Cactus juice-based post-generation fermentation product as well as preparation method and application thereof

Abstract

The invention provides a cactus fruit juice-based post-generation fermentation product and a preparation method and application thereof, and the preparation method of the cactus fruit juice-based post-generation fermentation product comprises the following steps: inoculating a bacterial suspension of plant lactobacillus HNU082 into pasteurized cactus fruit juice, and culturing in a bacterial incubator at 37 DEG C for 48 hours to obtain the cactus fruit juice-based post-generation fermentation product. And then carrying out instantaneous high-temperature sterilization on the cactus fruit fermented juice to prepare the cactus fruit juice-based post-generation fermented product. The postbiotic fermentation product can regulate the uric acid homeostasis in the HUA mouse body by regulating the generation and excretion of uric acid, and reduces kidney injury caused by HUA; the HUA of mice can be improved by increasing the level of short-chain fatty acid and adjusting disordered metabolic profiles and amino acid metabolic pathways. The metabiotics fermentation product provided by the invention is high in safety and strong in stability, can be absorbed and utilized by an organism, can be applied and developed into corresponding functional products, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of hyperuricemia treatment, and relates to a cactus juice-based postbiotic fermentation product and a preparation method and application thereof. Background Art

[0002] Hyperuricemia (HUA) is a serious metabolic disorder that causes chronic inflammation through the deposition of urate crystals in tissues, and causes chronic kidney disease and gout. In addition, HUA is closely related to a variety of metabolic syndromes such as obesity, hypertension, diabetes and atherosclerosis. High-purine foods, including seafood, animal offal and beer, increase the risk of HUA.

[0003] Uric acid (UA) is the final product of purine metabolism and is excreted through the kidneys (two-thirds) and intestines (one-third) to maintain the homeostasis of purine metabolism in the human body. Intestinal metabolites play a vital role in human health and disease. These metabolites are mainly produced by the intestinal microbiota and affect the physiological functions and immune system of the host through various mechanisms. Studies have shown that intestinal metabolites have great potential in the prevention and treatment of various diseases. For example, short-chain fatty acids and tryptophan metabolites can enhance anti-tumor immune responses and slow down tumor growth. In addition, intestinal metabolites are also involved in regulating the occurrence and development of various metabolic diseases such as obesity, diabetes, cardiovascular disease and HUA. Its mechanism of action involves multiple aspects such as immune regulation, lipid metabolism, inflammation control, and barrier function maintenance. Summary of the invention

[0004] Based on the above, the present invention aims to provide a cactus juice-based postbiotic fermentation product and a preparation method and application thereof. The postbiotic fermentation product has high safety, strong stability, can be better absorbed and utilized by the body, can be used to develop corresponding functional products, and has broad application prospects.

[0005] In order to achieve the technical purpose of the present invention, the present invention adopts the following technical solutions:

[0006] The invention provides a preparation method of a cactus juice-based postbiotic fermentation product, comprising: inoculating a bacterial suspension of Lactobacillus plantarum HNU082 into pasteurized cactus juice (Opuntia ficus-indica Juice, OFIJ), culturing in a 37°C bacterial incubator for 48 hours, and then instantaneously high-temperature sterilizing the cactus fruit fermented juice to obtain the cactus juice-based postbiotic fermentation product.

[0007] Preferably, the bacterial suspension of Lactobacillus plantarum HNU082 is inoculated into the pasteurized cactus juice at an inoculation rate of 4% (v / v, volume percentage); the pasteurization conditions are: 85° C., 15 min; the instantaneous high temperature sterilization conditions are 105° C., 20 min.

[0008] The present invention also provides a cactus juice-based postbiotic fermentation product, which is prepared by any of the above preparation methods.

[0009] The present invention further provides the use of the cactus juice-based postbiotic fermentation product in preparing a product for alleviating or treating hyperuricemia.

[0010] More preferably, the product includes but is not limited to a medicine.

[0011] Preferably, the product reduces serum uric acid (SUA), serum urea nitrogen (BUN) and serum creatinine (SCr) levels by means of cactus juice-based postbiotic ferment.

