CN115851508B - Streptococcus thermophilus JYST-26 for reducing oxalic acid and improving kidney stones, and product and application thereof - Google Patents

Abstract

The invention relates to the technical field of probiotics, and specifically relates to Streptococcus thermophilus JYST-26 that reduces oxalic acid and improves kidney stones, and its products and applications. Streptococcus thermophilus JYST-26 has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 27, 2019. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The number is CGMCC NO.18045, and the 16S rRNA sequence of Streptococcus thermophilus JYST‑26 is shown in SEQ ID NO.3. The Streptococcus thermophilus JYST‑26 has strong intestinal colonization ability, can reduce the formation of calcium oxalate kidney stones and the ability to dissolve calcium oxalate kidney stones, and can avoid uncomfortable symptoms caused by medication and surgery without any side effects.

Description

Streptococcus thermophilus JYST-26 that reduces oxalate and improves kidney stones and its products, application

Technical field

The invention relates to the technical field of probiotics, and specifically relates to a Streptococcus thermophilus JYST-26 that reduces oxalic acid and improves kidney stones, and its products and applications.

Background technique

Urolithiasis is one of the common diseases of the urinary system. Urolithiasis can be divided into upper urinary tract stones (kidney and ureter stones) and lower urinary tract stones (bladder and urethra stones). Calcium oxalate kidney stones are the most common type of kidney stones. The stones are relatively dense and hard in texture. It is often caused by excessive oxalic acid content in the body, which combines with calcium in the urine to form calcium oxalate stones over time. At this stage, the main treatments for calcium oxalate kidney stones include Western medicine, Chinese medicine, acupuncture, and surgery.

Western medicine treatment: Patients with long-term urolithiasis often develop severe urinary tract or bladder skin inflammation due to bacterial infection, and even cause pyelonephritis, renal failure and sepsis. Therefore, the treatment of urinary stones must be combined with local and systemic antibiotics, such as ampicillin, trimethoprim-sulfamethoxazole, or pyrantine drugs such as pyrantemidine. But antibiotics have serious side effects.

Traditional Chinese medicine treatment: Traditional Chinese medicine treatment is mainly based on clearing away heat, diuresis, treating stranguria and dispersing stones, supplemented by regulating qi, activating blood circulation, and softening and dispersing stagnation. Among them, Bazheng Powder is mostly used, and other prescriptions such as Shuangpai Stone Soup, Fossil Soup, Pig Soup, and Fossil Powder are used. Traditional Chinese medicine itself can only assist and cannot provide rapid treatment for severe cases.

Acupuncture treatment: Acupuncture at corresponding acupoints can significantly expand the smooth muscles of the renal pelvis, ureter, and urethra, enhance kidney function, increase renal blood flow, promote renal secretion function, and increase urine output. The two-way adjustment effect of acupuncture causes the ureter to contract and then relax, which can accordingly enhance the peristalsis of the kidney and ureter and expand the ureteral cavity, thereby lowering the stone and accelerating its discharge. However, medications are still needed for treatment.

Surgical treatment: The effect of surgical treatment is rapid, but the side effects are serious, and some patients cannot bear the surgical process. Moreover, surgery has a high recurrence rate and cannot fundamentally solve the problem.

Contents of the invention

In view of the technical problems of existing treatment methods for calcium oxalate kidney stones, such as large side effects and high cost, the present invention provides a Streptococcus thermophilus JYST-26 that reduces oxalate and improves kidney stones, and its products and applications. The Streptococcus thermophilus JYST-26 strain provided by the invention has the functions of reducing oxalic acid and improving kidney stones.

In the first aspect, the present invention provides a strain of Streptococcus thermophilus JYST-26, which has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 27, 2019. The deposit address is Beichen, Chaoyang District, Beijing. No. 3, Yard No. 1, West Road, the deposit number is CGMCC NO. 18045. The 16SrRNA sequence of Streptococcus thermophilus JYST-26 is shown in SEQ ID NO. 3.

In a second aspect, the present invention provides the application of Streptococcus thermophilus JYST-26 in preparing products that reduce oxalic acid and improve kidney stones.

