Preparation method and application of fermented rhizoma smilacis glabrae extract

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a preparation method and application of a fermented rhizoma smilacis glabrae extract. The invention relates to a fermented rhizoma smilacis glabrae extract, which is characterized in that a lactobacillus rhamnosus LR.M8 strain is utilized, the inhibition effect of the rhizoma smilacis glabrae extract on xanthine oxidase is improved through fermentation, and the treatment effect of the rhizoma smilacis glabrae extract on hyperuricemia is improved. The solution for improving the effect of the extract through biological fermentation is provided for solving the problems that the content of effective components in common medicinal and edible extracts is low, and the human body utilization rate is not high. The lactobacillus rhamnosus LR.M8 can effectively improve the adjusting effect of the rhizoma smilacis glabrae medicine and food homologous plant extract on uric acid balance, can reduce damage of inflammation to cells, and promotes in-vivo purine metabolism to recover balance. The fermentation method provided by the invention is simple and remarkable in effect, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological fermentation, and particularly relates to a preparation method and application of a fermented Smilax glabra Roxb. extract. BACKGROUND

[0002] In the process of uric acid metabolism, xanthine oxidase (XOD) plays a key role. This enzyme, mainly distributed in the liver, can catalyze the oxidation of hypoxanthine and xanthine to generate uric acid, and is a rate-limiting enzyme for regulating the biosynthesis of uric acid. Currently, the commonly used uric acid-lowering drugs such as allopurinol and febuxostat can effectively inhibit the activity of XOD and reduce the generation of uric acid, but they generally have adverse reactions such as gastrointestinal discomfort, skin allergy, abnormal liver function, blood system toxicity, and rare severe hypersensitivity reactions. Safer and less side-effect anti-gout functional foods can improve this problem.

[0003] The polyphenols (quercetin) and polysaccharides in Smilax glabra Roxb. can inhibit the activity of XOD and promote the excretion of uric acid, thereby reducing the levels of uric acid, creatinine and urea nitrogen. However, the active substances extracted by water extraction have no obvious effect on reducing uric acid. Under the catalysis of the enzyme system (esterase, hydrolase) produced by lactic acid bacteria, the components in Smilax glabra Roxb. can react to produce new active ingredients. At the same time, the macromolecular substances in Smilax glabra Roxb. are decomposed into small molecular active ingredients after fermentation, which are more easily absorbed and utilized by the human body.

[0004] Lactobacillus plantarum LR.M8 is isolated from food, which has been disclosed in the invention patent with the publication number CN 101671634 A Lactobacillus plantarum M8, Lactobacillus plantarum SLP and a preparation method thereof. SUMMARY

[0005] In view of the above problems, the application provides a preparation method and application of a fermented Smilax glabra Roxb. extract, which increases the key active substances in Smilax glabra Roxb. that play a role in reducing uric acid through lactic acid bacteria fermentation, and solves the health problem that macromolecular substances in Smilax glabra Roxb. are not easily absorbed and utilized by the human body.

[0006] The application discloses a preparation method of a Smilax glabra Roxb. fermentation product, which comprises the following steps:

[0007] (1) Lactobacillus rhamnosus LR. M8 strain activation: Lactobacillus rhamnosus LR. M8 strain is inoculated in MRS liquid medium, and is cultured at 37°C for 24 hours to obtain Lactobacillus rhamnosus LR. M8 liquid; the preservation number of the Lactobacillus rhamnosus LR. M8 is CGMCC No. 3002, which is preserved in China General Microbiological Culture Collection Center on April 7, 2009.

[0008] (2) Preparation of Smilax riparia liquid fermentation substrate: after the raw material of Smilax riparia is crushed, water is added, and the mass ratio of the raw material of Smilax riparia to water is 1:6-7; 60°C water extraction is repeated for 3 times for 1 hour to obtain preliminary extraction liquid, which is concentrated into extract semi-finished product, and the specific gravity d of the extract semi-finished product is 1.14-1.17; auxiliary material malt dextrin is added, and the mass of the malt dextrin is 30% of the mass of the extract semi-finished product; the Smilax riparia extraction powder is prepared by powder spraying drying, crushing and sieving; and the Smilax riparia extraction powder is mixed with water and sterilized to obtain the liquid fermentation substrate;

[0009] (3) Preparation of Smilax riparia fermentation product: the Lactobacillus rhamnosus LR. M8 liquid is inoculated in the liquid fermentation substrate, and the Smilax riparia fermentation product is obtained by fermentation.