[0012] Preferably, the product inhibits the production of uric acid by inhibiting the activities of adenosine deaminase (ADA) and xanthine oxidase (XOD) by the cactus juice-based postbiotic ferment.

[0013] Preferably, the product reduces kidney inflammation and kidney damage via cactus juice-based postbiotic ferment.

[0014] Preferably, the product restores the expression of uric acid transporter mRNA through cactus juice-based postbiotic fermentation products to maintain uric acid homeostasis.

[0015] Preferably, the product promotes the production of SCFAs by cactus juice-based postbiotic fermentation.

[0016] Preferably, the product regulates the disturbed metabolic profile and amino acid metabolic pathway through cactus juice-based postbiotic fermentation.

[0017] The beneficial effects of the present invention are:

[0018] A cactus juice-based postbiotic fermentation product prepared by the present invention can regulate uric acid homeostasis in HUA mice by regulating the production and excretion of uric acid, thereby reducing kidney damage caused by HUA. In addition, postbiotic fermentation broth supplements improve HUA in mice by increasing SCFAs levels, regulating disordered metabolic profiles and amino acid metabolic pathways. Compared with traditional drug treatments, postbiotic fermentation products are highly safe, stable, and more absorbable by the body. They can be used to develop corresponding functional products and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1OFIBP intervention in the present invention reduces serum uric acid, urea nitrogen and creatinine levels in HUA mice.

[0020] Figure 2 In the embodiment of the present invention, OFIBP intervention reduces the activity of enzymes related to uric acid synthesis in HUA mice.

[0021] Figure 3 In the embodiment of the present invention, OFIBP intervention alleviates the renal inflammatory response of HUA mice.

[0022] Figure 4 In the examples of the present invention, OFIBP intervention alleviates renal damage in HUA mice.

[0023] Figure 5 In the embodiment of the present invention, OFIBP intervention regulates the expression of genes related to uric acid metabolism homeostasis in HUA mice.

[0024] Figure 6 In the examples of the present invention, OFIBP intervention promotes the production of SCFAs in HUA mice.

[0025] Figure 7 Metabolic profile of HUA mice regulated by OFIBP intervention in the examples of the present invention.

[0026] Figure 8 In the embodiment of the present invention, OFIBP intervention predicted the amino acid metabolic pathway of HUA mice.

[0027] Fig. 9 In the embodiments of the present invention, OFIBP intervenes and regulates the amino acid metabolism of HUA mice. DETAILED DESCRIPTION

[0028] 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.

[0029] Example

[0030] 1. Preparation of postbiotic fermentation broth

[0031] Lactobacillus plantarum HNU082 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on March 9, 2021, with a deposit number of GDMCC NO: 61552, and a deposit address of 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, China. The taxonomic name is Lactobacillus plantarum. For specific public records, see patent CN202110619723.X, a strain of Lactobacillus plantarum HNU082 and its uses.

[0032] After activation, Lactobacillus plantarum HNU082 was inoculated into fresh MRS liquid culture medium at a 2% inoculum volume and cultured in a bacterial incubator at 37°C for 24 hours. The activated culture medium was centrifuged at 4000 rpm for 5 minutes to remove the supernatant, washed three times with 0.9% sterile saline and prepared into 1*10 9 The bacterial suspension of CFU / mL was inoculated into the pasteurized (85°C, 15min) cactus juice at an inoculation rate of 4% (v / v), and cultured in a 37°C bacterial incubator for 48h. The cactus fruit fermented juice was then sterilized at high temperature instantaneously to obtain the cactus juice-based postbiotic fermentation product.

[0033] MRS liquid culture medium: The formula of each liter of culture medium is: 10.0g casein digest; 10.0g beef extract powder; 4.0g yeast extract powder; 2.0g triammonium citrate; 5.0g sodium acetate; 0.2g magnesium sulfate heptahydrate; 0.05g manganese sulfate tetrahydrate; 2.0g dipotassium hydrogen phosphate; 20.0g glucose; 1.0g Tween-80.

