
(4) The concentration of the suspension is regulated to be 1.0-2.0X10 10 cfu/mL to obtain bacterial suspension, and freeze-drying the bacterial suspension to obtain bacterial powder.
Example 3
The adhesion colonization of the human colorectal adenocarcinoma cell line HT-29 by Streptococcus thermophilus JYST-26 was examined, and the human colorectal adenocarcinoma cell line HT-29 was purchased from the institute of basic medicine of the national academy of medical science, while LGG strain (ATCC 53103) was used as a control. The detection method comprises the following steps:
1. culturing:
(1) The strain JYST-26 and LGG were activated in MRS liquid medium and cultured for 16h at 37 ℃.
(2) HT-29 cells were cultured in DMEM medium (10% foetal calf serum) at 37deg.C in 5% CO 2 Culturing in a concentration incubator, and when the cells are fully differentiated, digesting with 0.25% pancreatin, and then passaging. Cells grew on the wall, and the culture medium was changed every day. After 3 passages, after the cell morphology is stable, the subsequent experiment can be carried out.
2. Adhesion experiments:
(1) The cultured HT-29 cells were digested and resuspended in DMEM medium at a cell concentration of 1X 10 6 cfu/mL。
(2) Inoculating into 24-well cell culture plate with sterile cell climbing sheet, inoculating 1mL into each well, and inoculating at 37deg.C with 5% CO 2 Culturing in a concentration incubator until the cells form a fully differentiated single cell layer.
(3) The medium was discarded and washed three times with sterile PBS.
(4) Inoculating activated Streptococcus thermophilus JYST-26 and LGG strain into MRS liquid culture medium, culturing for 16 hr, centrifuging the fermentation broth under 4000r/min to collect thallus, and regulating bacterial concentration to 10 with PBS buffer solution 8 cfu/mL。
(5) Adding the prepared bacterial liquid into 24-well plate, adding 1mL of each well, and adding 5% CO at 37deg.C 2 Concentration cultureIncubating in the box for 1h.
(6) Unbound cells were separated out, washed 5 times with PBS and fixed with methanol for 10min.
(7) The slide was removed for gram staining and counted under a microscope.
3. Analysis of results:
as shown in FIG. 1, the adhesion amount is expressed as total bacterial count per 100 cells in twenty random fields, and the adhesion rate of the Streptococcus thermophilus JYST-26 is significantly higher compared with that of the LGG strain, indicating that the Streptococcus thermophilus JYST-26 has a stronger intestinal tract colonization ability.
Example 4
The capability of the streptococcus thermophilus JYST-26 to decompose oxalic acid in vitro is detected, and the detection method is as follows:
1. culturing:
and (3) inoculating the streptococcus thermophilus JYST-26 into an MRS liquid culture medium for activation culture for 16 hours, and inoculating 5% fermentation liquor into the MRS-sodium oxalate culture medium for culture for 16 hours after the culture is finished. Meanwhile, a blank MRS-sodium oxalate culture medium without inoculated with streptococcus thermophilus JYST-26 is used as a control group.
2. Sample treatment:
centrifuging the fermentation liquor and blank MRS-sodium oxalate culture medium in step 1 at 4deg.C and 12000r/min, respectively, collecting supernatant, and filtering with 0.2 μm filter membrane.
3. Oxalic acid concentration detection:
the measurement was carried out using an ion chromatograph, the ambient temperature was 25 ℃, the carrier gas was nitrogen, the pressure was 0.2MPa, the highest pressure of the pump was set to 21MPa, the flow rate was 1mL/min, and the sample injection volume was 100. Mu.L.
A Dionex IonPac AS23 (4X 250 mm) anion guard column with high hydrophilicity and high column capacity is adopted, and an IonPac AG11 analytical column (4X 50 mm) anion guard column is adopted; the eluates were 16mmol/L sodium carbonate and 4mmol/L sodium bicarbonate.
1000mg/L oxalic acid solution was prepared, filtered through a 0.2 μm filter membrane, and diluted with ultrapure water to give standard solutions having concentrations of 16, 8, 4, 2, 1, 0.5 and 0.25 mg/L.
The sample was filtered through a 0.2 μm filter and diluted 100-fold with ultrapure waterOn-Guard H column and C 18 And (5) sample injection analysis after pretreatment of the small column.
4. Analysis of results:
as shown in FIG. 2, the streptococcus thermophilus JYST-26 has strong oxalic acid degradation capability and has certain potential in reducing oxalic acid and improving kidney stones.
Example 5
The effect of the streptococcus thermophilus JYST-26 on inhibiting the formation of calcium oxalate stones in the Drosophila Male tube is verified, and the test method is as follows:
1. culturing female drosophila melanogaster:
the W1118 drosophila (purchased from Bloomington Drosophila Stock Center) was fed into drosophila tubes filled with common standard food, 20 drosophila tubes, 10 male and female animals each, were cultured at 25 ℃ and 50% humidity in 12 hours day and night alternate climatic chambers, and after 10 days, the eclosion female adults were collected.