[0010] Preferably, in step (1), the inoculation amount of the Lactobacillus rhamnosus LR. M8 strain is 1-2% of the mass of the MRS liquid medium; the components of the MRS liquid medium are as follows: casein peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 5.0 g, sodium acetate 5.0 g, citric acid diamine 2.0 g, Tween 80 1.0 g, potassium phosphate dibasic 2.0 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate heptahydrate 0.05 g, calcium carbonate 20.0 g, and distilled water 1.0 L, and the pH is 6.8.

[0011] Preferably, in step (2), the mass ratio of the Smilax riparia extraction powder to water is 1:2.

[0012] Preferably, in step (2), the mass ratio of the Lactobacillus rhamnosus LR. M8 to the liquid fermentation substrate is 3:100.

[0013] Preferably, the fermentation temperature is 37 DEG C; the fermentation time is 96h.

[0014] Preferably, the effective component of the Smilax glabra fermentation product includes 5, 7-dimethoxy coumarin.

[0015] Application of a Smilax glabra fermentation product in improving the uric acid lowering performance of health food.

[0016] Application of a Smilax glabra fermentation product in preparing uric acid lowering medicine.

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

[0018] The present application provides a Lactobacillus rhamnosus LR.M8, which can effectively improve the uric acid lowering performance of Smilax glabra extract by fermenting Smilax glabra, and the reason is that the Lactobacillus rhamnosus LR.M8 can effectively improve the inhibition of xanthine oxidase by the extract during fermentation, according to non-target metabolic detection, the fermented Smilax glabra extract exhibits a significant uric acid lowering effect, and effectively enhances the uric acid lowering performance of a kind of medicinal and edible plant. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The inhibition rate of unfermented and fermented Smilax glabra extract on XOD.

[0020] Figure 2 The OPLS-DA score plot of Smilax glabra before and after lactic acid bacteria fermentation of the present application; wherein the horizontal coordinate PC1 represents the first principal component score value, and the vertical coordinate OC2 represents the first orthogonal component score value. The point represents the experimental sample, and the color represents different groups. Among them, T13 is Smilax glabra, and T46 is fermented Smilax glabra.

[0021] Figure 3 The volcano plot of the differential metabolites of Smilax glabra before and after lactic acid bacteria fermentation of the present application, wherein the horizontal coordinate represents the logarithm value of the Log2 of the quantitative value difference of a certain metabolite in two samples; the vertical coordinate represents the-log10 of the P value, and each point in the figure represents a metabolite. The larger the absolute value of the horizontal coordinate, the greater the fold difference of the expression amount of the certain metabolite between the two samples; the larger the vertical coordinate value, the more significant the difference expression, and the more reliable the screened differential expression metabolites. The size of the point represents the size of the VIP value, and the red point represents the differential up-regulation, the blue point represents the differential down-regulation, and the gray point represents the metabolite that does not meet the differential screening condition.

[0022] Figure 4is a differential metabolite clustering heat map of the Smilax glabra before and after lactic acid bacteria fermentation of the present application, wherein the columns represent samples, the rows represent metabolites, the clustering tree on the left is the differential metabolite clustering tree, and the top is the sample clustering tree. The gradient color represents the size of the quantitative value, the redder the color, the higher the expression, and the bluer the color, the lower the expression. The abscissa represents different samples, divided into T13 and T46 two categories, and each category has multiple repeated samples marked as T13_1, T13_2, etc.; the ordinate represents different metabolites, and the color of the heat map can directly observe the content of each substance in different samples, wherein T13 is Smilax glabra, and T46 is Smilax glabra after fermentation.

