Production process of urolithin A

Abstract

The invention relates to the technical field of extraction and utilization of chemical substances in walnut green seedcases, in particular to a production process of urolithin A. The production process specifically comprises the following steps: obtaining the walnut green seedcases; walnut green seedcase is mashed into pulp, a stabilizing solution is added, ultrasonic treatment is performed after uniform stirring, the fermentation substrate is obtained, and the stabilizing solution is an aqueous solution of a compound of sulfydryl-beta-cyclodextrin and chitosan; primary fermentation: inoculating a microbial bacterial liquid A for anaerobic fermentation, then filtering and collecting filtrate, the microbial bacterial liquid A being obtained by respectively inoculating bacillus velezensis and bacillus coagulans into a culture medium A for shake cultivation and activation and then mixing the bacillus velezensis and the bacillus coagulans; secondary fermentation: inoculating the filtrate with a microbial bacterial liquid B for anaerobic fermentation again, wherein the microbial bacterial liquid B is obtained by inoculating enterococcus faecium into a culture medium B for culture; and extracting. According to the method, the stabilizing solution is added into the walnut green seedcase, and two-stage fermentation is performed, so that the yield and the conversion rate of urolithin A are remarkably improved, and the method is safe, environmentally friendly and capable of being industrialized.

Description

Technical Field

[0001] The invention relates to the technical field of extraction and utilization of chemical substances in walnut green peel, in particular to a production process of urolithin A. Background Art

[0002] Ellagic acid is a polyphenol dilactone with the molecular formula C 14 H 16 O 18 , is a dimeric derivative of gallic acid, which is widely found in various soft fruits, nuts and other plant tissues. Ellagic acid has antioxidant and anti-aging, anti-inflammatory and anti-cancer effects, antihypertensive, hypoglycemic, sedative and other effects. It is a high-value-added health and beauty product, mainly used as an additive for medicines, health foods and cosmetics. Urolithin A, which is generated by the transformation of ellagic acid by intestinal microorganisms, is a star anti-aging product. Urolithin A, with a molecular formula of C 13 H8O4 can improve the cardiac function of ischemia / reperfusion mice by increasing the antioxidant activity of cardiomyocytes. It can also induce selective autophagy of mitochondria by activating the PINKI / Parkin ubiquitin-dependent pathway or BNIP3 receptor, thereby alleviating the damage of mitochondrial function caused by aging. It also plays an important role in improving human health.

[0003] An important source of ellagic acid is extracted from pomegranate peel, gallnut, etc. In recent years, some researchers have found that walnut green peel also has a high content of ellagic acid, so a small number of researchers have explored extraction from walnut green peel, but the process is relatively immature and has not been able to achieve scale and industrialization. Limited by the special structure of pomegranate, the processing and extraction of pomegranate peel in the prior art often sacrifices pomegranate seeds, and the process is relatively complicated, so the processing cost is high; while walnut green peel is completely agricultural waste, and its processing will not have any adverse effects on the more valuable walnut fruit, so the processing cost is low. Generally speaking, one pound of green peel walnut contains about 4-5 taels of green peel, and the content of ellagic acid in the green peel dry matter is about 1%, and one pound of pomegranate contains about 2 taels of pomegranate peel, and the content of ellagic acid in the pomegranate peel dry matter is about 1%-2%. The output of walnuts in my country is much greater than that of pomegranates, so under the background of environmental protection and sustainable development, the processing and utilization of walnut green peel has broader prospects and potential. Lin'an, Hangzhou is an important production area of ​​pecans. Its suitable climate and soil environment provide unique natural conditions for the growth of pecans. The green-skinned pecans produced here not only have full walnut kernels and high nutritional value, but also have bright green skins, moderate thickness, and rich active ingredients, making them extremely valuable for deep processing.

[0004] At present, urolithin A used in food and health products is obtained by microbial transformation, which is safe and environmentally friendly, but has the problem of low conversion rate. To this end, the prior art is committed to screening strains with high conversion rates in order to solve the above technical problems. For example, Chinese invention patent application No. 202211134634.7 and application publication No. CN115725451A (defined as prior art 1) disclose Enterococcus faecium FUA027 and its method and application for producing urolithin A. The strain is isolated from the human intestine, and its seed liquid is inoculated at an inoculation rate of 2% in a fermentation medium supplemented with 1% ellagic acid for anaerobic fermentation culture. Urolithin A can be detected after 40 hours, and the maximum concentration reaches 10.8μM after 50 hours. For example, Chinese invention patent application No. 202211409720.4 and application publication No. CN 115992074A disclose a strain of Lactobacillus plantarum and its use in producing urolithin A. The strain is derived from the feces of healthy people and is named Lactobacillus plantarum CCFM1290. It is inoculated with an inoculum dose of 2% into a culture medium containing 1.5 g / L of ellagitannins and fermented for 48 hours to obtain various urolithin substances, among which the content of urolithin A is 24.70±0.82 μM and the conversion rate is 8.59±0.62%. For example, Chinese invention patent application No. 202211404309.8 and application publication No. CN115786190A disclose a strain of Lactobacillus plantarum that can produce urolithin A for anti-aging and its application. The strain is derived from the feces of a healthy human body and is included in the catalog of edible fungi. It is inoculated with an inoculum dose of 2% into a fermentation culture medium containing commercially available NFC pomegranate juice and fermented for 48 hours to obtain a yield of 32.01±0.97μM urolithin A, and the conversion rate reaches 10.73±0.38%.

