CN116515684A

CN116515684A - Probiotic agent for improving hyperphosphatemia and application thereof

Info

Publication number: CN116515684A
Application number: CN202310318367.7A
Authority: CN
Inventors: 方曙光; 董瑶; 钱莉敏; 顾佳悦; 盖忠辉
Original assignee: WeCare Probiotics Co Ltd
Current assignee: WeCare Probiotics Co Ltd
Priority date: 2023-03-29
Filing date: 2023-03-29
Publication date: 2023-08-01
Anticipated expiration: 2043-03-29
Also published as: CN116515684B

Abstract

The invention relates to a probiotic agent for improving hyperphosphatemia and application thereof, wherein strains in the probiotic agent for improving hyperphosphatemia comprise lactobacillus helveticus Lactobacillus helveticus LH strain with a preservation number of CGMCC No.18796 and lactobacillus acidophilus Lactobacillus acidophilus LA strain with a preservation number of CGMCC No. 1.12735. The two strains can be matched and promoted mutually, the effect of improving the hyperphosphatemia is synergistic synergistically, under the condition of consistent using bacterial amount, compared with a single LH76 strain or a single LA85 strain, the compound of the two strains is obviously improved in the exertion of the effect of the hyperphosphatemia, and the probiotic provides a new strategy for treating the hyperphosphatemia, can be used for preparing related products for preventing, relieving or treating the hyperphosphatemia, and has wide application prospect.

Description

Probiotic agent for improving hyperphosphatemia and application thereof

Technical Field

The invention belongs to the technical field of probiotics, relates to a probiotic for improving hyperphosphatemia and application thereof, and in particular relates to a probiotic for improving hyperphosphatemia and application thereof in preparing medicines for preventing, relieving or treating hyperphosphatemia, renal fibrosis caused by hyperphosphatemia and exercise intolerance caused by hyperphosphatemia.

Background

Phosphorus is a mineral element that plays an important role in many biological processes, including cell signaling and energy metabolism. Inorganic phosphates are widely used as preservatives and odorants in western diets. Previous studies have shown that high serum phosphate concentrations are significantly associated with cardiovascular disease and mortality risk in the general population and in patients with Hyperphosphatemia. These findings indicate that the prevention and treatment of hyperphosphatemia may be helpful in improving cardiovascular prognosis in certain populations.

In addition to these reports showing that hyperphosphatemia is closely related to cardiovascular adverse outcomes, several studies have also shown that serum high phosphate concentrations are associated with rapid decline in renal function in patients with chronic nephritis CKD, suggesting that excessive phosphorus accumulation may impair renal function and cardiovascular health.

In recent years, the number of patients suffering from hyperphosphatemia due to diabetes, hypertension and aging is rapidly increasing. Hyperphosphatemia is a major problem in CKD patients. Maintaining normal phosphate balance is critical for many physiological and pathological processes, including bone mineralization and vascular calcification VC. In particular, the development of VC is a major cause of morbidity and mortality in CKD patients.

In addition, high phosphorus diets can also lead to reduced physical exercise capacity, resulting in exercise intolerance. Currently, phosphate cement is also the primary oral phosphorus reduction therapy in addition to phosphate-limiting dietetic therapy. These agents bind to phosphorus in the intestinal tract and are not absorbed by the body but are excreted via the faeces. All phosphate binders were effective in reducing serum phosphate concentration. Phosphate cement treatment is expected to remove 200-300mg of phosphate on average per day. However, these pills are large and numerous and have side effects, such as gastrointestinal discomfort, which reduce patient compliance.

The human gastrointestinal tract contains more than 100 trillion microorganisms, including more than 1000 bacteria. This diverse microflora is known as the microflora and promotes host health by promoting gut integrity, regulating host immunity, preventing pathogens, and providing nutritional and bioactive molecules. Intestinal biological disorders play a key role in a variety of pathological processes such as inflammatory bowel disease, diabetes, and obesity. If a microbial preparation capable of effectively absorbing phosphate can be developed and used as a new target for the effect of hyperphosphatemia, the problems of intestinal flora regulation, organism glycolipid metabolism regulation, chronic inflammation and the like are solved, the microbial preparation is safe and has no toxic or side effect, can even replace the conventional antibiotic treatment, and has great significance.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a probiotic agent for improving hyperphosphatemia and application thereof, in particular to a probiotic agent for improving hyperphosphatemia and application thereof in preparing medicines for preventing, relieving or treating hyperphosphatemia, renal fibrosis caused by hyperphosphatemia and exercise intolerance caused by hyperphosphatemia.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the invention provides a probiotic for improving hyperphosphatemia, wherein the bacterial strain in the probiotic for improving hyperphosphatemia comprises lactobacillus helveticus Lactobacillus helveticus LH bacterial strain with the preservation number of CGMCC No.18796 and lactobacillus acidophilus Lactobacillus acidophilus LA bacterial strain with the preservation number of CGMCC No. 1.12735.

