Thermal inactivation substance bifidobacterium subsp. Lactis metaplasm as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: centrifuging an activated culture solution of animal bifidobacterium subsp. Lactis HNU212 to remove a supernatant, washing with 0.9% normal saline, adjusting the strain concentration to 1 * 10 < 9 > CFU / mL, and inactivating at 90 DEG C for 15 minutes, thereby obtaining the thermal inactivation bifidobacterium subsp. Lactis metagen. The invention also provides a preparation method of the thermal inactivation bifidobacterium subsp. Lactis metagen, and the preparation method of the thermal inactivation bifidobacterium subsp. Lactis metagen comprises the following steps of: preparing the thermal inactivation bifidobacterium subsp. Lactis metagen and the preparation method of the thermal inactivation bifidobacterium subsp. Lactis metagen. When applied to an HUA animal model, the thermal inactivation substance bifidobacterium subsp. Lactis metaplasm prepared by the invention can effectively reduce the serum uric acid level, relieve kidney and intestinal injury and regulate host intestinal flora, and has the effect of relieving and treating HUA.

Description

Technical Field

[0001] The invention belongs to the technical field of hyperuricemia treatment, and relates to a heat-killed animal Bifidobacterium lactis subsp. postbiotic, and a preparation method and application thereof. Background Art

[0002] Hyperuricemia (HUA) is a common metabolic syndrome characterized by excessive uric acid production and insufficient uric acid excretion. Uric acid is the final product of purine metabolism. Elevated uric acid levels are a risk factor for metabolic diseases such as gout, hypertension, and chronic kidney disease. It has now become the "fourth highest" disease after "hypertension, hyperlipidemia, and hyperglycemia." Economic prosperity and rapid development of social welfare have led to changes in lifestyle and greatly changed people's dietary patterns. The intake of purine-rich foods such as broth, seafood, offal, and beer has also increased, and the prevalence of hyperuricemia and gout has gradually increased worldwide. In addition to diet, other factors such as gender, age, weather, region, and race are also related to the prevalence of hyperuricemia and gout. According to epidemiological statistics, the global prevalence of hyperuricemia is between 11.4% and 24.5%, with a larger proportion of men.

[0003] Drugs currently on the market, such as allopurinol, febuxostat, and benzbromarone, relieve hyperuricemia by inhibiting uric acid production and promoting uric acid excretion, respectively. Even though these drugs can effectively reduce uric acid to a certain extent, they still have limitations in terms of safety, effectiveness, and long-term treatment needs. Dietary control is also one of the methods for treating hyperuricemia, but the limitations of dietary composition and people's pursuit of a diverse diet structure limit the promotion of dietary therapy. Therefore, there is an urgent need for a natural, healthy, and non-toxic product to meet people's needs for lowering uric acid. Summary of the invention

[0004] In order to overcome the shortcomings of existing research, the purpose of the present invention is to provide a heat-killed animal Bifidobacterium lactis subsp. lactis postbiotic and its preparation method and application. The heat-killed animal Bifidobacterium lactis subsp. lactis postbiotic can effectively reduce serum uric acid levels, alleviate kidney and intestinal damage, and regulate the host intestinal flora in the application of HUA animal model, and has the effect of alleviating and treating HUA.

[0005] The technical solution adopted by the present invention to achieve the technical purpose is:

[0006] The present invention provides a method for preparing heat-killed animal Bifidobacterium lactis subsp. postbiotics, comprising:

[0007] The activated culture medium of animal Bifidobacterium lactis subsp. HNU212 was centrifuged to remove the supernatant, and the bacterial concentration was adjusted to 1×10 9CFU / mL, inactivated at 90°C for 15 min, which is the heat-killed animal Bifidobacterium lactis subsp.

[0008] The animal Bifidobacterium lactis subsp. HNU212 has been deposited in the Guangdong Provincial Microbial Culture Collection Center GDMCC, the storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage date is December 13, 2024, the storage number is GDMCC No: 65615, and the classification name is: Bifidobacterium animalissubsp.lactis.