In a third aspect, the present invention provides an oxalate-reducing and kidney-stone-improving product prepared from Streptococcus thermophilus JYST-26. The preparation method of the oxalic-acid-reducing and kidney-stone-improving product includes the following steps:

(1) Streptococcus thermophilus JYST-26 is first activated on MRS plate culture medium;

(2) Inoculate the activated Streptococcus thermophilus JYST-26 onto the MRS liquid medium and culture it to obtain bacterial liquid;

(3) Centrifuge the bacterial solution to collect the bacterial cells, wash them with sterile saline and resuspend them in 15wt% reconstituted skim milk to obtain a suspension;

(4) Adjust the concentration of the suspension to 1.0-2.0×10 ¹⁰ cfu/mL to obtain a bacterial suspension. After freeze-drying the bacterial suspension, a product that reduces oxalic acid and improves kidney stones is obtained.

Further, the raw materials of step (1) MRS plate culture medium include: 10g peptone, 5g beef meal, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween 80, 0.05g manganese sulfate tetrahydrate, citric acid 2g triammonium, 20g glucose, 0.2g magnesium sulfate heptahydrate, 15g agar, 1000mL distilled water;

The preparation method is to mix the above raw materials, stir under natural pH, and then sterilize at 121°C and 0.1MPa for 20 minutes. Pour the sterilized culture medium into a flat dish and let it cool for use.

Further, the raw materials of step (2) MRS liquid culture medium include: 10g peptone, 5g beef meal, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween 80, 0.05g manganese sulfate tetrahydrate, citric acid 2g triammonium, 20g glucose, 0.2g magnesium sulfate heptahydrate, 1000mL distilled water;

The preparation method is to mix the above raw materials, adjust the pH value to 6.8, stir, and then sterilize at 121°C and 0.1MPa for 20 minutes.

Further, the culture conditions in step (2) are 37°C and 24h.

Furthermore, the product for reducing oxalate and improving kidney stones also includes glucose.

Further, the number of Streptococcus thermophilus JYST-26 in the product for reducing oxalate and improving kidney stones is 10 ¹⁰ cfu/g.

The beneficial effects of the present invention are:

The Streptococcus thermophilus JYST-26 has strong intestinal colonization ability, can reduce the formation of calcium oxalate kidney stones and the ability to dissolve calcium oxalate kidney stones, and can avoid uncomfortable symptoms caused by medication and surgery without any side effects.

Description of the drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those of ordinary skill in the art, It is said that other drawings can also be obtained based on these drawings without exerting creative work.

Figure 1 is the adhesion test results in Example 3.

Figure 2 is the results of the in vitro oxalic acid decomposition ability test in Example 4.

Figure 3 is a comparative chart of the effects of inhibiting the formation of calcium oxalate stones in Example 5.

Figure 4 is a comparative chart of the dissolution effects of stones in the Malpighian ducts of Drosophila in Example 6.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described The embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present invention.

The glucose used in the specific embodiments of the present invention was purchased from Shandong Xiwang Group Co., Ltd.

The raw materials of the MRS plate culture medium used in the specific embodiment of the present invention include: 10g peptone, 5g beef meal, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween 80, 0.05g manganese sulfate tetrahydrate, lemon 2g triammonium acid, 20g glucose, 0.2g magnesium sulfate heptahydrate, 15g agar, 1000mL distilled water;

The preparation method is to mix the above raw materials, stir under natural pH, and then sterilize at 121°C and 0.1MPa for 20 minutes. Pour the sterilized culture medium into a flat dish and let it cool for use.

The raw materials of the MRS liquid culture medium used in the specific embodiment of the present invention include: 10g peptone, 5g beef meal, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween 80, 0.05g manganese sulfate tetrahydrate, lemon 2g triammonium acid, 20g glucose, 0.2g magnesium sulfate heptahydrate, 1000mL distilled water;

The preparation method is to mix the above raw materials, adjust the pH value to 6.8, stir, and then sterilize at 121°C and 0.1MPa for 20 minutes.