The invention creatively develops a novel probiotic compound mode, and compounds the Lactobacillus helveticus Lactobacillus helveticus LH strain and the Lactobacillus acidophilus Lactobacillus acidophilus LA strain, so that the Lactobacillus helveticus Lactobacillus helveticus LH strain and the Lactobacillus acidophilus Lactobacillus acidophilus LA strain can be mutually matched and mutually promoted, and the effects of improving the hyperphosphatemia are synergistically enhanced, and under the condition of consistent use bacterial load, compared with the single LH76 strain or the single LA85 strain, the compound of the two strains can obviously improve the exertion of the effects, and the technical effects which are difficult to be foreseen by the technicians in the field are obtained. The specific expression is as follows: (1) Significantly modulating serum urea nitrogen and phosphorus levels in patients with hyperphosphatemia; (2) Significantly improving renal fibrosis and inflammation levels in patients with hyperphosphatemia; (3) Improving the exercise intolerance condition of patients with hyperphosphatemia and the non-esterified fatty acid level during exercise. Therefore, the probiotic provides a new strategy for treating the hyperphosphatemia, can be used for preparing related products for preventing, relieving or treating the hyperphosphatemia, and has wide application prospect.

In addition, lactobacillus helveticus and lactobacillus acidophilus, so that the lactobacillus helveticus and lactobacillus helveticus are high in safety and difficult to generate drug resistance when being used for preparing related products for preventing, relieving or treating hyperphosphatemia.

Preferably, in the probiotic agent, the live bacterial count of the Lactobacillus helveticus Lactobacillus helveticus LH strain is not less than 1×10 ⁹ CFU/mL, e.g. 1X 10 ⁹ CFU/mL、3×10 ⁹ CFU/mL、5×10 ⁹ CFU/mL、8×10 ⁹ CFU/mL、1×10 ¹⁰ CFU/mL、5×10 ¹⁰ CFU/mL、1×10 ¹¹ CFU/mL、1×10 ¹² CFU/mL, etc.; the viable count of Lactobacillus acidophilus Lactobacillus acidophilus LA strain is not less than 1×10 ⁹ CFU/mL, e.g. 1X 10 ⁹ CFU/mL、3×10 ⁹ CFU/mL、5×10 ⁹ CFU/mL、8×10 ⁹ CFU/mL、1×10 ¹⁰ CFU/mL、5×10 ¹⁰ CFU/mL、1×10 ¹¹ CFU/mL、1×10 ¹² CFU/mL, etc.; other specific point values in the numerical ranges are selectable, and will not be described in detail herein.

Preferably, the ratio of the viable count of lactobacillus helveticus Lactobacillus helveticus LH strain to lactobacillus acidophilus Lactobacillus acidophilus LA strain is (3-6): 2, for example, 3:2, 7:4, 2:1, 9:4, 5:2, 6:2, etc., and other specific values within the above numerical ranges may be selected, which will not be described herein.

Based on the potential interaction relation between the LH76 strain and the LA85 strain, the invention also discovers that when the two strains are compounded according to the specific viable bacteria count ratio, the effects of the two strains on the aspects of regulating serum urea nitrogen and phosphorus levels of patients with hyperphosphatemia, improving renal fibrosis and inflammation levels of patients with hyperphosphatemia, improving exercise intolerance conditions of patients with hyperphosphatemia and non-esterified fatty acid levels in exercise are more remarkable.

Preferably, the probiotic agent is in a form of freeze-dried powder, capsule, tablet or granule.

The formulation of the probiotics related to the invention is not limited, and comprises the most commonly used freeze-dried powder, or further prepared capsules, tablets or granules. The lyophilized powder can be prepared by the following method:

inoculating lactobacillus acidophilus LA85 strain and lactobacillus helveticus LH76 strain into a culture medium respectively for culture to obtain a culture solution; centrifuging the culture solution to obtain thalli; re-suspending the thalli by using a freeze-drying protective agent to obtain re-suspension; lyophilizing the resuspension to obtain the final product, and mixing the two solutions at a certain ratio.