[0034] 2. Construction of HUA Mouse Model

[0035] 40 6-8 week old C57BL / 6J male mice (20-22g) were selected and raised under specific pathogen-free conditions. The mice were given standard feed, free drinking water and artificial light for 12 hours (12 hours of darkness and 12 hours of light alternately) every day. The environmental conditions were controlled at a temperature of 24±1°C and a relative humidity of 55±5%. After 7 days of adaptive feeding, the 40 mice were randomly divided into 5 groups (n=8 / group): normal group (CON), model group (MOD), cactus juice intervention group (OFI), cactus juice-based postbiotic intervention group (OFIBP) and allopurinol intervention group (ALP). The modeling and intervention schemes are as follows:

[0036] (1) Normal control group (CON): gavage with 0.5% CMC-Na solution at 9:30 am every day, followed by gavage with an equal volume of saline 1 hour later for 4 weeks;

[0037] (2) HUA model group (MOD): The mice were gavaged with potassium oxonate (300 mg / kg·BW) and adenine (100 mg / kg·BW) solution at 9:30 am every day, and then gavaged with normal saline 1 hour later for 4 weeks;

[0038] (3) Cactus juice intervention group (OFI): Oral gavage of potassium oxonate (300 mg / kg·BW) and adenine (100 mg / kg·BW) solution at 9:30 am every day, and then oral gavage of an equal volume of OFI 1 hour later for 4 weeks;

[0039] (4) Cactus juice-based postbiotic intervention group (OFIBP): Oral administration of potassium oxadiazine (300 mg / kg·BW) and adenine (100 mg / kg·BW) solution at 9:30 am every day, followed by oral administration of an equal volume of OFIBP 1 hour later, for 4 weeks;

[0040] (5) Allopurinol intervention group (ALP): mice were gavaged with potassium oxonate (300 mg / kg·BW) and adenine (100 mg / kg·BW) solution at 9:00 a.m. every day, and then gavaged with an equal volume of allopurinol (10 mg / kg·BW) 1 h later for 4 weeks. After the intervention period, mice were euthanized, and serum, cecal contents, feces, liver tissue, colon and kidney tissue were collected and stored at -80°C for later use.

[0041] 3. Detection of the intervention effect of cactus juice-based postbiotic fermentation on HUA mice

[0042] 1. Detection of serum indicators

[0043] The blood of mice was collected and centrifuged at 3000 r / min and 4°C for 15-20 min to obtain serum. The serum uric acid, creatinine and urea nitrogen levels of HUA mice were detected using commercial kits to evaluate the uric acid-lowering effect of OFIBP supplementation on HUA mice.

[0044] 2. Renal tissue pathological analysis and inflammatory factor detection

[0045] Kidney tissues were collected and fixed with 4% paraformaldehyde solution, then dehydrated with graded ethanol, embedded in paraffin, made into 4 μm paraffin sections, stained with hematoxylin-eosin (H&E) and Masson's trichrome, and the morphology of tissue sections was observed under a microscope.

[0046] The kidney tissue was quickly frozen with liquid nitrogen for later use. After the tissue was thawed, the temperature was maintained at 2-8°C. 9 volumes of PBS (pH 7.4) were added at a weight-to-volume ratio of 1g:9ml. The tissue was thoroughly homogenized with a homogenizer and then centrifuged at 2000-3000r / min for 20min. The supernatant was collected and renal inflammatory factors were detected using a commercial kit to analyze the alleviating effect of OFIBP supplementation on renal injury in HUA mice.

[0047] 3. RT-qPCR detection

[0048] Total RNA was extracted from kidney tissue, RNA concentration and purity were tested, and total RNA was reverse transcribed into cDNA. RT-qPCR was performed using the SYBR Green method, with β-actin as the internal reference gene and 2- ΔΔCT The mRNA expression level of the target gene was calculated by this method.

[0049] 4. Detection of short-chain fatty acids (SCFAs) concentration

[0050] After freeze-drying the colon contents and feces, saturated sodium chloride was added for homogenization. After homogenization, 10% sulfuric acid (v / v) was added for acidification. Subsequently, 800 μL of n-hexane was added to the sample to extract SCFAs. After standing for 30 minutes, centrifuged at 12000 rpm for 15 minutes, anhydrous sodium sulfate was added to remove residual water, and the supernatant was centrifuged and filtered through a 0.22 μm filter membrane. The SCFAs content of the sample was detected by gas chromatography.