[0005] However, the above-mentioned prior art can improve the conversion rate by simply relying on the strain, but the degree of improvement is limited, and the problem of low conversion rate cannot be fundamentally solved. The reason why the conversion rate of urolithin A is low is mainly due to two aspects: first, the hydrophilicity of ellagic acid is poor and unstable in aqueous solution, resulting in a low concentration of effective substances in the initial substrate; second, after being limited to the generation of urolithin A, the increasing concentration of urolithin A will produce a product inhibition effect, which will affect the activity of microorganisms, reduce the conversion efficiency, and then affect the generation of urolithin A. In addition, the above-mentioned prior art uses ellagitannins, ellagic acid or commercially available products as raw materials, and the cost of the raw material itself is relatively high, resulting in that even if it has a higher conversion rate, it is difficult to industrialize. For this reason, this application is proposed. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a process for producing urolithin A.

[0007] A process for producing urolithin A comprises the following steps: S1. Obtaining walnut green skin: Select healthy, pest-free and intact green skin walnuts, wash them, pile them in a ventilated and cool place to dry them naturally, and peel them after the green skin becomes soft and the moisture content is less than 8% to obtain the walnut green skin; the relevant treatment of step S1 helps to maximize the retention of ellagic acid in the walnut green skin and promote its stable existence without oxidation. The requirement of moisture content is on the one hand to concentrate the concentration of effective substances and ensure the accuracy and reliability of subsequent results, and on the other hand to prevent the walnut green skin from rotting and maintaining quality; in addition, walnuts should not be picked too late, because the green skin will naturally crack if picked too late, and part of it will be oxidized, resulting in a decrease in the content of effective substances. Green skin walnuts should be collected during the peak season of walnut harvesting as much as possible. At this time, the freshness of the walnut green skin can be guaranteed, the accuracy of the experimental data can be ensured, and the walnut green skin can be utilized to reduce environmental pollution; S2, preparing a fermentation substrate: crushing the walnut peel into a pulp and adding a stabilizing liquid, stirring the mixture evenly, and then ultrasonically treating the mixture, using the cavitation effect, mechanical effect and thermal effect of ultrasound to exert pressure on the cell wall, causing the cell wall to rupture, and increasing the frequency and speed of the movement of polyphenol molecules, so as to accelerate the efficient release of polyphenols in the walnut peel. The stabilizing liquid is an aqueous solution of a compound of thiol-β-cyclodextrin and chitosan. The effective ingredients of the stabilizing liquid, thiol-β-cyclodextrin and chitosan, have a significant adsorption effect on polyphenols, and continuously adsorb the released polyphenols to promote the continuous release of the remaining polyphenols in the walnut peel. S3, the first fermentation: inoculating microbial liquid A into the fermentation substrate for anaerobic fermentation, performing solid-liquid separation and collecting the filtrate after the fermentation is completed, wherein the microbial liquid A is obtained by inoculating Bacillus Velez and Bacillus coagulans into culture medium A respectively, activating them by shaking culture, and then mixing them; on the basis of ultrasonic treatment, combined with the first fermentation, polyphenols are hydrolyzed to generate ellagic acid, the extraction rate of ellagic acid is high, the extraction effect is good, and most of the ellagic acid in walnut green peel is enriched in the filtrate; the outer edge of β-cyclodextrin is hydrophilic while the inner cavity is hydrophobic, and the hydrophobic cavity structure can form an inclusion compound with ellagic acid, thereby reducing the contact between ellagic acid and the external environment, which not only reduces its The risk of oxidation and hydrolysis is eliminated, and the water solubility of ellagic acid is improved to a certain extent, and it also helps microorganisms to better contact ellagic acid molecules, thereby increasing its conversion rate; when β-cyclodextrin introduces thiol groups, the thiol groups can also form hydrogen bonds with functional groups such as hydroxyl groups in ellagic acid, further stabilizing the structure of ellagic acid and protecting its biological activity from being affected; under the dual effects of inclusion and hydrogen bonding, ellagic acid exists in water in large quantities and stably; chitosan is cationic, and thiol-β-cyclodextrin is anionic. After the two are mixed, they can form a complex through electrostatic interaction, and the complex helps to further improve the water solubility, stability and bioavailability of ellagic acid; S4, second fermentation: inoculating microbial liquid B into the filtrate for anaerobic fermentation again, wherein the microbial liquid B is obtained by inoculating Enterococcus faecium into culture medium B; when Enterococcus faecium is added, ellagic acid connected to the complex through inclusion, hydrogen bonding and the like is converted into urolithin A, and at the same time, the released complex in turn generates hydrogen bonding and inclusion bonding with urolithin A, so that the structure of urolithin A is stabilized and its solubility in water is increased; the present invention unexpectedly discovered that the presence of the complex significantly improves the conversion rate of urolithin A, and the reason for this may be that the combination of the complex and urolithin A can not only stabilize the structure of urolithin A to prevent it from being oxidized or decomposed, but also reduce the inhibitory effect of product concentration on microbial conversion to a certain extent, i.e., the so-called product inhibition effect, thereby avoiding the influence of increased urolithin A concentration on microbial activity, and the conversion rate of urolithin A is significantly increased under the dual effects; S5. Extraction: After the fermentation is completed, urolithin A is extracted.

[0008] Compared with the prior art, the present invention increases the concentration of ellagic acid, an effective substance in the fermentation substrate, and increases the concentration of reactants when preparing the fermentation substrate. This increases the concentration of the reactants, laying a foundation for the subsequent yield of urolithin A. On the other hand, the effect of the increase in the concentration of urolithin A on the activity of microorganisms can be reduced, and the amount of the product can be increased. The sulfhydryl-β-cyclodextrin and chitosan interact with each other and synergize to significantly improve the conversion rate and product yield of urolithin A. The present invention is safe, environmentally friendly, simple to operate, and has a simple process flow, and has great industrial prospects.

