Preparation method and application of fermented pawpaw extract

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a preparation method and application of a fermented papaya extract. The fermented papaya extract is prepared by taking water and a papaya extract as main base materials, and adding lactobacillus rhamnosus LR.M8, so that the inhibition effect on xanthine oxidase is improved. The lactobacillus rhamnosus LR.M8 can effectively improve the improvement effect of the papaya medicinal and edible homologous plant extract on hyperuricemia, and can repair kidney injury in a human body and regulate uric acid balance. The xanthine oxidase activity inhibition capability of the fermented papaya is obviously improved; the treatment effect on the hyperuricemia is stronger.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for preparing fermented papaya extract and its application. Background Technology

[0002] Saponins, papaya polysaccharides, water-soluble vitamins, and terpenoids in papaya can promote uric acid excretion by inhibiting XOD activity, thereby reducing uric acid, creatinine, and urea nitrogen levels. However, the active substances extracted using only hot water extraction have limited uric acid-lowering effects. Under the catalytic action of enzyme systems (cellulase, β-glucosidase) produced by lactic acid bacteria, the components in papaya can undergo deglycosylation or decarboxylation reactions, producing new active ingredients. Furthermore, the fermented active ingredients are more easily absorbed by the human body, improving their bioavailability.

[0003] CN114766677A - A *Lactobacillus rhamnosus* JM039 strain, its composition, and its application have significant therapeutic effects on irritable bowel syndrome, reducing serum endotoxins, decreasing pro-inflammatory factors, increasing anti-inflammatory factor levels, alleviating oxidative stress, and regulating intestinal flora imbalance, thereby promoting a healthy intestinal flora. CN116987617A - A *Lactobacillus rhamnosus* UA260 strain and its application in regulating blood uric acid. However, the prior art does not disclose the application of *Lactobacillus rhamnosus* fermented papaya to lower uric acid. Summary of the Invention

[0004] To address the above technical problems, this invention provides a method for preparing fermented papaya extract and its application. By fermenting with Lactobacillus rhamnosus, the key active substances in papaya that play a role in lowering uric acid are increased, while simultaneously solving the health problem that large molecules such as polysaccharides are not easily absorbed and utilized by the human body.

[0005] This invention provides the following technical solutions:

[0006] A method for preparing papaya ferment includes the following steps:

[0007] (1) Activation of Lactobacillus rhamnosus LR.M8 strain: Lactobacillus rhamnosus LR.M8 strain was inoculated into MRS liquid medium and cultured at 37℃ for 24 hours to obtain Lactobacillus rhamnosus LR.M8 bacterial suspension; the Lactobacillus rhamnosus Lr. M8 has the accession number CGMCC No.3002, is deposited at the China General Microbiological Culture Collection Center, China General Microbiological Culture Collection Center, deposit date: 2009-04-07, deposit address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing.

[0008] (2) Preparation of Papaya Liquid Fermentation Substrate: Papaya raw material is crushed and water is added. The mass ratio of papaya 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. Papaya extract powder is prepared by spray drying, crushing and sieving. Papaya extract powder is mixed with water and sterilized to obtain a liquid fermentation substrate.

[0009] (3) Preparation of papaya fermentation product: The Lactobacillus rhamnosus LR. M8 bacterial solution was inoculated onto the liquid fermentation substrate and fermented to obtain the papaya fermentation product.

[0010] Preferably, the inoculation amount in step (1) 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.

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

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

[0013] Preferably, the fermentation temperature is 37°C and the fermentation time is 96 hours.

[0014] Preferably, the active ingredient in the papaya ferment includes bergamot lactone.

[0015] Application of a papaya ferment in improving the uric acid-lowering properties of health foods.

[0016] Application of a papaya ferment in the preparation of uric acid-lowering drugs.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention provides a strain of Lactobacillus rhamnosus LR.M8, which can effectively improve the uric acid-lowering performance of papaya extract through fermentation. The reason for this is that Lactobacillus rhamnosus LR.M8 can effectively enhance the inhibitory effect of the extract on xanthine oxidase during fermentation. According to non-target metabolism detection, the fermented papaya extract showed a significant uric acid-lowering effect, effectively enhancing the uric acid-lowering performance of a medicinal and edible plant. Attached Figure Description

[0019] Figure 1 The inhibition rates of XOD by unfermented and fermented papaya extracts are shown.

[0020] Figure 2 This is a principal component analysis (PCA) score graph of papaya before and after lactic acid bacteria fermentation according to the present invention; where the horizontal axis PC1 represents the score of the first principal component, and the vertical axis PC2 represents the score of the second principal component. Dots represent samples, circles represent 95% confidence intervals, and colors represent different groups. M13 represents papaya, and M46 represents fermented papaya.