The raw materials of the MRS-sodium oxalate medium used in the specific embodiment of the present invention include: 10g peptone, 5g beef meal, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween 80, and 0.05g manganese sulfate tetrahydrate. , 2g triammonium citrate, 20g glucose, 0.2g magnesium sulfate heptahydrate, 2.68g sodium oxalate, 1000mL distilled water;

The preparation method is to mix the above raw materials, stir under natural pH, and then sterilize at 121°C and 0.1MPa for 20 minutes to prepare an MRS-oxalic acid medium with a sodium oxalate content of 20 mmol/L.

The PBS buffer used in the specific embodiment of the present invention is prepared as follows: 0.27g potassium dihydrogen phosphate, 1.42g disodium hydrogen phosphate, 8g sodium chloride, and 0.2g potassium chloride. Add about 800 mL of deionized water and stir thoroughly to dissolve. , add concentrated hydrochloric acid to adjust the pH to 7.4, add deionized water to adjust the volume to 1L, and sterilize at 121°C for 20 minutes.

The ordinary standard food used in the specific implementation of this method is prepared as follows: 40g of sucrose, 25g of yeast, 67g of corn flour, 10g of soybean flour, 10g of agar powder, 1g of sodium benzoate, 40g of maltose and 1000mL of distilled water are mixed in a 33°C water bath 1 hour, then boil. When the temperature drops to 60-70°C, add 7 mL of propionic acid, mix well, and aliquot.

The rat food used in the specific implementation of this 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, and brewer's yeast. 1g, 1g salt, 6.6g starch, 3.4g glycine, 0.5g methionine, 0.5g calcium carbonate.

Example 1

1. Screening of bacterial strains

1. Source of bacteria:

In July 2012, naturally fermented cheese was displayed in the homes of herders in Dalat Banner, Inner Mongolia.

2. Prepare samples:

(1) Take sterilized physiological saline (0.85%) into a sterile Erlenmeyer flask, then add 1g of bacterial source sample into it, shake, and set aside;

(2) Dilute the solution in step (1) to prepare samples with different concentration gradients, namely 10 ^-1 , 10 ^-2 , 10 -3 , 10 ^-4 , 10 ^-5 , ^{10 -6 ,} and 10 ^-7 , labeled They are 1#, 2#, 3#, 4#, 5#, 6# and 7# respectively, ready for use;

3. Cultivation:

Use a spreader to apply solutions 1#, 2#, 3#, 4#, 5#, 6#, and 7# to the MRS plate culture medium respectively, and culture them at 37°C under anaerobic conditions for 48 hours;

4. Select colonies:

The characteristics of the colonies are 1-2 mm in diameter, rounded colonies, neat edges, and slightly white with bulges in the middle. Pick 5 single colonies.

5. Separation and purification:

The single colonies picked were inoculated into the screening medium using the streaking method, and cultured at 37°C under anaerobic conditions for 48 hours. The single colonies were picked and stored in glycerol tubes at -70°C.

2. Identification of bacterial species

Send the separated and purified single colonies for identification. The identification unit is: Sangon Bioengineering (Shanghai) Co., Ltd.

1. During the identification process, the primers used are as follows:

27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

1492R: 5'-GGTTACCTTGTTACGACTT-3'.

2. The identified gene sequences are as follows:

CGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGTCCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGA CGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGGTAATACGTA TCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAG TCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGTGCATGGTTGTCGTCAG CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCGAGCTAATTCCTTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAG TCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGC.

3. After identification, the bacterium is Streptococcus thermophilus , and its taxonomic units are: Bacteria; Firmicutes; Bacilli; Lactobacilales; Streptococaceae; Streptococcus.

4. Name the strain Streptococcus thermophilus JYST-26 and send it to the General Microbiology Center of China Committee for the Collection of Microbial Cultures for preservation. The preservation information is as follows;

The Streptococcus thermophilus JYST-26 was deposited on June 27, 2019 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures. Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, deposit number: CGMCCNo.18045.

Example 2

According to the fact that the bacterial count of Streptococcus thermophilus JYST-26 in the product is 10 ¹⁰ cfu/g, the Streptococcus thermophilus JYST-26 bacteria powder and glucose are compounded.