[0009] It should be noted that: (1) The walnut green peel is preferably crushed in a low temperature, light-proof environment. If mechanical equipment is used for crushing, a slow, low-temperature crusher is preferably used and the exposure time is reduced to reduce the loss of polyphenols. The walnut green peel is crushed into pulp and used immediately. If it cannot be used immediately, it should be isolated from air and dried and stored in a sealed, light-proof, low-temperature environment to prevent the polyphenols from being oxidized.

[0010] (2) Mercapto-β-cyclodextrin is an existing technology and can be purchased commercially or prepared in-house.

[0011] (3) The stabilizing liquid is an aqueous solution of a compound of mercapto-β-cyclodextrin and chitosan. There are hydrogen bonds, electrostatic attraction and other forces between mercapto-β-cyclodextrin and chitosan, but there is no chemical bond, which is completely different from the chitosan derivatives chemically modified by mercapto-β-cyclodextrin. The present application unexpectedly found that appropriately controlling the compounding ratio of mercapto-β-cyclodextrin and chitosan and the concentration of the stabilizing liquid can help adsorb the effective substances in the walnut green peel without causing flocculation.

[0012] (4) Purified water, i.e., deionized water, is preferably used to prepare the stabilizing solution. Purified water that has been freed of impurities such as organic matter, ions, and microorganisms can effectively prevent the above substances from interfering with the experimental results.

[0013] Preferably, in step S2, the chitosan is water-soluble chitosan, and mercapto-β-cyclodextrin and water-soluble chitosan are mixed in a mass ratio of (0.1-0.4): 1 to form the composite. Water-soluble chitosan is generally modified chitosan, such as carboxyl-modified chitosan, which has good water solubility and can load mercapto-β-cyclodextrin to form a complex through electrostatic interaction after being dissolved in water, and finally a stable solution with uniform composition and uniform dispersion is obtained.

[0014] Preferably, in step S2, the mass percentage of the stabilizing liquid is 5%-20%, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0015] There is not only electrostatic interaction between chitosan and thiol-β-cyclodextrin, but also hydrogen bonding, and an association structure is formed through hydrogen bonding. The greater the amount of chitosan and thiol-β-cyclodextrin added, the greater the concentration of the stabilizing solution, the more association structures, and the greater the viscosity of the aqueous solution. When the viscosity reaches a certain level, the viscous aqueous solution will hinder the transmission of substances, and the microorganisms will be hindered from obtaining nutrients and discharging waste during fermentation, which is not conducive to the normal growth of microorganisms and the synthesis of metabolites, so the reaction rate will be reduced, thereby affecting the fermentation efficiency. Moreover, if the concentration of the stabilizing solution is too high, excessive adsorption of chitosan and thiol-β-cyclodextrin to effective substances will produce flocculation, causing the effective substances to be unable to stably exist in the filtrate, but will have an adverse effect on the enrichment of effective substances. The present invention has found through a large number of innovative experiments that when the mass percentage of the stabilizing solution is 5%-20%, the content of chitosan and thiol-β-cyclodextrin is moderate, which can fully exert the stabilizing effect without affecting the normal growth of microorganisms.

[0016] Preferably, in step S2, the material-liquid ratio is 1:(12-20), the ultrasonic frequency is 12-15kHz, the ultrasonic power is 150-240W, the treatment time is 15-35min, during which the temperature of the mixture is maintained at 58-65°C, and after obtaining the fermentation substrate, it is naturally cooled to room temperature before proceeding to the next step.

[0017] In order to fully extract and dissolve the ellagic acid in the walnut green peel, the extraction temperature, extraction time, solid-liquid ratio and ultrasonic technical parameters all need to be optimized. The present invention has been confirmed through a large number of innovative experiments that in an aqueous solution added with a compound of thiol-β-cyclodextrin and chitosan, at a solid-liquid ratio of 1: (12-20), an ultrasonic frequency of 12-15kHz, an ultrasonic power of 150-240W, and an extraction temperature of 58-65°C, most of the ellagic acid can be extracted into the aqueous solution in 15-35 minutes.

[0018] In this embodiment, if the solid-liquid ratio is greater than 1:12, the solvent is too little, and the extraction efficiency of ellagic acid is low; if the solid-liquid ratio is less than 1:20, the solvent is too much, and although the extraction efficiency of ellagic acid is high, it will cause difficulty in subsequent processing, increase processing costs and processing time; when the solid-liquid ratio is 1:(12-20), such as 1:12, 1:15, 1:18, 1:20, etc., the extraction efficiency of ellagic acid is high and the subsequent processing is relatively simple. On this basis, the solid-liquid ratio is further preferably 1:15, at which time the extraction efficiency is the highest.