[0021] Figure 3 This is a p-value plot of principal component analysis (PCA) of papaya before and after lactic acid bacteria fermentation according to the present invention. The horizontal axis (log2(FC)) represents the logarithmic transformation of the fold change in the relative expression level or content of a substance between the two groups of samples; the vertical axis represents the logarithmic value of the p-value (-log10). Each point in the plot represents a metabolite. A larger absolute value on the horizontal axis indicates a greater fold change in the expression level of a metabolite between the two samples; a larger value on the vertical axis indicates more significant differential expression, and the more reliable the differentially expressed metabolites are. The size of the point represents the VIP value, with red points representing upregulation, blue points representing downregulation, and gray points representing metabolites that did not meet the differential screening criteria.

[0022] Figure 4This is a heatmap of principal component analysis (PCA) of papaya before and after lactic acid bacteria fermentation according to the present invention. Columns represent samples, rows represent metabolites, the clustering tree on the left is the differential metabolite clustering tree, and the top is the sample clustering tree. Gradient colors indicate the magnitude of quantitative values; the redder the color, the higher the expression level, and the bluer the color, the lower the expression level. Metabolite names are not displayed for samples with more than 150 metabolites. The horizontal axis represents different samples, divided into two categories: M13 and M46. Each category contains multiple replicate samples labeled as M13_1, M13_2, etc. The vertical axis represents different metabolites. The heatmap colors visually show the levels of each substance in different samples, where M13 represents papaya and M46 represents fermented papaya.

[0023] Figure 5 This is a quantitative graph showing the levels of bergapten, an important metabolite in papaya before and after lactic acid bacteria fermentation, according to the present invention. The horizontal axis represents different groups, and the vertical axis represents the range of quantitative values ​​for the metabolite. M13 represents papaya, and M46 represents fermented papaya. Asterisks indicate significant differences between the two groups: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0024] Figure 6 This is a differential enrichment score diagram of papaya before and after lactic acid bacteria fermentation according to the present invention. The horizontal axis is the DA-score value, and the formula is DA-score = (number of upregulated substances - number of downregulated substances) / total number of differential substances in the pathway. The vertical axis is the metabolic pathway, and the size of the top point of the bar indicates the number of differentially enriched metabolites in the pathway. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0026] The Lactobacillus plantarum LR.M8 involved in this invention was isolated from food and purified by streak plate separation. It was identified as Lactobacillus plantarum by 16S rRNA and has been disclosed in CN101671634 A.

[0027] Example 1

[0028] A method for preparing a fermentation product includes the following steps:

[0029] (1) Activation of strain: Take Lactobacillus rhamnosus strain and inoculate it into MRS liquid medium at an inoculation amount of 1%-2% (the components of MRS liquid medium are: 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, 1.0 L distilled water, pH (6.8), and incubate at 37℃ for 24 hours to obtain Lactobacillus rhamnosus bacterial solution.

[0030] (2) Preparation of Papaya Liquid Fermentation Substrate: Papaya raw material is crushed and water is added. The mass ratio of papaya 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. Papaya extract powder is prepared by spray drying, crushing and sieving. Papaya extract powder is mixed with water and sterilized to obtain a liquid fermentation substrate. (3) Preparation of Fermentation Product: Activated Lactobacillus plantarum is used to prepare the fermentation product. Lactobacillus rhamnosus LR.M8 was inoculated onto a liquid fermentation substrate, with a mass ratio of 3:100 between Lactobacillus rhamnosus LR.M8 and the liquid fermentation substrate. The liquid fermentation substrate inoculated with Lactobacillus rhamnosus LR.M8 was placed in a constant temperature incubator for fermentation to obtain fermented product. The fermentation time was 96 hours and the fermentation temperature was 37°C. The fermented product was then placed in a vacuum freeze dryer for drying. The dried fermented product was then pulverized through a 60-mesh sieve to obtain fermented product powder.

[0031] Comparative Example 1

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

[0033] Test section

[0034] Take 1g of the fermentation powder obtained in Example 1, add ultrapure water (water:sample = 100:1, v / w), and bring the volume to 100 mL. Sonicate the mixture for 15 min at room temperature using an ultrasonic machine. After mixing, obtain a papaya sample solution A1 with a concentration of 10 mg / mL.

[0035] Take 1g of the sterile extract powder from Comparative Example 1, add ultrapure water (water:sample = 100:1, v / w), bring the volume to 100 mL, sonicate at room temperature for 15 min, and mix well to obtain 10 mg / mL papaya sample solution B1.