[0051] The contents of SCFAs in the colonic contents and feces of mice were analyzed by an Agilent 8890 gas chromatography system (Agilent, Santa Clara, CA, USA) equipped with a HP-INNOWAX column (30 m × 0.320 mm × 0.50 μm, Agilent, Santa Clara, CA, USA) and a flame ionization detector (Agilent, Santa Clara, CA, USA).

[0052] 5. Non-targeted metabolomics analysis

[0053] The cecal contents were homogenized and centrifuged to collect the supernatant, which was analyzed using a Vanquish UHPLC system equipped with an Orbitrap Q ExactiveTM HF-X mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Metabolites were predicted by molecular ion peaks and fragment ions, and then matched with databases (including mzVault and MassList) to obtain accurate qualitative results.

[0054] 6. Targeted amino acid metabolomics analysis

[0055] The cecal contents were homogenized and centrifuged to collect the supernatant, and the amino acids in the samples were qualitatively and quantitatively determined using LC-ESI-MS / MS (UHPLC-Qtrap) (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic column was dvanceBio MS Spent Media (2.1×50mm, 2.7μm), the column temperature was 40°C, and the injection volume was 1μL. The mobile phases were mobile phase A (0.1% formic acid, 10mM ammonium formate, 95% aqueous solution) and mobile phase B (0.1% formic acid, 10mM ammonium formate, 95% acetonitrile solution), with a flow rate of 5min / mL. The elution gradient was: 0-3min, 10% A; 3-4min, 40% A; 4-6min, 10% A. The mechanism of OFIBP supplementation in alleviating HUA mice was explored from the perspective of intestinal metabolites.

[0056] IV. Results Analysis

[0057] 1. OFIBP intervention reduces the levels of serum uric acid and kidney injury indicators in HUA mice

[0058] Depend on Figure 1 As shown in A, the serum uric acid (SUA) level of HUA mice in the CON group (51.88±8.9μmol / L) was significantly lower than that in the MOD group (106.02±22.02μmol / L), indicating that the HUA model was successfully established. In addition, OFIBP and OFI intervention significantly reduced the UA level in HUA mice (p<0.05). Serum urea nitrogen (BUN) and serum creatinine (SCr) are mainly used to evaluate renal function. When their contents are abnormal, renal function is impaired or metabolism is abnormal. Compared with the MOD group, the intake of OFIBP (reduced by 41.91% and 43.06%), OFI (reduced by 45.78% and 35.24%), and allopurinol (reduced by 71.14% and 54.61%) reduced SCr and BUN levels, respectively. The results showed that supplementation of OFIBP can alleviate HUA induced by potassium oxonate and adenine ( Figure 1 ).

[0059] 2. OFIBP intervention reduces the activity of uric acid synthesis-related enzymes in HUA mice

[0060] Adenosine deaminase (ADA) and xanthine oxidase (XOD) are key enzymes in the purine metabolism process and affect the production of UA. Compared with the CON group, the serum ADA and liver XOD activities of HUA mice in the MOD group were significantly increased (p<0.05), and the enzyme activities were significantly decreased after supplementation with OFIBP. Compared with the OFI group, the OFIBP group had a more significant inhibitory effect on ADA and XOD activities (p<0.05). The results showed that supplementation with OFIBP could inhibit the production of UA by inhibiting ADA and XOD activities ( Figure 2 ).

[0061] 3. OFIBP intervention alleviates the inflammatory response in HUA mice

[0062] Lipopolysaccharide (LPS), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) play a key role in inflammatory response and immune regulation, and can reflect the degree of inflammation in the body. Compared with the CON group, the levels of LPS, IL-6 and TNF-α in HUA mice in the MOD group increased by 21.42%, 14.86% and 31.34%, respectively (p<0.05). Compared with the MOD group, the levels of LPS, IL-6 and TNF-α in the OFIBP group decreased by 22.77%, 31.32% and 28.86%, respectively (p<0.05). The OFIBP group had a more significant effect than the OFI group in alleviating renal injury. The results showed that OFIBP can reduce renal inflammation and injury in HUA mice ( Figure 3 ).