[0019] In the present embodiment, the technical parameters of ultrasonic treatment are strictly limited to: ultrasonic frequency 12-15kHz, ultrasonic power 150-240W. If the ultrasonic frequency is too small and the ultrasonic power is too small, the cavitation effect, mechanical effect and thermal effect of the ultrasonic wave are insufficient, and the cell wall of the walnut green peel cannot be effectively destroyed, and the effect of accelerating the diffusion and dissolution of ellagic acid from the cell into the solvent is limited, so the ellagic acid contained in the fermentation substrate cannot be fully extracted, resulting in a low concentration of effective substances in the fermentation substrate, which is not conducive to improving the yield of urolithin A. If the ultrasonic frequency is too large and the ultrasonic power is too large, the cavitation effect, mechanical effect and thermal effect of the ultrasonic wave are excessive, although there is a positive effect on accelerating the diffusion of ellagic acid, it will have an adverse effect on the inclusion effect of the compound on ellagic acid and the hydrogen bonding between the compound and ellagic acid, so that the solubility of ellagic acid in the solvent is limited and the stability is poor, which is also not conducive to obtaining a fermentation substrate with a high concentration of effective substances, and is equally unhelpful to improving the yield of urolithin A.

[0020] In this embodiment, the ultrasonic time can be controlled at 15-35 minutes, specifically 15, 20, 25, 30, 35 minutes, etc. If the ultrasonic time is too short, the diffusion and dissolution of ellagic acid will not be complete. If the ultrasonic time is too long, it will lead to energy waste on the one hand, and on the other hand, the stability of the dissolved ellagic acid will be affected.

[0021] In this embodiment, the extraction temperature is 58-65°C, specifically 58, 60, 62, 63, 64, 65°C, etc. This temperature range can accelerate the release of ellagic acid from walnut green peel and promote the dissolution of ellagic acid in water without causing the decomposition of ellagic acid or adversely affecting the inclusion or hydrogen bonding between ellagic acid and the compound.

[0022] Preferably, in step S3, culture medium A is an MRS liquid culture medium modified by corn syrup, Bacillus velez and culture medium A are cultured and activated at (0.5-0.8) g / 100 ml to obtain Bacillus velez liquid, and Bacillus coagulans and culture medium A are cultured and activated at (0.25-0.55) g / 100 ml to obtain Bacillus coagulans liquid. After the culture, the Bacillus velez liquid and the Bacillus coagulans liquid are mixed at a volume ratio of 1:1 to obtain microbial liquid A, and the inoculation amount of the microbial liquid A in the fermentation substrate is 2 v%.

[0023] Compared with glucose, corn syrup provides a wider range of carbon source options for microorganisms. Bacillus Velezii and Bacillus coagulans are both Bacillus with a wide range of carbon source utilization capabilities. The present invention found that when corn syrup is used to replace glucose in MRS liquid culture medium in equal amounts, the above two Bacillus exhibit better growth and metabolic performance.

[0024] In this embodiment, both Bacillus Velez and Bacillus coagulans are cultured and activated in a shaking table, and the specific technical parameters are: temperature 36° C., rotation speed 200 rpm, and culture time 36 h.

[0025] Preferably, in step S3, the temperature of anaerobic fermentation is 30-35° C. and the time is 24-48 hours.

[0026] After the first fermentation is completed, not only is ellagic acid enriched, but many biochemical changes produced by microbial fermentation can also modify plant components. At the same time, under the buffer environment provided by the stabilizing solution, ellagic acid has high biological activity.

[0027] Preferably, in step S4, the Enterococcus faecium is purchased from the China General Microbiological Culture Collection Center with a collection number of CGMCCNO.24964, and the specific components of medium B are: peptone 16.0 g / l, yeast extract 7.0 g / l, potassium chloride 5.0 g / l, starch 1.0 g / l, glucose 1.0 g / l, sodium pyruvate 1.0 g / l, arginine 1.0 g / l, sodium succinate 0.5 g / l, cysteine Hydrochloride 0.5g / l, sodium bicarbonate 0.4g / l, ferric pyrophosphate 0.5g / l, hemin chloride 0.005g / l, vitamin K 0.0005g / l, sodium thioglycolate 0.5g / l, dithiothreitol 1.0g / l, the microbial culture B is obtained by inoculating the Enterococcus faecium into the culture medium B and culturing at pH 6.8-7.0 and 37°C for 24h, and the inoculation amount of the microbial culture B in the filtrate is 2v%.

[0028] Preferably, in step S4, the temperature of anaerobic fermentation is 37° C., the time is 48-55 h, and the pH is 6.8-7.0.

[0029] In this embodiment, it is more appropriate to control the duration of the second fermentation to 48-55h. If the time is too short, the fermentation is incomplete, which is not conducive to the full participation of ellagic acid in the reaction, and the yield of urolithin A is low. If the time is too long, since the formation reaction of urolithin A has been basically completed, simply extending the fermentation time will not only not increase the content of urolithin A, but also as the fermentation process continues to advance, a large amount of by-products will be generated in the system, which will have an adverse effect on the stability and purity of urolithin A.

[0030] Preferably, step S3 and step S4 are continuously stirred at a low speed, and the rate of the low speed stirring is 80-120 rpm, such as 80, 85, 90, 95, 100, 105, 110, 115, 120 rpm.

[0031] In this embodiment, continuous low-speed stirring can avoid the generation of bubbles, reduce microbial damage, and protect the microbial growth environment. On the other hand, it can promote the full mixing of the fermentation substrate and the microorganisms, ensure that the microorganisms can fully contact the fermentation substrate, and maintain the uniform distribution of the microorganisms in the fermentation substrate, so as to promote the interaction and synergistic fermentation between the microorganisms, thereby improving the fermentation efficiency and the fermentation effect. In addition, low-speed stirring can reduce the violent shearing effect of the substance, avoid the degradation of the fermentation product and the weakening of the catalytic effect, thereby ensuring the quality and yield of the product.