[0023] Figure 5 is a quantitative diagram of the important metabolite 5,7-dimethoxycoumarin (Citropten) in Smilax glabra before and after lactic acid bacteria fermentation of the present application, wherein the abscissa is different groups, and the ordinate is the quantitative value range of the metabolite. The star between the two groups indicates the significance of the difference between the two groups, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0024] Figure 6 is a differential enrichment score diagram of Smilax glabra before and after lactic acid bacteria fermentation of the present application, wherein the abscissa is the DA-score value, the formula is DA-score=(up-regulated substance number-down-regulated substance number) / total number of differential substances in the pathway, and the ordinate is the metabolic pathway. The size of the point at the top of the column indicates the number of differential metabolites enriched in the pathway. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] Example 1

[0027] A preparation method of a fermentation product, comprising the following steps:

[0028] (1) Bacterial activation: Lactobacillus rhamnosus LR.M8 strain was inoculated in MRS liquid medium (the composition of MRS liquid medium: casein peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 5.0 g, sodium acetate 5.0 g, diamine citric acid 2.0 g, Tween 80 1.0 g, potassium phosphate dibasic 2.0 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate heptahydrate 0.05 g, calcium carbonate 20.0 g, distilled water 1.0 L, pH (6.8), and cultured at 37°C for 24 hours to obtain Lactobacillus rhamnosus LR.M8 bacterial liquid.

[0029] (2) Preparation of Smilax riparia liquid fermentation substrate: The raw material of Smilax riparia was crushed and added with water, and the mass ratio of the raw material of Smilax riparia to water was 1:6-7; 60°C water extraction for 1 hour, repeated 3 times to obtain the preliminary extract, and the preliminary extract was concentrated into extract semi-finished product, the specific gravity d of the extract semi-finished product was 1.14-1.17, and then auxiliary material malt dextrin was added, the mass of the malt dextrin was 30% of the mass of the extract semi-finished product; the Smilax riparia extract powder was prepared by powder spraying drying, crushing and sieving; and the Smilax riparia extract powder was mixed with water and sterilized to obtain the liquid fermentation substrate;

[0030] (3) Preparation of fermentation product: the activated Lactobacillus rhamnosus LR.M8 was inoculated in the liquid fermentation substrate, and the mass ratio of Lactobacillus rhamnosus LR.M8 to liquid fermentation substrate was 3:100; the liquid fermentation substrate inoculated with Lactobacillus rhamnosus LR.M8 was placed in a thermostat for fermentation to prepare the fermentation product, the fermentation time was 96h, and the fermentation temperature was 37°C; the prepared fermentation product after fermentation was placed in a vacuum freeze dryer for drying, and the dried fermentation product was crushed by a crusher and passed through a 60-mesh sieve to obtain a fermentation product powder.

[0031] Comparative Example 1

[0032] The difference between the present comparative example and Example 1 is that no Lactobacillus rhamnosus LR.M8 in Example 1 is added or inoculated in the present comparative example.

[0033] Test Part

[0034] The fermentation product powder prepared in Example 1 was taken, 1 g of powder was weighed, ultrapure water (water: sample = 100: 1, v / w) was added, the volume was adjusted to 100 mL, ultrasonic machine was used for ultrasonic treatment at room temperature for 15 min, and after mixing, Smilax riparia sample solution A1 of 10 mg / mL was obtained.

[0035] Take the sterile extraction powder in Comparative Example 1, take 1 g of the powder, add ultrapure water (water: sample = 100: 1, v / w), make up to 100 mL, and ultrasonic for 15 min at room temperature. After mixing, a 10 mg / mL Smilax glabra sample solution B1 is obtained.

[0036] 1. In vitro XOD inhibition rate determination method

[0037] Principle: XOD is also known as xanthine oxidase, mainly concentrated in the liver, catalyzing the oxidation of hypoxanthine and xanthine to uric acid, and is a key enzyme for uric acid generation. Uric acid has a strong absorption at 293 nm, and its generation amount is proportional to enzyme activity within a certain time. When there is an enzyme inhibitor, the amount of uric acid generated is reduced, and its absorbance is quantitatively related to the degree of inhibition, so it can be quickly and quantitatively analyzed by spectrophotometer or enzyme label instrument.