2. Test grouping:
(1) Control group: w1118 female Drosophila at day 1 of birth was fed in normal culture.
(2) Model group: the 1-day-old W1118 female Drosophila was placed in a diet containing 1% sodium oxalate by mass.
(3) Low dose group: placing 1 day-old W1118 female Drosophila in food containing sodium oxalate 1% by weight, and adding JYST-26 bacterial powder of Streptococcus thermophilus of example 2 to 10 7 cfu/g。
Medium dose group: placing 1 day-old W1118 female Drosophila in food containing sodium oxalate 1% by weight, and adding JYST-26 bacterial powder of Streptococcus thermophilus of example 2 to 10 8 cfu/g。
High dose group: placing 1 day-old W1118 female Drosophila in food containing sodium oxalate 1% by weight, and adding JYST-26 bacterial powder of Streptococcus thermophilus of example 2 to 10 9 cfu/g。
3. Analysis of results:
after one week, the Drosophila Margaret is dissected, the calculus formation is observed under a polarized light microscope, and the area of the calculus is counted. As shown in FIG. 3, it can be seen that the low dose group has a remarkable effect of inhibiting the formation of stones (p < 0.05) compared with the model group, while the medium dose group and the high dose group have no formation of stones, which means that the addition amount of the streptococcus thermophilus JYST-26 in the medium dose group can achieve the effect of completely inhibiting the formation of stones.
Example 6
The dissolution effect of the streptococcus thermophilus JYST-26 on stones in the Drosophila Markov tube is verified, and the test method is as follows:
1. culturing female drosophila melanogaster:
the W1118 drosophila (purchased from Bloomington Drosophila Stock Center) was fed into drosophila tubes filled with common standard food, 20 drosophila tubes, 10 male and female animals each, were cultured at 25 ℃ and 50% humidity in 12 hours day and night alternate climatic chambers, and after 10 days, the eclosion female adults were collected.
2. Test grouping:
(1) Control group: w1118 female Drosophila at day 1 of birth was fed in normal culture.
(2) Model group: the 1-day-old W1118 female Drosophila was placed in a diet containing 1% sodium oxalate by mass.
(3) Low dose group: placing 1 day of birth W1118 female Drosophila in food containing 1% sodium oxalate by mass, dissecting Drosophila after one week, determining molding success, and transferring to food containing 1% sodium oxalate by mass and 10% sodium oxalate by mass 7 cfu/g food of example 2 Streptococcus thermophilus JYST-26 powder.
(4) Medium dose group: operating in the same low dose group, the food content of Streptococcus thermophilus JYST-26 was changed to 10 8 cfu/g。
(5) High dose group: operating in the same low dose group, the food content of Streptococcus thermophilus JYST-26 was changed to 10 9 cfu/g。
3. Analysis of results:
after one week of culture, the Drosophila Margaret is dissected, the formation of stones is observed under a polarized light microscope, and the areas of the stones are counted. As shown in FIG. 4, it can be seen that after adding Streptococcus thermophilus JYST-26, the stones in Drosophila Mahalanobis tube are obviously reduced (p < 0.05); along with the increase of the adding amount of the streptococcus thermophilus JYST-26, the calculus in the tube of the Mahalanobis also reduced, which indicates that the streptococcus thermophilus JYST-26 strain has a certain calculus dissolving effect.
Example 7
The effect of the streptococcus thermophilus JYST-26 on inhibiting the formation of rat stones is verified, and the test method is as follows:
1. rat culture:
SPF-grade male Sprague-Dawley rats (purchased from medical laboratory animal center, guangdong province) were selected for 39 animals, and the body weight was 180-220g. After 1 week of adaptive feeding, rats were randomly divided into 3 groups of 13 animals each, and weight recordings were weighed.
2. Test grouping:
(1) Control group: the mouse food is eaten, and distilled water is used for drinking water.
(2) Model group: 5% L-Hydroxyproline (HLP) is added into the rat food, and distilled water is used for drinking water.
(3) JYST-26 group: based on the model group, the stomach is irrigated 2X 10 per day 8 cfu/kg of example 2 Streptococcus thermophilus JYST-26 bacterial powder.
All rats were free to eat and were cultured at 25℃for 12h in a circadian cycle.
3. Rat index detection:
(1) Weight change%: after 28 days of the experiment, the rats were weighed and the change in body weight was calculated, body weight change= (post-experiment body weight-pre-experiment body weight)/pre-experiment body weight x 100%.
(2) Urine volume mL: the rats were then placed in metabolic cages, urine collected over 24 hours, and urine volume measured.
(3) Urooxalic acid mg: oxalic acid content in urine samples was measured by the method for oxalic acid detection in example 4. And 24h urooxalic acid amount, 24h urooxalic acid amount=urooxalic acid concentration×24h urine total amount was calculated.
(4) Kidney weight g: rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.35 mL/100 g), the abdominal cavity was cut open, the kidneys were flushed in situ with 4 ℃ normal saline through the abdominal aorta, the kidneys were removed cleanly, the water was drained with sterile filter paper, and the kidneys were weighed.