[0032] Preferably, in step S5, the specific operation of the extraction is: extracting the obtained fermentation broth with an extractant, wherein the extractant is a mixture of CH3CN:H2O:HCOOH in a volume ratio of 80:19.9:0.1, and the extract is freeze-dried and then re-dissolved with ethyl acetate and then filtered through a 0.22 μm filter membrane.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Walnut peel, a byproduct of walnut processing, is used as the source of ellagic acid, which has high yield and low cost. Walnut peel can be directly used after being crushed, without the need for post-processing such as drying and powdering. The pre-treatment is simple and efficient, which is conducive to sustainable development and can avoid environmental pollution. It meets the requirements of environmental protection and circular economy, and has a bright prospect for large-scale and production production. (2) The ellagic acid in the walnut peel is accelerated to diffuse outward from the walnut peel cells under the action of ultrasound, and after the first fermentation, it is stabilized and dissolved in water in large quantities by forming inclusion complexes and hydrogen bonds under the action of the complex of thiol-β-cyclodextrin and chitosan. The ellagic acid in this form is not only structurally stable, but also has high bioavailability, which provides a good prerequisite for subsequent microbial transformation; (3) When ellagic acid begins to convert into urolithin A, the mercapto-β-cyclodextrin and chitosan that were previously bound to ellagic acid instead bind to urolithin A and encapsulate it, which not only stabilizes the structure of urolithin A and increases the solubility of urolithin A in water, but also reduces the inhibitory effect of product concentration on microbial conversion to a certain extent, allowing the microorganisms to maintain a high activity and promote the continued production of urolithin A. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 The ultraviolet absorption spectra of the ellagic acid product and the ellagic acid standard collected after the completion of step S3 in Example 1; Figure 2 The HPLC chromatogram of the ellagic acid product and the ellagic acid standard collected after the completion of step S3 in Example 1; Figure 3 The ultraviolet absorption spectra of urolithin A and urolithin A standard substance generated in Example 1; Figure 4 This is the liquid chromatogram of urolithin A generated in Example 1. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0037] In the following examples and comparative examples, mercapto-β-cyclodextrin was prepared in-house. The specific preparation method was as follows: first, a 0.8 mol / L NaOH aqueous solution was added dropwise to a 0.15 mol / L β-cyclodextrin aqueous suspension to obtain a mixed solution 1, wherein the weight ratio of NaOH to β-cyclodextrin was 1:10, and then a 1.8 mol / L methylbenzenesulfonyl chloride acetonitrile solution was added dropwise to the mixed solution 1 to obtain a mixed solution 2, wherein the weight ratio of methylbenzenesulfonyl chloride acetonitrile to β-cyclodextrin was 1:6, and then the mixed solution 2 was stirred at room temperature until After at least 2 hours, the mixture was allowed to stand for 2 days and the precipitated solid was collected. After recrystallization and drying, sulfonylated β-cyclodextrin crystals were obtained. The crystals and thiourea were then dissolved in a water-methanol mixed solvent (specifically, methanol and water were mixed in a volume ratio of 80:20) and stirred evenly to obtain a mixed solution 3, wherein the addition ratio of the crystals, thiourea, and water-methanol mixed solvent was 2g:2g:80ml. Finally, 1,2-dichloroethane was added to the mixed solution 3, wherein the weight ratio of the crystals to 1,2-dichloroethane was 1:15, to obtain thiol-β-cyclodextrin.

[0038] In the following examples and comparative examples, green walnuts were harvested from walnut farmers in Lin'an, Hangzhou on September 7, 2024; water-soluble chitosan was purchased from Shijiazhuang Dingmin Pharmaceutical Technology Co., Ltd.; Bacillus Velez was purchased from Shanghai Xuanke Biotechnology Co., Ltd.; Bacillus coagulans was purchased from Shanghai Jiaguan Biotechnology Co., Ltd. The remaining raw materials not specified were purchased commercially, and no specific manufacturer, specifications, etc. were required. Ellagic acid standard was purchased from Merck Chemical Technology (Shanghai) Co., Ltd., item number HY-B0183.

[0039] In the following examples and comparative examples, culture medium A is an MRS liquid culture medium modified with corn syrup, specifically, the glucose in the MRS liquid culture medium is replaced with an equal amount of corn syrup. Bacillus velez is cultured with culture medium A at 0.6 g / 100 ml to obtain a Bacillus velez liquid, and Bacillus coagulans is cultured with culture medium A at 0.4 g / 100 ml to obtain a Bacillus coagulans liquid. After the culture is completed, the Bacillus velez liquid and the Bacillus coagulans liquid are mixed at a volume ratio of 1:1 to obtain a microbial liquid A, and the inoculation amount of the microbial liquid A in the fermentation substrate is 2 v%.

[0040] In the following examples and comparative examples, Enterococcus faecium was purchased from China General Microbiological Culture Collection Center with a collection number of CGMCCNO.24964. The specific components of medium B were: peptone 16.0 g / l, yeast extract 7.0 g / l, potassium chloride 5.0 g / l, starch 1.0 g / l, glucose 1.0 g / l, sodium pyruvate 1.0 g / l, arginine 1.0 g / l, sodium succinate 0.5 g / l, cysteine Hydrochloride 0.5g / l, sodium bicarbonate 0.4g / l, ferric pyrophosphate 0.5g / l, hemin chloride 0.005g / l, vitamin K 0.0005g / l, sodium thioglycolate 0.5g / l, dithiothreitol 1.0g / l, the microbial liquid B is obtained by inoculating the Enterococcus faecium into the culture medium B and culturing at pH 6.8 and 37°C for 24h, and the inoculation amount of the microbial liquid B in the filtrate is 2v%.