[0038] Experimental method:

[0039] Take 100 μL of Smilax glabra sample solution A1 and add it to well A1,

[0040] Take 100 μL of Smilax glabra sample solution A2 and add it to well B1,

[0041] Take 0.002 g of XOD (50 U / g) and dissolve it in PBS buffer (pH = 7.4) to make up to 100 mL, and prepare a 0.5 U / mL XOD solution. Add 50 μL of XOD solution (0.1 U / mL) to test tubes A1 and B1, respectively, and then shake well at 37°C for 15 min,

[0042] Take 0.091 g of xanthine, first add 5 mL of NaOH (0.1 mol / mL) solution, and ultrasonic-assisted dissolution. After the xanthine is completely dissolved, adjust the pH to neutral with PBS buffer (pH = 7.4), and make up to 100 mL to obtain a 0.6 mM xanthine solution. Add 50 μL of xanthine solution (0.6 mMol / mL) to test tubes A1 and B1, respectively, and measure the absorbance value at 293 nm,

[0043] Each experiment has three parallel groups, and each group is repeated three times.

[0044] Table 1 XOD inhibition capacity experimental reaction system

[0045] Unit (μL) A1 B1 Blank Morinda officinalis sample 100 100 0 pH 7.4 buffer solution 0 0 150 0.5 U / mL XOD solution 50 50 0 0.6 mM Xanthine solution 50 50 50

[0046] Inhibition rate (%) = (1 - (ΔA sample - ΔA blank) / (ΔA control - ΔA blank)) x 100%

[0047] Note: ΔA sample: absorbance change at 293 nm of the sample group containing the P. multiflora extract.

[0048] ΔA control: absorbance change at 293 nm of the sample group not containing the P. multiflora extract.

[0049] ΔA blank: absorbance change at 293 nm of the blank control group without enzyme.

[0050] Experimental results

[0051] As shown in Table 1, the P. multiflora extract after fermentation by L. rhamnosus LR.M8, its ability to inhibit XOD has been significantly improved, the inhibition rate of XOD from 29.28 ± 4.42% before fermentation to 59.16 ± 4.36% after fermentation, which shows that the fermentation treatment of L. rhamnosus LR.M8 can effectively improve the inhibition ability of P. multiflora extract to XOD. This enhancement may be due to the interaction between the metabolites produced by microorganisms during fermentation and plant components, thereby improving the inhibition ability of P. multiflora extract to XOD. Figure 1 2. Non-targeted metabolomics assay

[0052] Experimental principle: Non-targeted metabolomics is a technology for comprehensive analysis of all small molecule metabolites in biological samples, aiming to find differential metabolites and reveal potential biomarkers or metabolic pathway changes.

[0053] Experimental method

[0054] 2.1 Sample preparation

[0055] Take the P. multiflora extract solution without inoculating L. rhamnosus, and dry it in a vacuum freeze dryer. The dried fermentation product is crushed by a crusher to pass through a 60-mesh sieve to obtain fermentation powder. Named T13, three samples in parallel.

[0056] Take the P. multiflora extract solution inoculated with L. rhamnosus, and ferment it at 37°C for 96h. After fermentation is completed, the extract solution is dried in a vacuum freeze dryer. The dried fermentation product is crushed by a crusher to pass through a 60-mesh sieve to obtain fermentation powder. Named T46, three samples in parallel.

[0057] Through non-targeted metabolomics, differential metabolites of P. multiflora fermentation are found, potential biomarkers or metabolic pathway changes are revealed, and fermentation effect is evaluated.

[0058] 2.2 Metabolite extraction

[0059] The extraction reagents and equipment used in the metabolomics detection process are shown in Tables 2 and 3 below.