[0009] Preferably, the activated culture fluid is obtained by the following method: animal Bifidobacterium lactis subsp. HNU212 is inoculated with 2% inoculum into 5 mL of fresh liquid culture medium, cultured in an anaerobic incubator at 37° C. for 24 hours, and subcultured three times as culture fluid; the formula per liter of culture medium is: 10.0 g casein digest; 10.0 g beef extract powder; 4.0 g yeast extract powder; 2.0 g triammonium citrate; 5.0 g sodium acetate; 0.2 g magnesium sulfate heptahydrate; 0.05 g manganese sulfate tetrahydrate; 2.0 g dipotassium hydrogen phosphate; 20.0 g glucose; 1.0 g Tween-80; 1 g cysteine ​​hydrochloride; the final pH is 5.7±0.2.

[0010] Preferably, the activated culture solution is centrifuged at 4000 rpm for 5 min to remove the supernatant.

[0011] The present invention also provides a heat-killed animal Bifidobacterium lactis subsp. postbiotic, which is prepared by the above preparation method.

[0012] The present invention further provides the use of the heat-killed animal Bifidobacterium lactis subsp. postbiotics in the preparation of products for relieving or treating hyperuricemia.

[0013] More preferably, the product includes but is not limited to a medicine.

[0014] Preferably, the product reduces serum uric acid, creatinine and urea nitrogen levels, regulates uric acid excretion, and alleviates hyperuricemia by heat-killing animal Bifidobacterium lactis subsp.

[0015] Preferably, the product reduces the production of uric acid by inhibiting the activity of uric acid metabolizing enzymes including purine nucleoside phosphorylase, adenosine deaminase and xanthine oxidase through heat-killed animal Bifidobacterium lactis subsp.

[0016] Preferably, the product reduces the body's inflammatory response and alleviates hyperuricemia by heat-killing animal Bifidobacterium lactis subsp. lactis postbiotics, including reducing the concentrations of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β).

[0017] Preferably, the product reduces hyperuricemia by reducing kidney damage, inflammatory infiltration, kidney fibrosis and intestinal damage through heat-killed animal Bifidobacterium lactis subsp.

[0018] Preferably, the product modulates the structure and composition of the intestinal flora by heat-killed animal Bifidobacterium lactis subsp.

[0019] The beneficial effects of the present invention are:

[0020] The postbiotics of the present invention are prepared from heat-killed animal Bifidobacterium lactis subspecies HNU212, and the postbiotics are more suitable for storage than probiotics, without worrying about the inactivation of microorganisms. The present invention also evaluates the alleviating effect of this postbiotic on hyperuricemia through animal experiments of postbiotic intervention in hyperuricemia. The results showed that HUA mice intervened with postbiotics reduced the production of uric acid by regulating uric acid excretion and inhibiting the activity of uric acid metabolic enzymes, and postbiotics also showed excellent anti-inflammatory effects. In addition, postbiotic intervention also alleviates HUA by reducing kidney and intestinal damage and regulating changes in intestinal flora. The present invention clarifies the mechanism of action of postbiotics in alleviating hyperuricemia, and provides a green, safe, and easy-to-store and process postbiotic, which can be used to prepare related products for lowering uric acid, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Effects of HNU212 postbiotic intervention on serum uric acid, creatinine and urea nitrogen levels in HUA mice.

[0022] Figure 2 Effects of HNU212 postbiotic intervention on the activities of uric acid metabolism-related enzymes in HUA mice.

[0023] Figure 3 Effect of HNU212 postbiotic intervention on inflammatory response in HUA mice.

[0024] Figure 4 Effects of HNU212 postbiotic intervention on kidney and colon damage in HUA mice.

[0025] Figure 5 Effect of HNU212 postbiotic intervention on intestinal microbiota diversity in HUA mice.

[0026] Figure 6 Effect of HNU212 postbiotic intervention on the composition of intestinal flora in HUA mice. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0028] Example

[0029] 1. Preparation of heat-killed animal Bifidobacterium lactis subsp.