Among them, Streptococcus thermophilus JYST-26 bacterial powder is prepared by the following preparation method:

(1) Streptococcus thermophilus JYST-26 is first activated on MRS plate culture medium;

(2) Inoculate the activated Streptococcus thermophilus JYST-26 into the MRS liquid medium at an inoculum volume of 1% (v/v) and culture it at 37°C for 24 hours to obtain bacterial liquid;

(3) Centrifuge the bacterial solution to collect the bacterial cells, wash them with sterile saline and resuspend them in 15wt% reconstituted skim milk to obtain a suspension;

(4) Adjust the concentration of the suspension to 1.0-2.0×10 ¹⁰ cfu/mL to obtain a bacterial suspension. Freeze-dry the bacterial suspension to obtain bacterial powder.

Example 3

The adhesion and colonization effect of Streptococcus thermophilus JYST-26 on the human colorectal adenocarcinoma cell line HT-29 was detected. The human colorectal adenocarcinoma cell line HT-29 was purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, and the LGG strain ( ATCC53103) as a control. The detection method is as follows:

1. Cultivation:

(1) Streptococcus thermophilus JYST-26 and LGG strains were activated in MRS liquid medium and cultured at 37°C for 16 hours.

(2) HT-29 cells are cultured in DMEM medium (containing 10% fetal bovine serum) at 37°C in an incubator containing 5% _CO2 . When the cells are completely differentiated, digest them with 0.25% trypsin. , and then passaged. The cells grow adherently and the culture medium is changed every day. After 3 passages, when the cell morphology is stable, subsequent experiments can be carried out.

2. Adhesion experiment:

(1) Digest the cultured HT-29 cells and resuspend them in DMEM medium. Adjust the cell concentration to 1×10 ⁶ cfu/mL.

(2) Inoculate it into a 24-well cell culture plate with a built-in sterile cell slide, inoculate 1 mL into each well, and culture it in an incubator containing 5% _CO2 at 37°C until the cells form complete differentiation. single cell layer.

(3) Discard the culture medium and wash three times with sterile PBS.

(4) Inoculate the activated Streptococcus thermophilus JYST-26 and LGG strains into the MRS liquid medium and culture for 16 hours. Then, centrifuge the fermentation liquid at 4000r/min to collect the cells, and adjust the bacterial liquid with PBS buffer. The concentration is 10 ⁸ cfu/mL.

(5) Add the prepared bacterial solution to the 24-well plate, add 1 mL to each well, and incubate for 1 hour in an incubator at 37°C and containing 5% _CO2 .

(6) Separate unbound bacterial cells, then rinse 5 times with PBS and fix with methanol for 10 minutes.

(7) Take out the slides for Gram staining and count under a microscope.

3. Result analysis:

As shown in Figure 1, the adhesion amount is expressed as the total number of bacteria/100 cells in twenty random fields. Compared with the LGG strain, the adhesion rate of Streptococcus thermophilus JYST-26 is significantly higher, indicating that the thermophilic chain Cocci JYST-26 has strong intestinal colonization ability.

Example 4

To detect the ability of Streptococcus thermophilus JYST-26 to decompose oxalic acid in vitro, the detection method is as follows:

1. Cultivation:

Streptococcus thermophilus JYST-26 was inoculated into MRS liquid medium for activation and culture for 16 hours. After the culture, 5% of the fermentation broth was inoculated into MRS-sodium oxalate medium and cultured for 16 hours. At the same time, the blank MRS-sodium oxalate medium without inoculation of Streptococcus thermophilus JYST-26 was used as the control group.

2. Sample processing:

Centrifuge the fermentation broth and blank MRS-sodium oxalate culture medium in step 1 at 4°C and 12000 r/min respectively, take the supernatant, filter it through a 0.2 μm filter membrane, and set aside.

3. Oxalic acid concentration detection:

Use an ion chromatograph for measurement. The ambient temperature is 25°C, the carrier gas is nitrogen, the pressure is 0.2MPa, the maximum pressure of the pump is set to 21MPa, the flow rate is 1mL/min, and the injection volume is 100μL.