[0060] ​

[0061] Table 2 Reagents and standards for metabolomics detection

[0062] Name CAS Purity Brand Methanol 67-56-1 ≥99.0% Thermo 2-Chloro-L-phenylalanine (internal standard substance) 103616-89-3 98% Aladdin

[0063] Table 3 Instruments and equipment for metabolite extraction

[0064] Name Brand Model Refrigerated centrifuge Xiangyi H1850-R Mixing instrument Its Linbail BE-2600 Tissue grinder Meibi MB-96 Ultrasonic cleaner Shumei KQ-800DE Filter membrane Jinteng 0.22 μm PTFE

[0065] 1. Accurately weigh an appropriate amount of sample into a 2 mL centrifuge tube, add 600 μL of methanol containing 2-chloro-L-phenylalanine (4 ppm),

[0066] vortex for 30 s;

[0067] 2. Add steel beads and put into a tissue grinder, grind at 55 Hz for 60 s;

[0068] 3. Ultrasonic at room temperature for 15 min;

[0069] 4. Centrifuge at 12000 rpm at 4°C for 10 min, take the supernatant and filter through a 0.22 μm membrane, add the filtrate to the detection bottle, and use for

[0070] LC-MS detection.

[0071] 2.3 On-machine detection

[0072] The reagents and detection instruments used for metabolomics detection are shown in Tables 4 and 5 below.

[0073] Table 4 Main reagents for metabolomics detection

[0074] Name CAS Purity Brand Acetonitrile 75-05-8 ≥99.9% Thermo Formic acid 64-18-6 LC-MS grade TCI Ammonium formate 540-69-2 ≥99.9% Sigma H2O / / Millipore

[0075] Table 5 Instruments for metabolomics detection LC-MS

[0076] Name Brand Model Liquid chromatograph Thermo Vanquish Mass spectrometer Thermo Q Exactive

[0077] 2.3.1 Chromatographic conditions

[0078] Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid system, using ACQUITY UPLC® HSS T3 (2.1 x 100 mm, 1.8 µm) (Waters, Milford, MA, USA) chromatographic column, flow rate of 0.3 mL / min, column temperature of 40 ℃, sample size of 2 μL. Positive ion mode, mobile phase is 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2), gradient elution program: 0~1 min, 10% B2; 1~5 min, 10%~98% B2; 5~6.5 min, 98% B2; 6.5~6.6 min, 98%~10% B2; 6.6~8 min, 10% B2. Negative ion mode, mobile phase is acetonitrile (B3) and 5 mM ammonium carbonate water (A3), gradient elution program: 0~1 min, 10% B3; 1~5 min, 10%~98% B3; 5~6.5 min, 98% B3; 6.5~6.6 min, 98%~10% B3; 6.6~8 min, 10% B3.

[0079] 2.3.2 Mass spectrometry conditions

[0080] Thermo Q Exactive mass spectrometry detector (Thermo Fisher Scientific, USA), electrospray ion source (ESI), positive and negative ion modes respectively collect data. Positive ion spray voltage is 3.50 kV, negative ion spray voltage is -2.50 kV, sheath gas 40 arb, auxiliary gas 10 arb. Capillary temperature 325 ℃, with a resolution of 70000 for primary full scan, primary ion scan range m / z 100~1000, and HCD is used for secondary fragmentation, collision energy is 30 eV, secondary resolution is 17500, and the first 10 ions are collected for fragmentation, while dynamic exclusion is used to remove unnecessary MS / MS information.

[0081] Experimental results

[0082] Figure 2 T13 and T46 are significantly separated on the PC1 axis, and the unfermented and fermented groups have significant differences in principal components. As shown in Figure 3-4 Figure 2, during the fermentation of Smilax glabra, the metabolites changed significantly, and 248 differential metabolites were identified, of which 101 were up-regulated and 147 were down-regulated, and substances such as xylitol and 5,7-dimethoxy coumarin accumulated significantly. Figure 5Citropten (5,7-dimethoxycoumarin) of Poria cocos, the expression amount of which in the unfermented group T13 is significantly higher than that in the fermented group T46 (P <0.05), is related to the inhibition function of Poria cocos on XOD.