[0063] 4. OFIBP intervention alleviates renal damage in HUA mice

[0064] The histopathological analysis of OFIBP on the improvement of renal injury in HUA mice showed that the size and morphology of the glomeruli in the CON group mice were normal, with clear boundaries and intact brush border structures in the renal tubules ( Figure 4 ). The MOD group mice had severe renal tissue damage. The renal damage in each intervention group was improved, especially after supplementation with OFIBP. However, supplementation with OFI could not completely improve glomerular atrophy and the brush structure of the proximal tubules, and supplementation with ALP could not completely restore the infiltration of inflammatory cells in mice. Masson trichrome staining showed that severe renal fibrosis was observed in the tubular interstitium of the MOD group mice, which was improved after intake of OFBP. Therefore, supplementation with OFIBP can alleviate renal damage.

[0065] 5. OFIBP intervention regulates the expression of genes related to uric acid metabolism homeostasis in HUA mice

[0066] Glucose transporter 9 (GLUT9), ATP-binding cassette subfamily G member 2 (ABCG2), and organic anion transporter 1 (OAT1) are involved in the transport of UA in the body. Among them, abnormal expression of GLUT9 will promote the reabsorption of UA, leading to increased UA content, and abnormal expression of ABCG2 and OAT1 will also promote UA secretion. The results of RT-qPCR of renal tissue showed that compared with the CON group, the mRNA expression level of GLUT9 in the kidney of the MOD group was significantly increased ( Figure 5 A, p<0.05). In contrast, the mRNA expression levels of ABCG2 and OAT1 were significantly decreased ( Figure 5BC, p<0.05). OFIBP intervention restored the mRNA expression of GLUT9 in HUA mice to normal levels, and the mRNA expression levels of ABCG2 in OFIBP, OFI, and ALP groups increased by 49.80%, 40.94%, and 102.23% compared with the MOD group. The results showed that OFIBP could restore the expression of UA transporter mRNA and maintain UA homeostasis.

[0067] 6. OFIBP intervention promotes SCFAs production in HUA mice

[0068] SCFAs play an important role in maintaining intestinal health, regulating immune and metabolic functions, and preventing a variety of diseases. Figure 6 As shown in the figure, the total SCFAs level in the colon contents of HUA mice in the MOD group was significantly lower than that in the CON group. Compared with the MOD group, the total SCFAs levels in the OFIBP and ALP groups were significantly increased, among which the levels of propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, and hexanoic acid in the OFIBP group were significantly increased. The trend of SCFAs levels in feces was consistent with that in colon contents, but the total SCFAs content decreased slightly. The results showed that supplementation with OFIBP could promote the production of SCFAs in the intestine of HUA mice.

[0069] 7. OFIBP intervention regulates the metabolic profile of HUA mice

[0070] The cecal contents of mice were collected and their metabolite components were identified. PCA, partial least squares discriminant analysis (PLS-DA) and orthogonal projection latent structure discriminant analysis (OPLS-DA) were used to analyze the metabolite categories. OPLS-DA was used as a supervised model to screen differential metabolites through 7-fold cross validation and variable impact on projection (VIP)>1. Based on the KEGG database, the important differential metabolites in the metabolic pathways were analyzed.

[0071] The results of the present invention show that in ESI+ and ESI- ions, PCA and PLS-DA analysis found that there were significant differences in metabolites between the groups ( Figure 7 AB). To further determine the overall metabolic changes between the MOD and OFIBP groups, OPLS-DA analysis was performed, and the results showed that the two groups of samples were significantly separated, indicating that OFIBP intervention significantly altered the metabolic profile of HUA mice ( Figure 7 C). S-plot analysis to identify differential metabolites in the model and normal mice, each dot represents a metabolite ( Figure 7 D) As can be seen from the figure, the metabolic profile of HUA model mice induced by potassium oxonate and adenine was significantly changed.