[0041] Example 1 A process for producing urolithin A comprises the following steps: S1. Obtaining walnut green skin: Select healthy, pest-free and intact green skin walnuts, wash them, place them in a pile in a ventilated and cool place to let the green skin dry naturally, and peel them off after they become soft to obtain the walnut green skin; S2, preparing a fermentation substrate: crushing the walnut peel into a pulp and adding a stabilizing solution, stirring the mixture evenly, and then ultrasonically treating the resulting mixture to obtain a fermentation substrate, wherein the stabilizing solution is an aqueous solution of a compound of thiol-β-cyclodextrin and water-soluble chitosan; wherein thiol-β-cyclodextrin and water-soluble chitosan are mixed in a mass ratio of 0.25:1, the mass percentage of the stabilizing solution is 12%, the solid-liquid ratio of the walnut peel to the stabilizing solution is 1:15, and the technical parameters of the ultrasonic treatment are: ultrasonic frequency 13kHz, ultrasonic power 180W, time 30min, temperature 60°C; S3, first fermentation: after the temperature of the fermented substrate drops to room temperature, microbial liquid A is inoculated into the fermentation substrate for anaerobic fermentation, the temperature of the anaerobic fermentation is 33° C., the time is 48 h and the whole process is stirred at a speed of 100 rpm, after the fermentation is completed, solid-liquid separation is performed and the filtrate is collected, the microbial liquid A is obtained by inoculating Bacillus Velez and Bacillus coagulans into culture medium A respectively, activating them by shaking culture and then mixing them; S4, second fermentation: inoculating microbial solution B into the filtrate for anaerobic fermentation again, the anaerobic fermentation temperature is 37°C, the time is 50 hours, the pH is 6.8 and the whole process is stirred at a speed of 100 rpm, the microbial solution B is obtained by inoculating Enterococcus faecium into culture medium B; S5. Extraction: After the fermentation is completed, the obtained fermentation liquid is extracted with an extractant, wherein the extractant is a mixture of CH3CN:H2O:HCOOH in a volume ratio of 80:19.9:0.1. The extract is freeze-dried and re-dissolved with ethyl acetate, and then filtered through a 0.22 μm filter membrane.

[0042] Example 2 Compared with Example 1, in step S2, mercapto-β-cyclodextrin and water-soluble chitosan are mixed at a mass ratio of 0.1:1. The rest are the same as in Example 1.

[0043] Example 3 Compared with Example 1, in step S2, mercapto-β-cyclodextrin and water-soluble chitosan are mixed at a mass ratio of 0.2:1. The rest are the same as in Example 1.

[0044] Example 4 Compared with Example 1, in step S2, mercapto-β-cyclodextrin and water-soluble chitosan are mixed at a mass ratio of 0.4:1. The rest are the same as in Example 1.

[0045] Example 5 Compared with Example 1, in step S2, the mass percentage of the stabilizing liquid is 5%. The rest is the same as Example 1.

[0046] Example 6 Compared with Example 1, in step S2, the mass percentage of the stabilizing liquid is 10%. The rest are the same as in Example 1.

[0047] Example 7 Compared with Example 1, in step S2, the mass percentage of the stabilizing liquid is 20%. The rest are the same as in Example 1.

[0048] Example 8 Compared with Example 1, in step S2, the technical parameters of ultrasonic treatment are: ultrasonic frequency 15 kHz, ultrasonic power 180 W, time 15 min. The rest are consistent with Example 1.

[0049] Example 9 Compared with Example 1, in step S2, the technical parameters of ultrasonic treatment are: ultrasonic frequency 12 kHz, ultrasonic power 240 W, time 25 min. The rest are consistent with Example 1.

[0050] Example 10 Compared with Example 1, in step S2, the technical parameters of the ultrasonic treatment are: ultrasonic frequency 14 kHz, ultrasonic power 150 W, and time 35 min. The rest are consistent with Example 1.

[0051] Comparative Example 1 Compared with Example 1, no stabilizing liquid was added, and the rest was the same as Example 1.

[0052] Comparative Example 2 Compared with Example 1, the stabilizing solution does not contain mercapto-β-cyclodextrin, and the rest is consistent with Example 1.

[0053] Comparative Example 3 Compared with Example 1, the stabilizing liquid does not contain water-soluble chitosan, and the rest is consistent with Example 1.

[0054] Comparative Example 4 Compared with Example 1, in step S2, mercapto-β-cyclodextrin and water-soluble chitosan are mixed at a mass ratio of 0.6:1. The rest are the same as in Example 1.

[0055] Comparative Example 5 Compared with Example 1, in step S2, mercapto-β-cyclodextrin and water-soluble chitosan are mixed at a mass ratio of 0.02:1. The rest are the same as in Example 1.

[0056] Comparative Example 6 Compared with Example 1, in step S2, the mass percentage of the stabilizing liquid is 2%. The rest is the same as Example 1.

[0057] Comparative Example 7 Compared with Example 1, in step S2, the mass percentage of the stabilizing liquid is 25%. The rest are the same as in Example 1.

[0058] Comparative Example 8 Compared with Example 1, in step S2, the ultrasonic frequency is adjusted to 25 kHz. The rest is consistent with Example 1.

[0059] Comparative Example 9 Compared with Example 1, in step S2, the ultrasonic frequency is adjusted to 8 kHz. The rest is consistent with Example 1.

[0060] Comparative Example 10 Compared with Example 1, in step S2, the ultrasonic power is adjusted to 500 W. The rest is consistent with Example 1.

[0061] Comparative Example 11 Compared with Example 1, in step S2, the ultrasonic power is adjusted to 50 W. The rest is consistent with Example 1.