Preferably, the medium includes an MRS medium.

Preferably, the MRS medium includes, in concentration: 8-12g/L peptone, 8-12g/L beef extract, 15-25g/L glucose, 1-3g/L sodium acetate, 3-7g/L yeast powder, 1-3g/L, K of diammonium citrate ₂ PO ₄ ·3H ₂ O 2-3g/L、MgSO ₄ ·7H ₂ O 0.05-0.2g/L、MnSO ₄ 0.01-0.1g/L, tween 80 0.5-2mL/L, cysteine hydrochloride 0.1-1g/L.

Preferably, the lyophilization is by vacuum freezing.

Preferably, the probiotic agent for improving hyperphosphatemia further comprises a lyoprotectant and/or a functional adjuvant.

Preferably, the lyoprotectant comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

Preferably, the functional auxiliary agent comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soy oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.

The functional auxiliary agent, namely the prebiotic, can be matched with the strain to play a role, so that the effect of the probiotic in improving chronic alcohol-induced liver diseases is further improved.

In a second aspect, the invention provides the use of a probiotic for improving hyperphosphatemia according to the first aspect in the manufacture of a medicament for preventing, alleviating or treating hyperphosphatemia.

In a third aspect, the present invention provides the use of a probiotic for ameliorating hyperphosphatemia according to the first aspect in the manufacture of a medicament for preventing, alleviating or treating renal fibrosis caused by a hyperphosphatemia diet.

In a fourth aspect, the present invention provides the use of a probiotic for ameliorating hyperphosphatemia according to the first aspect in the manufacture of a medicament for preventing, alleviating or treating exercise intolerance caused by a high phosphorus diet.

Compared with the prior art, the invention has the following beneficial effects:

Drawings

FIG. 1 is a graph comparing the statistical results of serum urea nitrogen levels for each group of mice;

FIG. 2 is a graph comparing the statistical results of serum phosphorus levels of mice in each group;

FIG. 3 is a graph comparing serum calcium level statistics for each group of mice;

FIG. 4 is a graph showing comparison of statistical results of relative nucleic acid expression levels of type I collagen in kidney cortex of mice in each group;

FIG. 5 is a graph showing a comparison of the statistical results of the relative nucleic acid expression levels of fibronectin in the renal cortex of each group of mice;

FIG. 6 is a graph showing comparison of statistical results of relative nucleic acid expression levels of plasminogen activator inhibitor-1 in kidney cortex of each group of mice;

FIG. 7 is a graph showing comparison of statistical results of relative nucleic acid expression levels of TNF- α in renal cortex of mice in each group;

FIG. 8 is a graph showing comparison of statistical results of relative nucleic acid expression levels of IL-1β in renal cortex of mice in each group;

FIG. 9 is a graph showing comparison of statistical results of relative nucleic acid expression levels of ICAM-1 in the renal cortex of each group of mice;

FIG. 10 is the maximum oxygen consumption (VO) during the exercise endurance test (ETT) for each group of mice ₂ ) Is a statistical result comparison graph of the (a);

FIG. 11 is a graph comparing the statistics of the maximum exercise duration of the exercise endurance test (ETT) for each group of mice;

FIG. 12 is a graph comparing the statistical results of serum non-esterified fatty acid (NEFA) levels after exercise endurance test (ETT) for each group of mice.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

Peptone, beef extract, glucose, sodium acetate, yeast powder, diammonium hydrogen citrate, K, as referred to in the examples below ₂ PO ₄ ·3H2O、MgSO ₄ ·7H ₂ O、MnSO ₄ Tween 80 and cysteine hydrochloride were purchased from national pharmaceutical group chemical Co.

The following examples relate to the following media:

MRS Medium (g/L): 10g/L peptone, 10g/L beef extract, 25g/L glucose, 2g/L sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate ₂ PO ₄ ·3H ₂ O 2.6g/L、MgSO ₄ ·7H ₂ O 0.1g/L、MnSO ₄ 0.05g/L, tween 80 1mL/L, cysteine amino acid salt 0.5g/L.

The lactobacillus helveticus related to the following embodiment is named as lactobacillus helveticus Lactobacillus helveticus LH strain, the preservation unit is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), the preservation time is 2019, 11 and 04 days, the preservation number is CGMCC No.18796, and the address is: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

The lactobacillus acidophilus according to the following examples is named lactobacillus acidophilus Lactobacillus acidophilus LA strain, the preservation unit is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), the preservation time is 2020 and 20 days, the preservation number is CGMCC No.1.12735, and the address is: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

The bacterial suspensions referred to in the following examples: inoculating each strain into skimmed milk, and culturing at 37deg.C for 20 hr for activation to obtain activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 4% (v/v), and culturing for 20h at 37 ℃ to obtain bacterial solution; diluting to obtain the final product.