[0072] 8. OFIBP intervention regulates amino acid metabolism in HUA mice

[0073] like Figure 8 As shown in A, to further determine the overall metabolic changes between the MOD group and the OFIBP group, OPLS-DA analysis showed that there were significant differences between the two groups, and supplementation of OFIBP significantly changed the metabolic profile of HUA mice. According to KEGG annotation and enrichment analysis, the differential metabolites were mainly concentrated in amino acid metabolism. A total of 33 differential metabolites were screened out from the enriched amino acid metabolites, of which 14 differential metabolites were upregulated in the MOD group and 19 differential metabolites were upregulated in the OFIBP group. Further analysis of the enrichment pathways of the differential metabolites showed that they were mainly involved in tyrosine metabolism, glycine, serine and threonine metabolism, valine, leucine and isoleucine biosynthesis, and tryptophan metabolism, etc. ( Figure 8 B).

[0074] To further investigate the effects of amino acid metabolism on HUA, 21 amino acids were quantitatively analyzed. Fig. 9 As shown in A, OPLS-DA analysis found significant differences among the CON, MOD, and OFIBP groups. In addition, S-plot analysis showed differential expression of 21 amino acids, and asparagine was considered to be the most significantly different metabolite among the three groups ( Fig. 9 B). Compared with the CON group, the total amino acid content of the MOD group was increased. In contrast, after the intake of OFIBP, the total amino acid content of HUA mice decreased by 18.07% ( Fig. 9 CD). Among them, except L-aspartate, 18 amino acids were enriched in MOD mice and changed after OFIBP intervention. These results indicate that OFIBP intervention can regulate the disordered metabolic profile and amino acid metabolic pathways in HUA mice.

[0075] In conclusion, the present invention shows that supplementation with OFIBP can alleviate HUA by inhibiting the production of UA by inhibiting the activity of XOD and ADA, regulating the expression of UA transporters and promoting the excretion of UA. Meanwhile, supplementation with OFIBP also regulates intestinal-related amino acid metabolism to alleviate HUA in mice. OFIBP supplementation is more effective than OFI supplementation in alleviating HUA.

[0076] 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 cactus juice-based postbiotic fermentation product, comprising: The bacterial suspension of Lactobacillus plantarum HNU082 was inoculated into the pasteurized cactus juice and cultured in a bacterial incubator at 37°C for 48 hours. The cactus fruit fermented juice was then sterilized at high temperature instantaneously to obtain a cactus juice-based postbiotic fermentation product.

2. The preparation method according to claim 1, characterized in that: The bacterial suspension of Lactobacillus plantarum HNU082 was inoculated into the pasteurized cactus juice at an inoculation rate of 4%; the pasteurization conditions were: 85° C., 15 min; the instantaneous high-temperature sterilization conditions were 105° C., 20 min.

3. A cactus juice-based postbiotic fermentation product, prepared by the preparation method according to claim 1 or 2.

4. Use of the cactus juice-based postbiotic fermentation product according to claim 3 in the preparation of a product for alleviating or treating hyperuricemia.

5. The use according to claim 4, characterized in that: The product reduces serum uric acid, serum urea nitrogen and serum creatinine levels through cactus juice-based postbiotic fermentation products.

6. The use according to claim 4, characterized in that: The product inhibits the production of uric acid by inhibiting the activities of adenosine deaminase and xanthine oxidase through cactus juice-based postbiotic fermentation.

7. The use according to claim 4, characterized in that: The product reduces kidney inflammation and kidney damage through cactus juice-based postbiotic ferments.

8. The use according to claim 4, characterized in that: The product restores the expression of uric acid transporter mRNA through cactus juice-based postbiotic fermentation products to maintain uric acid homeostasis.

9. The use according to claim 4, characterized in that: The product promotes the production of SCFAs through cactus juice-based postbiotic fermentation.

10. The use according to claim 4, characterized in that: The product regulates the disordered metabolic profile and amino acid metabolic pathway through cactus juice-based postbiotic fermentation products.

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