[0030] 1. Fermentation medium ingredients

[0031] Culture medium formula (per liter): 10.0 g casein digest; 10.0 g beef extract powder; 4.0 g yeast extract powder; 2.0 g ammonium citrate trihydrate; 5.0 g sodium acetate; 0.2 g magnesium sulfate heptahydrate; 0.05 g manganese sulfate tetrahydrate; 2.0 g potassium dihydrogen phosphate; 20.0 g glucose; 1.0 g Tween-80; 1 g cysteine ​​hydrochloride; final pH 5.7±0.2.

[0032] Usage: Weigh 54g of this product, add 1L of distilled water or deionized water, stir, heat and boil until completely dissolved, divide into test tubes or Erlenmeyer flasks, and sterilize at 121℃ for 15min.

[0033] 2. Activation of Bifidobacterium animalis subsp. lactis HNU212

[0034] Probiotics were inoculated into 5 mL of fresh liquid culture medium at 2% inoculation volume and cultured in an anaerobic incubator at 37°C for 24 h, and subcultured three times as culture medium. The activated culture medium was centrifuged at 4000 rpm for 5 min to remove the supernatant, and the culture concentration was adjusted to 1×10 9 CFU / mL, and obtain HNU212 bacterial suspension.

[0035] 3. Preparation of heat-killed animal Bifidobacterium lactis subsp.

[0036] The HNU212 bacterial suspension prepared in step 2 is inactivated at 90° C. for 15 min to obtain the heat-killed animal Bifidobacterium lactis subsp. postbiotic (HNU212 postbiotic).

[0037] 2. Construction and grouping of HUA mouse models

[0038] 40 6-8 week old SPF grade C57BL / 6J male mice (20-22g) were raised under specific pathogen-free conditions. The mice were fed with a standard laboratory diet, artificial light for 12 hours per day (12 hours of darkness and 12 hours of light alternately), and free access to water. Environmental conditions were controlled at a relative humidity of 55±5% and a temperature of 24+1°C. After one week of adaptive feeding, the mice were randomly divided into a normal group (NC), a model group (MD), an active probiotic intervention group (HNU212), and a postbiotic intervention group (P_HNU212). The grouping scheme is as follows:

[0039] (1) Normal control group (NC): gavage with 0.5% CMC-Na at 8:30 am every day, followed by gavage with an equal volume of saline 1 hour later for 4 weeks;

[0040] (2) HUA model group (MD): potassium oxonate (250 mg / kg) and inosine (375 mg / kg), guanosine (375 mg / kg) were gavaged at 8:30 every day to induce the high nucleoside HUA model (dissolved in 0.5% CMC-Na), and then an equal volume of normal saline was gavaged 1 hour later for 4 weeks;

[0041] (3) Active probiotic intervention group (HNU212): potassium oxonate (250 mg / kg) and inosine (375 mg / kg) and guanosine (375 mg / kg) were administered orally at 8:30 am every day to induce the high nucleoside HUA model. One hour later, 1×10 9 CFU / mL probiotics, for 4 weeks;

[0042] (4) Postbiotic intervention group (P_HNU212): potassium oxonate (250 mg / kg) and inosine (375 mg / kg) and guanosine (375 mg / kg) were administered orally at 8:30 am every day to induce the high nucleoside HUA model. One hour later, 1×10 9 CFU / mL HNU212 postbiotics for 4 weeks.

[0043] After the experiment, the mice were euthanized, and their serum, liver, kidney, intestinal tissue, and feces were collected for relevant measurements.

[0044] Effects of HNU212 postbiotic intervention on HUA mice

[0045] 1. Effect of HNU212 postbiotic intervention on uric acid excretion in HUA

[0046] The mouse blood was centrifuged at 4°C, 3000r / min for 20min to obtain serum, and the serum uric acid, urea nitrogen and creatinine levels were measured using commercial kits. Compared with the model group, HNU212 postbiotic intervention significantly reduced the serum uric acid, creatinine and urea nitrogen levels of HUA mice ( Figure 1, p<0.05). Among them, serum uric acid decreased from 108.10 μmol / L to 54.83 μmol / L, and showed the same action trend as active probiotics. Blood urea nitrogen and creatinine are both commonly used clinical indicators to reflect renal function. When renal function is impaired, both levels increase and uric acid metabolism is unbalanced. The present invention shows that heat-killed animal Bifidobacterium lactis subsp. postbiotics have significant ability to reduce uric acid and regulate uric acid metabolic balance.