(5) Degree of stone formation: making kidney tissue into kidney longitudinal soft tissue directional slice, observing with polarized light microscope, according to rapid calcium oxalate animal model five-stage classification method, the crystal is classified into (+++) according to the presence or absence of crystals, the number, the degree of polymerization and the site (++), (+ -) and (-) (++), (+), (±), (-).
4. Analysis of results:
the effects of Streptococcus thermophilus JYST-26 on rat body weight, urine volume, kidney weight, and urooxalic acid are shown in Table 1. It can be seen that, for the four indicators of rat body weight, urine volume, kidney weight, and urooxalic acid, JYST-26 group was not significantly different from the control group (p > 0.05), while model group was significantly different from the control group (p < 0.01). It shows that the streptococcus thermophilus JYST-26 has better inhibition effect on the formation of rat calculus.
TABLE 1 JYST-26 effect on rat weight, urine volume, kidney weight, and urooxalic acid
The evaluation table of the degree of stone formation of the kidney tissue of the rat is shown in Table 2. It can be seen that JYST-26 had a significant decrease in stone formation rate (p < 0.01) compared with the model group, indicating that JYST-26 had a significant inhibitory effect on the formation of rat stones.
TABLE 2 evaluation of the degree of calculus formation in rat kidney tissue
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (7)
1. A streptococcus thermophilus JYST-26 for reducing oxalic acid and improving kidney stones is characterized in that the streptococcus thermophilus is @ mStreptococcus thermophilus) JYST-26 has been preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the year of 27 of 2019, the preservation address is No.3 of North Chen Silu No.1 of the Korean region of Beijing city, the preservation number is CGMCC No.18045, and the 16S rRNA sequence of JYST-26 of Streptococcus thermophilus is shown as SEQ ID No. 3.
2. Use of a streptococcus thermophilus JYST-26 according to claim 1 for preparing oxalic acid reducing and kidney stone improving bacterial powder.
3. An oxalic acid reducing and kidney stone improving bacterial powder prepared from streptococcus thermophilus JYST-26 as claimed in claim 1, wherein the preparation method of the oxalic acid reducing and kidney stone improving bacterial powder comprises the following steps:
(1) Streptococcus thermophilus JYST-26 was first activated on MRS plate medium;
(2) Inoculating the activated streptococcus thermophilus JYST-26 to an MRS liquid culture medium for culture to obtain bacterial liquid;
(3) Centrifugally collecting bacterial liquid, washing the bacterial liquid with sterile physiological saline, and then re-suspending the bacterial liquid in 15wt% reconstituted skim milk to obtain suspension;
(4) The concentration of the suspension is regulated to be 1.0-2.0X10 10 cfu/mL to obtain bacterial suspension, and freeze-drying the bacterial suspension to obtain oxalic acid reducing and kidney stone improving bacterial powder.
4. The oxalic acid reducing and kidney stone improving bacterial powder according to claim 3, wherein the raw materials of the MRS plate culture medium in the step (1) comprise: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar and 1000mL of distilled water;
the preparation method comprises mixing the above materials, stirring at natural pH, sterilizing at 121deg.C under 0.1MPa for 20min, pouring sterilized culture medium into a dish, and cooling.
5. The oxalic acid reducing and kidney stone improving bacterial powder according to claim 3, wherein the raw materials of the MRS liquid culture medium in the step (2) comprise: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water;
the preparation method comprises mixing the above materials, adjusting pH to 6.8, stirring, and sterilizing at 121deg.C under 0.1MPa for 20 min.
6. The oxalic acid reducing and kidney stone improving bacterial powder according to claim 3, wherein the culture condition in the step (2) is 37 ℃ for 24 hours.
7. The fungus powder for reducing oxalic acid and improving kidney stones according to claim 3, wherein the fungus powder for reducing oxalic acid and improving kidney stones has a fungus body number of 10 of streptococcus thermophilus JYST-26 10 cfu/g。
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| CN101485359A (en) * | 2007-11-22 | 2009-07-22 | 上海交通大学医学院附属第三人民医院 | Application of commercially available yoghourt in preventing urinary calculus |
| CN113249280A (en) * | 2021-07-16 | 2021-08-13 | 中科嘉亿营养医学(山东)微生态研究院有限公司 | Streptococcus thermophilus STN26, bacterium powder and application in uric acid reducing product |
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| CN101485688A (en) * | 2007-11-22 | 2009-07-22 | 上海交通大学医学院附属第三人民医院 | Application of edible lactobacillus in preventing urinary calculus |
| CN101485359A (en) * | 2007-11-22 | 2009-07-22 | 上海交通大学医学院附属第三人民医院 | Application of commercially available yoghourt in preventing urinary calculus |
| CN113249280A (en) * | 2021-07-16 | 2021-08-13 | 中科嘉亿营养医学(山东)微生态研究院有限公司 | Streptococcus thermophilus STN26, bacterium powder and application in uric acid reducing product |
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