Dionex IonPac AS23 (4×250mm) anion guard column with high hydrophilicity and high column capacity and IonPac AG11 analytical column (4×50mm) anion guard column are used; the eluent is 16mmol/L sodium carbonate and 4mmol/L sodium carbonate. Sodium bicarbonate.

Prepare a 1000 mg/L oxalic acid solution, filter it with a 0.2 μm filter membrane, and dilute it with ultrapure water into standard solutions with concentrations of 16, 8, 4, 2, 1, 0.5, and 0.25 mg/L.

After filtering the sample through a 0.2 μm filter membrane, dilute it 100 times with ultrapure water, pass it through an On-Guard H column and a C ₁₈ pretreatment cartridge before injecting the sample for analysis.

4. Result analysis:

As shown in Figure 2, Streptococcus thermophilus JYST-26 has a strong ability to degrade oxalic acid and has certain potential in reducing oxalic acid and improving kidney stones.

Example 5

To verify the effect of Streptococcus thermophilus JYST-26 on inhibiting the formation of calcium oxalate stones in the Malpighian ducts of Drosophila, the test method is as follows:

1. Cultivate female fruit flies:

W1118 fruit flies (purchased from Bloomington Drosophila Stock Center) were fed in fruit fly tubes containing ordinary standard food, 20 per tube, 10 males and 10 males each, and cultured in a climate chamber at 25°C, 50% humidity, and a 12-hour day and night cycle. , collected the emerging female adults after 10 days.

2. Test grouping:

(1) Control group: 1-day-old W1118 female fruit flies were cultured and fed normally.

(2) Model group: One-day-old W1118 female fruit flies were placed in food containing 1% mass fraction of sodium oxalate.

(3) Low-dose group: One-day-old W1118 female fruit flies were placed in food containing 1% sodium oxalate by mass, and the Streptococcus thermophilus JYST-26 bacterial powder of Example 2 was added to the food to 10 ⁷ cfu /g.

Medium-dose group: One-day-old W1118 female fruit flies were placed in food containing 1% sodium oxalate by mass fraction, and Streptococcus thermophilus JYST-26 bacterial powder of Example 2 was added to the food to 10 ⁸ cfu/g.

High-dose group: One-day-old W1118 female fruit flies were placed in food containing 1% sodium oxalate by mass fraction, and Streptococcus thermophilus JYST-26 bacterial powder of Example 2 was added to the food to 10 ⁹ cfu/g.

3. Result analysis:

One week later, the Malpighian ducts of the fruit flies were dissected, the stone formation was observed under a polarized light microscope, and the stone area was counted. The results are shown in Figure 3. It can be seen that the low-dose group has a significant inhibitory effect on stone formation compared with the model group (p<0.05), while there is no stone formation in the medium-dose group and the high-dose group, indicating that the medium-dose group is addicted to stone formation. The added amount of Streptococcus thermos JYST-26 can completely inhibit stone formation.

Example 6

To verify the dissolving effect of Streptococcus thermophilus JYST-26 on stones in the Malpighian ducts of Drosophila, the test method is as follows:

1. Cultivate female fruit flies:

W1118 fruit flies (purchased from Bloomington Drosophila Stock Center) were fed in fruit fly tubes containing ordinary standard food, 20 per tube, 10 males and 10 males each, and cultured in a climate chamber at 25°C, 50% humidity, and a 12-hour day and night cycle. , collected the emerging female adults after 10 days.

2. Test grouping:

(1) Control group: 1-day-old W1118 female fruit flies were cultured and fed normally.

(2) Model group: One-day-old W1118 female fruit flies were placed in food containing 1% mass fraction of sodium oxalate.

(3) Low-dose group: One-day-old W1118 female fruit flies were placed in food containing 1% mass fraction of sodium oxalate. One week later, the fruit flies were dissected. After confirming that the modeling was successful, they were transferred to food containing 1% mass fraction of oxalic acid. Sodium and 10 ⁷ cfu/g of Example 2 Streptococcus thermophilus JYST-26 powder in food.