[0047] 2. Effect of HNU212 postbiotic intervention on uric acid metabolic enzyme activities in HUA mice

[0048] The liver and the extract were homogenized and centrifuged at a ratio of 1:9 to obtain the supernatant, and the activities of purine nucleoside phosphorylase, adenosine deaminase and xanthine oxidase were determined using commercial kits. Purine nucleoside phosphorylase, adenosine deaminase and xanthine oxidase are involved in the body's purine metabolism and ultimately catalyze the production of uric acid. Inhibiting their active expression can reduce the accumulation of uric acid in the body. In the assay of purine metabolic enzyme activity in the serum and liver of HUA mice, the results showed that compared with the model group, the activity of uric acid metabolic enzymes was significantly reduced after HNU212 postbiotic intervention, among which HNU212 postbiotic intervention reduced adenosine deaminase activity by 67.48%, showing an inhibitory effect better than that of active probiotics (reduction of 48.87%). The results of the present invention show that HNU212 postbiotic intervention can directly / indirectly inhibit the production of uric acid. ( Figure 2 , p<0.05).

[0049] 3. Effect of HNU212 postbiotic intervention on inflammatory response in HUA mice

[0050] Serum lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β) were measured using commercial kits. High levels of uric acid can promote the expression of inflammatory proteins by triggering complex proinflammatory cascades that damage cells and tissues, thereby inducing an inflammatory response in the body. Compared with the model group, after HNU212 postbiotic intervention, the inflammatory expression of HUA mice decreased, including lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β) concentrations decreased by 18.77%, 22.98%, and 13.69%, respectively ( Figure 3 , p<0.05).

[0051] 4. Effects of HNU212 postbiotic intervention on kidney and colon damage in HUA mice

[0052] The animal kidney and colon tissues were fixed in 4% paraformaldehyde fixative at 4°C, stained with hematoxylin and eosin, and the stained sections were scanned to observe tissue damage. The kidney plays an important role in maintaining serum uric acid levels through the reabsorption and excretion of uric acid through a complex transport system. When the renal excretion pathway is damaged, uric acid is mainly eliminated through the extrarenal excretion pathway as a compensatory mechanism. From the results of renal H&E and MASSON staining, it can be found that HNU212 postbiotic intervention can reduce kidney damage, inflammatory infiltration and renal fibrosis in HUA mice. The same results were also shown in colon H&E staining. Postbiotic intervention alleviated intestinal damage in HUA ( Figure 4 ).

[0053] 5. Effect of HNU212 postbiotic intervention on intestinal flora diversity in HUA mice

[0054] Fecal samples of mice were collected, and total microbial genomic DNA was extracted from the fecal samples, followed by 16SrRNA gene sequencing. The intestine is responsible for 1 / 3 of the total uric acid excretion. When uric acid is secreted into the intestine, it is rapidly metabolized by components of the intestinal microbiota. Studies have shown that intestinal microbiota plays an important role in alleviating HUA by promoting the catabolism of purine and uric acid and reducing the intestinal absorption of purine and uric acid. Compared with the model group, after postbiotic intervention, the experimental groups and the control group (NC) were divided into different clusters in the principal component analysis (PCA) at the genus level, and the clusters of the postbiotic group (P_HNU212) tended to the NC group, indicating that the structure of the intestinal flora changed between different groups. The results of partial least squares discriminant analysis (PLS-DA) showed that the samples could be clearly distinguished and clustered into different clusters, indicating that the composition of the intestinal flora of mice in different experimental groups was significantly different, and the changes in the community structure of the intestinal flora of HUA mice with postbiotic intervention tended to the NC group ( Figure 5 ).

[0055] 6. Effect of HNU212 postbiotic intervention on the composition of intestinal flora in HUA mice

[0056] Stability and resilience are the characteristics of healthy intestinal flora. When external factors exceed tolerance, ecological imbalance may occur. HUA is one of the external factors. The results of the present invention showed that compared with the NC group, the HUA caused by high nucleosides in the model group increased the relative abundance of Bacteroidetes at the phylum level and reduced Firmicutes. This change was reversed after postbiotic intervention ( Figure 6 ). In addition, the composition of the intestinal microbiota of each group was analyzed at the genus level, and it was found that norank_f_Muribaculaceae and Prevotellaceae UCG-001, as the dominant species, affected the intestinal flora of HUA mice ( Figure 6 ).