[0036] 1. Method for determining the in vitro XOD inhibition rate

[0037] Experimental Principle: XOD, also known as xanthine oxidase, is mainly concentrated in the liver. It catalyzes the oxidation of hypoxanthine and xanthine to uric acid, and is a key enzyme in uric acid production. Uric acid has a strong absorption at 293 nm, and its production within a certain time is directly proportional to enzyme activity. When enzyme inhibitors are present, uric acid production decreases, and its absorbance has a quantitative relationship with the degree of inhibition. Therefore, rapid quantitative analysis can be performed using a spectrophotometer or ELISA reader. The IC50 value refers to the concentration at which a compound or drug can inhibit a biological process or achieve 50% activity under certain conditions; the lower the value, the stronger the inhibitory effect.

[0038] Experimental methods:

[0039] Add 100 μL of papaya sample solution A1 to well A1.

[0040] Add 100 μL of papaya sample solution A2 to well B1.

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

[0042] Weigh 0.091 g of xanthine and add it to 5 mL of NaOH (0.1 mol / mL) solution, then sonicate to dissolve it. After the xanthine is completely dissolved, adjust the pH to neutral with PBS buffer (pH=7.4) and bring the volume to 100 mL to obtain a 0.6 mM xanthine solution. Add 50 μL of the xanthine solution (0.6 mMol / mL) to test tubes A1 and B1 respectively, and measure the absorbance at 293 nm.

[0043] Each experiment was conducted in triplicate, with three groups performing the experiments three times.

[0044] Table 1 Experimental reaction system for XOD inhibition ability

[0045] Unit (μL) A1 B1 blank Papaya 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)) × 100%

[0047] Note: ΔA sample: Absorbance change at 293 nm for the sample group containing papaya extract.

[0048] ΔA control: Absorbance change at 293 nm for the sample group without papaya extract.

[0049] ΔA blank: The change in absorbance at 293 nm in the enzyme-free blank control group.

[0050] Experimental results

[0051] like Figure 1 As shown, the XOD inhibition ability of papaya extract was significantly enhanced after fermentation with Lactobacillus rhamnosus LR.M8, increasing from 19.18±0.94% before fermentation to 41.74±1.70% after fermentation. This indicates that fermentation with Lactobacillus rhamnosus LR.M8 can effectively enhance the XOD inhibition ability of papaya extract. This enhancement may be attributed to the interaction between the metabolites produced by microorganisms and plant components during fermentation, thereby improving the XOD inhibition ability of papaya extract.

[0052] 2. Non-targeted metabolomics assays

[0053] Experimental principle: Untargeted metabolomics is a technique for comprehensively analyzing all small molecule metabolites in biological samples, with the aim of discovering differential metabolites and revealing potential biomarkers or changes in metabolic pathways.

[0054] Experimental methods

[0055] 2.1 Sample Preparation

[0056] The extract of papaya without Lactobacillus rhamnosus was dried in a vacuum freeze dryer. The dried fermentation product was then pulverized through a 60-mesh sieve to obtain fermentation product powder. This powder was named M13, and three replicates were taken.

[0057] Papaya extract inoculated with Lactobacillus rhamnosus was fermented at 37°C for 96 hours. After fermentation, the extract was dried in a vacuum freeze dryer. The dried fermentation product was then pulverized through a 60-mesh sieve to obtain fermentation powder. This powder was named M46, and three replicates were taken.

[0058] By using non-targeted metabolomics to discover differential metabolites in papaya fermentation, we can reveal potential biomarkers or changes in metabolic pathways and assess fermentation effects.

[0059] 2.2 Metabolite Extraction

[0060] The extraction reagents and equipment used in the metabolomics detection process are shown in Tables 2 and 3 below.

[0061] Table 2. Main reagents and standards for metabolomics detection

[0062] name CAS purity brand methanol 67-56-1 ≥99.0% Thermo 2-Chloro-L-phenylalanine (internal standard) 103616-89-3 98% Aladdin

[0063] Table 3. Instruments and equipment for metabolite extraction

[0064] name brand model Refrigerated centrifuge Xiangyi H1850-R Mixer Qilinbell BE-2600 tissue grinder Meibi MB-96 ultrasonic cleaner Shumei KQ-800DE Filter membrane Jin Teng 0.22 µm PTFE

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

[0066] Vortex oscillation for 30 s;

[0067] 2. Add steel balls, place in a tissue homogenizer, and homogenize at 55 Hz for 60 s;

[0068] 3. Ultrasound at room temperature for 15 minutes;

[0069] 4. Centrifuge at 12000 rpm for 10 min at 4℃, collect the supernatant and filter through a 0.22 μm membrane. Add the filtrate to the test bottle for further processing.