Examples

The present example explores the effect of probiotics on blood biochemistry, kidney fibrosis, serum inflammatory factors, exercise endurance, non-esterified fatty acid levels during exercise in a mouse model of hyperphosphatemia:

(1) Animals were grouped and a model of hyperphosphatemia mice was established:

all mice (weighing about 25 g) were free of specific pathogens. The mice are kept in a cage, the environment is clean and quiet, the temperature is 23-25 ℃, and the humidity is 50-70%. Male C57BL/6 mice (7 weeks old) were supplied by Shanghai laboratory animal center. All procedures involving mice were in accordance with guidelines provided by the Shanghai laboratory animal Care and animal Experimental center (license number 2022122007).

Male SPF-class C57BL/6 mice of 7 weeks of age were randomly divided into 5 groups of 10 animals each, which were respectively a normal control group (CTL group), a high-phosphorus diet model group (HP group), lactobacillus acidophilus LA85 group (LA 85 group), lactobacillus helveticus LH76 group (LH 76 group) and a complex probiotic group (LA85+LH76, viable count ratio 2:1). After 1 week of adaptive feeding, mice were divided into 5 groups, and the model group and the probiotic intervention group were fed with high-phosphorus feed (adenine 0.2%, calcium 0.6% and phosphorus 1.0% in conventional feed), and the control group was fed with feed (calcium 0.8% and phosphorus 0.6% in conventional feed), and the probiotic intervention group was given probiotic bacteria simultaneously (at a dose of 1×10) ¹⁰ CFU/day) for 12 weeks.

(2) Collection and processing of blood samples: serum was isolated from whole blood of mice by the orbital inner canthus venous plexus method, and serum urea nitrogen (BUN), serum Ca, serum P levels were measured using a full-automatic biochemical analyzer (AU 5400, japan).

As shown in fig. 1-3, the serum urea nitrogen content and serum phosphorus level of the high phosphorus diet model group HP mice were significantly increased compared to the control group, and had a reversed trend of decrease after probiotic dry prognosis, and the trend was substantially similar to that of the control group when probiotic LA85 and LH76 were used in combination. In addition, there was no significant difference in serum calcium levels between groups, i.e., it was shown that the test successfully established a model of hyperphosphatemia mice, and that there was substantially no other internal environmental disturbance due to reduced renal function that interfered with the test (note: p <0.01 compared to the HP group, NS. compared to the CTL group).

(3) RT-PCR (reverse transcription-polymerase chain reaction) detection of renal fibrosis and inflammation indexes: extracting total RNA of kidney cortex by TRIzol method, detecting RNA concentration and purity by Nano Dmp 2000 ultramicro spectrophotometer, carrying out reverse transcription according to the specification of a reverse transcription kit, and carrying out qRT-PCR experiment by using Step OneP lus real-time fluorescence quantitative PCR instrument. The kidney fibrosis index includes type I collagen (CollagenI), fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1).

As shown in fig. 4-6, the expression of collgen I, FN and PAI-1 was significantly increased in the HP group compared to the control group, i.e. the kidneys showed fibrosis, whereas the symptoms of the probiotic LA85, LH76 and la85+lh76 were reversed in the mice with hyperphosphatemia, especially in the la85+lh76 group, and the relative nucleic acid expression of collgen I, FN and PAI-1 was significantly reduced and substantially restored to normal level (note: compared to the HP group, p <0.01 was expressed).

Then, the relative nucleic acid expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and intercellular adhesion molecule-1 (ICAM-1) was examined as an index of inflammation.

Inflammatory response is the initiating factor for interstitial fibrosis, and there are a variety of inflammatory factors involved in the process of interstitial fibrosis. The results are shown in FIGS. 7-9, and the elevated expression of the pro-inflammatory factors TNF- α, IL-1β, ICAM-1, which are the inflammatory indicators, in the HP group of mice in the high phosphorus diet model group, as compared to the control group, indicates a significant inflammatory response in the kidney tissue. The inflammatory response of each group was down-regulated after probiotic intervention, wherein the effect of the complex probiotic group was most pronounced and was able to substantially restore normal levels (note: p <0.01 compared to HP group).