[0057] The above results indicate that HNU212 postbiotic intervention can change the structure and composition of the intestinal flora of HUA mice, participate in the pathogenesis of hyperuricemia through multiple mechanisms, and may become one of the targets for alleviating and treating hyperuricemia and related diseases.

[0058] The heat-killed animal Bifidobacterium lactis subspecies HNU212 postbiotic prepared by the present invention has been shown in in vivo animal experiments to be able to alleviate the symptoms of hyperuricemia, mainly by regulating uric acid excretion and inhibiting the activity of uric acid metabolic enzymes; reducing the expression of anti-inflammatory factors; alleviating kidney inflammation, fibrosis and intestinal damage; and improving intestinal flora disorders.

[0059] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a heat-killed animal Bifidobacterium lactis subsp. postbiotic, comprising: The activated culture medium of animal Bifidobacterium lactis subsp. HNU212 was centrifuged to remove the supernatant, and the bacterial concentration was adjusted to 1×10 9 CFU / mL, inactivated at 90°C for 15 min, which is the heat-killed animal Bifidobacterium lactis subsp. The animal Bifidobacterium lactis subsp. HNU212 has been deposited in the Guangdong Provincial Microbial Culture Collection Center GDMCC, the storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage date is December 13, 2024, the storage number is GDMCC No: 65615, and the classification name is: Bifidobacterium animalissubsp.lactis.

2. The preparation method according to claim 1, characterized in that: The activated culture fluid is obtained by the following method: animal Bifidobacterium lactis subsp. HNU212 is inoculated in 5 mL of fresh liquid culture medium at a 2% inoculation rate, cultured in an anaerobic incubator at 37° C. for 24 hours, and subcultured three times as culture fluid; the formula of each liter of culture medium is: 10.0 g of casein digest; 10.0 g of beef extract powder; 4.0 g of yeast extract powder; 2.0 g of triammonium citrate; 5.0 g of sodium acetate; 0.2 g of magnesium sulfate heptahydrate; 0.05 g of manganese sulfate tetrahydrate; 2.0 g of potassium hydrogen phosphate; 20.0 g of glucose; 1.0 g of Tween-80; 1 g of cysteine ​​hydrochloride; and the final pH is 5.7±0.

2.

3. The preparation method according to claim 1, characterized in that: The activated culture solution was centrifuged at 4000 rpm for 5 min to remove the supernatant.

4. A heat-killed animal Bifidobacterium lactis subsp. postbiotic, obtained by the preparation method according to any one of claims 1 to 3.

5. Use of the heat-killed animal Bifidobacterium lactis subsp. lactis postbiotics according to claim 4 in the preparation of a product for alleviating or treating hyperuricemia.

6. The use according to claim 5, characterized in that: The product reduces serum uric acid, creatinine and urea nitrogen levels, regulates uric acid excretion and relieves hyperuricemia through heat-killed animal Bifidobacterium lactis subsp.

7. The use according to claim 5, characterized in that: The product reduces the generation of uric acid by inhibiting the activity of uric acid metabolic enzymes, including purine nucleoside phosphorylase, adenosine deaminase and xanthine oxidase, through heat-killed animal Bifidobacterium lactis subspecies postbiotics.

8. The use according to claim 5, characterized in that: The product reduces the body's inflammatory response and relieves hyperuricemia by using heat-killed animal Bifidobacterium lactis subsp. lactis postbiotics, including reducing the concentrations of lipopolysaccharide, tumor necrosis factor-α and interleukin 1β.

9. The use according to claim 5, characterized in that: The product reduces hyperuricemia by reducing kidney damage, inflammatory infiltration, kidney fibrosis and intestinal damage through heat-killed animal Bifidobacterium lactis subsp.

10. The use according to claim 5, characterized in that: The product regulates the structure and composition of intestinal flora through heat-killed animal Bifidobacterium lactis subsp. postbiotics.

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