[0062] The filtrate collected after the completion of step S3 in each embodiment and comparative example was sampled, and separated and extracted using non-ionic macroporous resin Amberlite XAD-16, using deionized water as eluent and ethanol as eluent, and then freeze-dried to obtain an ellagic acid-enriched product. Figure 1 As shown: UV test was performed on the ellagic acid standard and the ellagic acid extracted in Example 1, and both had maximum absorption at a wavelength of 254nm. HPLC test was performed on the ellagic acid standard and the ellagic acid extracted in Example 1, and the main test conditions were: Waters 1525 liquid chromatograph was selected, the chromatographic column was XBridge RC18 (250×4.6mm) with a particle size of 5μm, and the column temperature was 40°C; the mobile phase included mobile phase A and mobile phase B, mobile phase A was a formic acid aqueous solution containing 2mM ammonium acetate, and mobile phase B was methanol, with an injection volume of 10μL; the elution conditions were: flow rate 1.0mL / min, gradient elution, and the specific operation was: 0-3min, the volume fraction of mobile phase B was 10%; 3-6min, the volume fraction of the mobile phase increased from 10% to 30%; 6-12min, the volume fraction of mobile phase B was maintained at 30%; 12-25min, the volume fraction of mobile phase B increased from 30% to 100%. As shown Figure 2 As shown: the peak time of Example 1 is the same as the peak time of the ellagic acid standard. Figure 1 and Figure 2 It fully demonstrates that the filtrate obtained after the first fermentation in Example 1 is enriched with ellagic acid (the test results of the other examples are Figure 1 , Figure 2 The test results of Example 1 are similar, so they are not attached separately). The ellagic acid product was weighed, and the yield of ellagic acid in the walnut green peel was calculated according to the input amount of walnut green peel and the output amount of ellagic acid (yield = ellagic acid mass / walnut green peel mass*100%). The specific results are shown in Table 1.

[0063] The liquid after removing the insoluble matter from the filter membrane of each embodiment and comparative example was used as the test sample for UV test. Figure 3 As shown: the ultraviolet absorption spectrum of Example 1 is very close to that of urolithin A, and the maximum absorption wavelengths of both are 296 nm. The other examples also have detection results close to those of Example 1. In addition, a Waters 1525 liquid chromatograph (chromatographic column is XBridge RC18250×4.6 mm with a particle size of 5 μm) is selected to perform high performance liquid chromatography (HPLC) test to determine the content of urolithin A. The test operation adopted is the same as that of prior art 1; the maximum absorption wavelength is determined to be 305 nm by spectral scanning; as shown Figure 4As shown: the peak time of Example 1 is the same as that of the prior art 1, which shows that urolithin A is indeed generated, and the peak area is larger than that of the prior art 1, which shows that the content of urolithin A is higher, and the other embodiments also have test results close to those of Example 1. The content of urolithin A is quantitatively calculated through the high-performance liquid chromatograms of each embodiment and comparative example, and the specific results are shown in Table 1.

[0064] Table 1 ; From Table 1, we can see that: (1) From the test results of Examples 1 to 10, it can be seen that under the process parameters specified in the present invention, the yield of ellagic acid is 0.75%-0.84%, and at least 80% of the ellagic acid in the walnut green peel is extracted, and the industrialization prospect is very good; under this premise, after the second fermentation, the concentration of urolithin A is at least 40.2 μM, and can reach up to 54.3 μM. At this time, the conversion rate of urolithin A is about 18%.

[0065] Combining Comparative Examples 1, 2 and 3, it can be seen that if the stabilizing solution contains only water-soluble chitosan or only mercapto-β-cyclodextrin, although it has a certain positive effect on increasing the yield of ellagic acid, the degree of improvement is very limited. When the two substances are compounded and added according to a specific mass ratio, the yield of ellagic acid is significantly increased, and the concentration of urolithin A obtained is significantly increased. Obviously, the increase in the yield of ellagic acid and the increase in the conversion rate and yield of urolithin A in the present invention are the result of the mutual influence and synergistic effect of mercapto-β-cyclodextrin and chitosan, and both are indispensable.

[0066] (2) From the test results of Examples 1 to 4, Comparative Example 4 and Comparative Example 5, it can be seen that the mass ratio of mercapto-β-cyclodextrin to water-soluble chitosan has a significant effect on the yield of ellagic acid and the concentration of urolithin A. If the mass ratio is greater than or less than the range specified in the present invention, the yield of ellagic acid and the concentration of urolithin A are significantly reduced. Obviously, if the mass ratio of the two is inappropriate, the formed complex may have limited adsorption capacity for effective substances due to uneven composition or dispersion, which is not conducive to the extraction of ellagic acid and the formation of urolithin A.

[0067] (3) From the test results of Example 1, Example 5-Example 7, Comparative Example 6, and Comparative Example 7, it can be seen that the mass percentage of the stabilizing solution has a significant effect on the yield of ellagic acid and the concentration of urolithin A. If the mass percentage is too large, the viscosity may be too large to cause flocculation of ellagic acid, which is not conducive to the formation of ellagic acid, and may hinder the formation of urolithin A due to the unfavorable normal growth and metabolism of microorganisms. The preferred mass percentage of the stabilizing solution is 5%-20%. Within this range, the yield of ellagic acid is high and the conversion rate of urolithin A is high.