(4) Determination of non-esterified fatty acid levels during exercise intolerance in high phosphorus diet mice: exercise endurance test (ETT): after 12 weeks of high phosphorus diet, mice were fasted for 2 hours and subjected to a graded treadmill exercise. All mice were familiar with the treadmill 2 days prior to exercise. All mice were exercise tested between 5-8 pm to avoid diurnal variation in athletic performance, glucose and fatty acid levels. The running machine starts to move at a speed of 5m/min,no inclination was found for 3 min. The speed is increased by 2.5m/min every 3min until the oxygen uptake VO of the mice is not increased ₂ Or refusing to run. Determination of maximum oxygen consumption during ETT (VO ₂ ) Maximum exercise duration to assess the exercise capacity of the mice.

The results are shown in FIGS. 10-11, during the treadmill exercise endurance test, oxygen intake VO of the HP model group ₂ And gradually increases with increasing workload. However, the HP group of high phosphorus diet mice had VO at each exercise level compared to the control group ₂ Significantly lower. In addition, the duration of exercise was also shortened in the high phosphorus diet HP group mice compared to the control group. However, after probiotic intervention, the exercise intolerance of the hyperphosphatemia mice is relieved, and especially the effect of the combined group of LA85+ LH76 is most obvious.

Serum non-esterified fatty acid (NEFA) levels were then determined enzymatically using commercial reagents (martial purity organisms).

As shown in fig. 12, it can be seen that there was substantially no difference in the resting level of NEFA in each group of mice, but that serum NEFA was significantly increased in each group after exercise. The increase in NEFA levels in mice in the high phosphorus diet group was significantly reduced after the treadmill exercise endurance test compared to the control group. This diminished increase in NEFA was associated with reduced levels of fat oxidation during baseline and exercise endurance tests. In addition, NEFA is low due to excessive consumption of carbohydrate fat metabolism in the body, and is generally common in patients with diabetic thyroid disease. After probiotic intervention, especially in the LA85 and LH76 complex, normal fat metabolism levels were essentially maintained.

The applicant states that the present invention is illustrated by the above examples as a probiotic for improving hyperphosphatemia and its use, but the invention is not limited to, i.e. it does not mean that the invention must be practiced in dependence on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. The probiotics for improving hyperphosphatemia is characterized in that strains in the probiotics for improving hyperphosphatemia comprise lactobacillus helveticus Lactobacillus helveticus LH strain with a preservation number of CGMCC No.18796 and lactobacillus acidophilus Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No. 1.12735.

2. The probiotic for improving hyperphosphatemia according to claim 1, wherein the number of viable bacteria of lactobacillus helveticus Lactobacillus helveticus LH strain is not less than 1 x 10 in the probiotic ⁹ CFU/mL; the viable count of Lactobacillus acidophilus Lactobacillus acidophilus LA strain is not less than 1×10 ⁹ CFU/mL。

3. The probiotic for improving hyperphosphatemia according to claim 1 or 2, characterized in that the ratio of the number of viable bacteria of lactobacillus helveticus Lactobacillus helveticus LH strain to lactobacillus acidophilus Lactobacillus acidophilus LA strain is (3-6): 2.

4. A probiotic for ameliorating hyperphosphatemia according to any of claims 1-3, wherein said probiotic is in a dosage form comprising a lyophilized powder, capsule, tablet or granule.

5. The hyperphosphatemia-ameliorating probiotic of any of claims 1-4, wherein said hyperphosphatemia-ameliorating probiotic further comprises a lyoprotectant and/or a functional adjuvant.

6. The probiotic for ameliorating hyperphosphatemia according to claim 5, wherein the lyoprotectant comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

7. The probiotic for improving hyperphosphatemia according to claim 5, wherein the functional auxiliary agent comprises any one or a combination of at least two of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soy oligosaccharides, inulin, spirulina, arthrospira, coriolus polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.

8. Use of a probiotic for ameliorating hyperphosphatemia according to any of claims 1-7 in the manufacture of a medicament for preventing, alleviating or treating hyperphosphatemia.

9. Use of a probiotic agent for improving hyperphosphatemia according to any of claims 1-7 in the manufacture of a medicament for preventing, alleviating or treating renal fibrosis caused by a hyperphosphatemia diet.

10. Use of a probiotic agent for ameliorating hyperphosphatemia according to any of claims 1-7 in the manufacture of a medicament for preventing, alleviating or treating exercise intolerance caused by a hyperphosphatemia diet.