[0070] LC-MS detection.

[0071] 2.3 On-machine testing

[0072] The reagents and 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 <![CDATA[H2O]]> / / Millipore

[0075] Table 5. Metabolomics Detection Instruments LC-MS

[0076] name brand model liquid chromatograph Thermo Vanquish mass spectrometer Thermo Q Exactive

[0077] 2.3.1 Chromatographic conditions

[0078] A Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid chromatography system was used, employing an ACQUITY UPLC® HSS T3 (2.1 × 100 mm, 1.8 µm) column (Waters, Milford, MA, USA), with a flow rate of 0.3 mL / min, a column temperature of 40 °C, and an injection volume of 2 μL. Positive ion mode was used, with a mobile phase of 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2). The gradient elution program was: 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. In negative ion mode, the mobile phase consisted of acetonitrile (B3) and 5 mM ammonium formate aqueous solution (A3), with the gradient elution program as follows: 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] A Thermo Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) was used with an electrospray ionization (ESI) source, acquiring data in both positive and negative ion modes. The positive ion spray voltage was 3.50 kV, and the negative ion spray voltage was -2.50 kV. The sheath gas concentration was 40 arb, and the auxiliary gas concentration was 10 arb. The capillary temperature was 325 °C. A first-stage full scan was performed at a resolution of 70,000 m / z, with a first-stage ion scan range of 100–1000 m / z. Second-stage fragmentation was performed using an HCD with a collision energy of 30 eV and a second-stage resolution of 17,500 m / z. The first 10 ions acquired were fragmented, and unnecessary MS / MS information was removed using dynamic exclusion.

[0081] Experimental results

[0082] Figure 2 The high PC1 explanatory power (62.6%) indicates that most data differences can be captured by PC1, making the analysis results reliable. M13 and M46 are clearly separated on the PC1 axis, indicating that metabolites underwent significant changes during papaya fermentation.

[0083] like Figure 3-6As shown, papaya metabolites underwent significant changes during fermentation, with 283 differentially expressed metabolites identified, of which 178 were upregulated and 105 were downregulated. The number of upregulated metabolites far exceeded the number of downregulated metabolites, indicating that fermentation significantly activated the papaya metabolic network, especially the biosynthesis of secondary metabolites. During fermentation, proteins were degraded into small peptides and free amino acids, and fructose was consumed; substances including organic acids, phenols, xylitol, and bergapten significantly accumulated. The increase in the total amount and variety of these substances indicates that fermentation promoted the decomposition of macromolecules and the accumulation of small-molecule active substances in papaya. Bergapten, a component of papaya, was expressed at a significantly higher level in the unfermented group M13 than in the fermented group M46 (P < 0.05), and its content was correlated with the inhibitory function of papaya against XOD.

[0084] The above results indicate that the fermentation process significantly enhances the nutritional value and functional activity of papaya through microbial degradation and biotransformation.

[0085] This invention measured the in vitro uric acid-lowering activity of *Lactobacillus rhamnosus* LR.M8 before and after fermentation of papaya. The experimental results demonstrated that papaya extract fermented with *Lactobacillus rhamnosus* LR.M8 exhibited enhanced uric acid-lowering properties, effectively repairing oxidative enzyme damage and regulating uric acid balance in the human body. The bergapten content in the fermented papaya extract was significantly increased, resulting in a more significant inhibitory effect on xanthine oxidase activity.

Claims

1. A method for preparing a papaya ferment, 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℃ for 24h to obtain Lactobacillus rhamnosus LR. M8 bacterial suspension; the preservation number of Lactobacillus rhamnosus Lr. M8 is CGMCCNo.3002; (2) Preparation of Papaya Liquid Fermentation Substrate: Papaya raw material is crushed and water is added. The mass ratio of papaya 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. Papaya extract powder is prepared by spray drying, crushing and sieving. Papaya extract powder is mixed with water and sterilized to obtain a liquid fermentation substrate. (3) Preparation of papaya fermentation product: The Lactobacillus rhamnosus LR. M8 bacterial solution was inoculated onto the liquid fermentation substrate and fermented to obtain the papaya 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 papaya 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 papaya 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 active ingredient in the papaya ferment includes bergamot lactone.

7. The application of the papaya ferment prepared by any one of the preparation methods according to claims 2-6 in improving the uric acid-lowering performance of health foods.

8. The use of papaya ferment prepared by any one of the preparation methods according to claims 2-6 in the preparation of uric acid-lowering drugs.

Images