[0068] (4) By comparing the test results of Example 1 with Comparative Examples 8 to 11, it can be seen that when preparing the fermentation substrate, the frequency and power of the ultrasonic treatment have strict requirements. Too large or too small is not conducive to improving the yield of ellagic acid and the concentration of urolithin A. In combination with Examples 8 to 10, it can be seen that the preferred ultrasonic frequency is 12-15 kHz and the ultrasonic power is 150-240 W.

[0069] In summary, the present invention increases the concentration of the effective substance ellagic acid in the fermentation substrate and increases the concentration of the reactants by adding mercapto-β-cyclodextrin and chitosan when preparing the fermentation substrate, and reduces the influence of the increase of urolithin A concentration on the microbial activity and increases the amount of the product. Mercapto-β-cyclodextrin and chitosan interact with each other and synergize to significantly improve the conversion rate and product yield of urolithin A. Compared with the prior art, the present invention has great industrial prospects, is safe, environmentally friendly, and has a simple process flow.

[0070] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. Those skilled in the art may change, modify, replace and deform the above embodiments within the scope of the present invention. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples without contradiction.

Claims

1. A process for producing urolithin A, characterized in that: The steps are as follows: S1. Obtaining walnut green skin: Select healthy, pest-free and intact green skin walnuts, wash them, place them in a pile in a ventilated and cool place to let the green skin dry naturally, and peel them off after the green skin becomes soft and the moisture content is less than 8% to obtain the walnut green skin; S2, preparing a fermentation substrate: crushing the walnut green peel into a pulp and adding a stabilizing liquid, stirring the mixture evenly and then ultrasonically treating the mixture to obtain a fermentation substrate, wherein the stabilizing liquid is an aqueous solution of a compound of thiol-β-cyclodextrin and chitosan; S3, first fermentation: inoculating microbial liquid A into the fermentation substrate for anaerobic fermentation, performing solid-liquid separation after the fermentation is completed and collecting the filtrate, wherein the microbial liquid A is obtained by inoculating Bacillus Velezii and Bacillus coagulans into culture medium A respectively, activating them by shaking culture, and then mixing them; S4, second fermentation: inoculating microbial solution B into the filtrate for anaerobic fermentation again, wherein the microbial solution B is obtained by inoculating Enterococcus faecium into culture medium B; S5. Extraction: After the fermentation is completed, urolithin A is extracted.

2. The process for producing urolithin A according to claim 1, characterized in that: In step S2, the chitosan is water-soluble chitosan, and mercapto-β-cyclodextrin and water-soluble chitosan are mixed in a mass ratio of (0.1-0.4):1 to form the composite.

3. The process for producing urolithin A according to claim 2, characterized in that: In step S2, the mass percentage of the stabilizing liquid is 5%-20%.

4. The process for producing urolithin A according to claim 3, characterized in that: In step S2, the material-liquid ratio is 1:(12-20), the ultrasonic frequency is 12-15kHz, the ultrasonic power is 150-240W, and the treatment time is 15-35min. During this period, the temperature of the mixture is maintained at 58-65°C. After obtaining the fermentation substrate, it is naturally cooled to room temperature before proceeding to the next step.

5. The process for producing urolithin A according to claim 1, characterized in that: In step S3, culture medium A is an MRS liquid culture medium modified by corn syrup, Bacillus velez and culture medium A are cultured at (0.5-0.8) g / 100 ml to obtain Bacillus velez liquid, and Bacillus coagulans and culture medium A are cultured at (0.25-0.55) g / 100 ml to obtain Bacillus coagulans liquid. After the culture, the Bacillus velez liquid and the Bacillus coagulans liquid are mixed at a volume ratio of 1:1 to obtain microbial liquid A, and the inoculation amount of the microbial liquid A in the fermentation substrate is 2 v%.

6. The process for producing urolithin A according to claim 5, characterized in that: In step S3, the temperature of anaerobic fermentation is 30-35°C and the time is 24-48 hours.

7. The process for producing urolithin A according to claim 1, characterized in that: In step S4, the Enterococcus faecium was purchased from the China General Microbiological Culture Collection Center with a collection number of CGMCCNO.24964. The specific components of culture medium B were: peptone 16.0 g / l, yeast extract 7.0 g / l, potassium chloride 5.0 g / l, starch 1.0 g / l, glucose 1.0 g / l, sodium pyruvate 1.0 g / l, arginine 1.0 g / l, sodium succinate 0.5 g / l, cysteine ​​hydrochloride Salt 0.5g / l, sodium bicarbonate 0.4g / l, ferric pyrophosphate 0.5g / l, hemin chloride 0.005g / l, vitamin K0.0005g / l, sodium thioglycolate 0.5g / l, dithiothreitol 1.0g / l, the microbial liquid B is obtained by inoculating the Enterococcus faecium into the culture medium B and culturing at pH 6.8-7.0 and 37°C for 24h, and the inoculation amount of the microbial liquid B in the filtrate is 2v%.

8. The process for producing urolithin A according to claim 7, characterized in that: In step S4, the temperature of anaerobic fermentation is 37° C., the time is 48-55 h, and the pH is 6.8-7.

0.

9. The process for producing urolithin A according to claims 1-8, characterized in that: In step S3 and step S4, low-speed stirring is continued, and the rate of the low-speed stirring is 80-120 rpm.

10. The process for producing urolithin A according to claim 9, characterized in that: In step S5, the specific operation of the extraction is: extracting the obtained fermentation liquid with an extractant, wherein the extractant is a mixture of CH3CN:H2O:HCOOH in a volume ratio of 80:19.9:0.1, and the extract is freeze-dried and re-dissolved with ethyl acetate, and then filtered through a 0.22 μm filter membrane.

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