(4) Medium-dose group: The operation is the same as that of the low-dose group, except that the content of Streptococcus thermophilus JYST-26 in food is changed to 10 ⁸ cfu/g.

(5) High-dose group: The operation is the same as that of the low-dose group, except that the content of Streptococcus thermophilus JYST-26 in food is changed to 10 ⁹ cfu/g.

3. Result analysis:

After one week of culture, the Drosophila Malpighian ducts were dissected, the stone formation was observed under a polarized light microscope, and the stone area was counted. The results are shown in Figure 4. It can be seen that after adding Streptococcus thermophilus JYST-26, the stones in the Malpighian ducts of Drosophila were significantly reduced (p<0.05); The number of stones in the tube also decreased, indicating that Streptococcus thermophilus JYST-26 strain has a certain effect on dissolving stones.

Example 7

To verify the effect of Streptococcus thermophilus JYST-26 on inhibiting stone formation in rats, the test method is as follows:

1. Rat culture:

Thirty-nine SPF grade male Sprague-Dawley rats (purchased from Guangdong Medical Experimental Animal Center) were selected, weighing 180-220g. After 1 week of adaptive feeding, the rats were randomly divided into 3 groups, 13 rats in each group, and their body weights were measured and recorded.

2. Test grouping:

(1) Control group: eat rat food and drink distilled water.

(2) Model group: 5% L-hydroxyproline (HLP) was added to the rat food, and distilled water was used for drinking water.

(3) JYST-26 group: On the basis of the model group, 2×10 ⁸ cfu/kg of Streptococcus thermophilus JYST-26 powder of Example 2 was administered daily.

All rats had free access to food and were cultured at 25°C with a 12-h day-night cycle.

3. Rat indicator detection:

(1) Body weight change %: After 28 days of experiment, the rats were weighed and the weight change was calculated. Weight change = (weight after experiment - weight before experiment) / weight before experiment × 100%.

(2) Urine volume mL: The rats were then placed in metabolic cages, urine was collected within 24 hours, and urine volume was measured.

(3) Urinary oxalic acid mg: Use the method for detecting oxalic acid in Example 4 to detect the oxalic acid content in the urine sample. And calculate the 24h urinary oxalic acid amount, 24h urinary oxalic acid amount = urinary oxalic acid concentration × 24h total urine amount.

(4) Kidney weight g: Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.35 mL/100g), the abdominal cavity was incised, and the kidneys were flushed in situ through the abdominal aorta with 4°C normal saline, and both kidneys were removed after they were clean. , sterile filter paper was used to control the moisture and weighed.

(5) Degree of stone formation: The kidney tissue is made into longitudinal soft tissue oriented slices and observed with a polarizing microscope. According to the five-level classification method of the rapid calcium oxalate animal model, it is divided into ( +++), (++), (+), (±), (-), among which (-) level is negative for stone formation, (+++), (++), (+), (±) The level is positive for stone formation.

4. Result analysis:

The effects of Streptococcus thermophilus JYST-26 on the body weight, urine output, kidney weight, and urinary oxalic acid of rats are shown in Table 1. It can be seen that for the four indicators of rat body weight, urine output, kidney weight, and urinary oxalic acid, there is no significant difference between the JYST-26 group and the control group (p>0.05), while there is a significant difference between the model group and the control group (p <0.01). This shows that Streptococcus thermophilus JYST-26 has a good inhibitory effect on stone formation in rats.

Table 1 Effects of JYST-26 on body weight, urine output, kidney weight, and urinary oxalic acid in rats

The evaluation table for the degree of stone formation in rat kidney tissue is shown in Table 2. It can be seen that the stone formation rate of the JYST-26 group is significantly reduced compared with the model group (p<0.01), indicating that Streptococcus thermophilus JYST-26 has a significant inhibitory effect on stone formation in rats.

Table 2 Evaluation table for the degree of stone formation in rat kidney tissue

Although the present invention has been described in detail with reference to the accompanying drawings in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the invention, and these modifications or substitutions should be within the scope of the invention/any Those skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention, and they should all be covered by the protection scope of the present invention.

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。