[0083] The fermentation process particularly activates the phenylpropanoid and flavonoid biosynthesis pathways Figure 6 ), so that the content of key active ingredients such as 5,7-dimethoxycoumarin (Citropten) is significantly improved. In combination with in vitro experiments, it is proved that the fermented Poria cocos extract shows stronger xanthine oxidase (XOD) inhibitory activity and antioxidant capacity, and the improvement of the uric acid-lowering effect is mainly due to the accumulation of small molecule metabolites such as Citropten and the synergistic effect of multiple pathways. The above results show that the fermentation of Poria cocos by Lactobacillus rhamnosus LR.M8 can directionally optimize the uric acid-lowering active ingredients of Poria cocos, and significantly improve the nutritional value and functional activity of Poria cocos.

[0084] The present application determines the in vitro uric acid-lowering activity of Poria cocos before and after fermentation by Lactobacillus rhamnosus LR.M8, and the experimental results prove that the Poria cocos extract has higher uric acid-lowering performance after fermentation by Lactobacillus rhamnosus LR.M8, can repair the damage of oxidase in the human body, and regulate the balance of uric acid. The content of 5,7-dimethoxycoumarin (Citropten) in the fermented Poria cocos extract is significantly improved, and the influence on the inhibition of xanthine oxidase activity is more significant.

Claims

1. A method for preparing a fermented product of Smilax glabra, characterized in that, The method includes the following steps: (1) Activation of Lactobacillus rhamnosus LR. M8 strain: Lactobacillus rhamnosus LR. M8 strain was inoculated into MRS liquid medium and cultured at 37°C for 24 hours to obtain Lactobacillus rhamnosus LR. M8 bacterial suspension; the preservation number of Lactobacillus rhamnosus Lr. M8 is CGMCCNo.3002; (2) Preparation of Smilax glabra liquid fermentation substrate: Smilax glabra raw material is crushed and water is added. The mass ratio of Smilax glabra raw material to water is 1:6-7. Water extraction is carried out at 60℃ for 1 hour, and repeated 3 times to obtain a preliminary extract. The preliminary extract is concentrated into a semi-finished extract with a specific gravity d=1.14-1.

17. Maltodextrin is added as an excipient. The mass of maltodextrin is 30% of the mass of the semi-finished extract. Smilax glabra extract powder is prepared by spray drying, crushing and sieving. The Smilax glabra extract powder is mixed with water and sterilized to obtain a liquid fermentation substrate. (3) Preparation of Smilax glabra fermentation product: The Lactobacillus rhamnosus LR. M8 bacterial solution was inoculated onto the liquid fermentation substrate and fermented to obtain the Smilax glabra fermentation product.

2. The preparation method according to claim 1, characterized in that, In step (1), the inoculation amount is 1-2% of the mass of Lactobacillus rhamnosus LR. M8 strain in the MRS liquid culture medium; the composition of the MRS liquid culture medium is: 10.0 g casein peptone, 10.0 g beef extract, 5.0 g yeast extract, 5.0 g glucose, 5.0 g sodium acetate, 2.0 g diammonium citrate, 1.0 g Tween 80, 2.0 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate heptahydrate, 20.0 g calcium carbonate, and 1.0 L distilled water, pH 6.

8.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of Smilax glabra extract powder to water is 1:

2.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of Lactobacillus rhamnosus LR. M8 to the liquid fermentation substrate is 3:

100.

5. The preparation method according to claim 1, characterized in that, The fermentation temperature is 37°C; the fermentation time is 96 hours.

6. The preparation method according to any one of claims 2-5, characterized in that, The effective components of the fermented Smilax glabra include 5,7-dimethoxycoumarin.

7. The application of the fermented Smilax glabra product prepared by any one of claims 2-6 in improving the uric acid-lowering properties of health foods.

8. The use of the fermented Smilax glabra product prepared by any one of the preparation methods according to claims 2-6 in the preparation of uric acid-lowering drugs.

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