Method for preparing small-molecular hyaluronic acid by enzyme membrane reactor and application of small-molecular hyaluronic acid

Abstract

The invention discloses a method for preparing micromolecular hyaluronic acid by an enzyme membrane reactor and application of the micromolecular hyaluronic acid. Wherein the small molecular weight hyaluronic acid comprises low molecular weight hyaluronic acid and hyaluronic acid oligosaccharide. According to the invention, the bioreactor and the membrane separation device are coupled, so that the one-step preparation of two kinds of micromolecular hyaluronic acid with different molecular weight distributions is realized, the molecular structure is complete, the preparation conditions are mild and efficient, and the method is easy to apply to industrial production. The micromolecular hyaluronic acid prepared by the method provided by the invention can effectively improve the capacity of removing DPPH free radicals and hydroxyl free radicals, has certain oxidation resistance, and can be used for improving the intestinal oxidative stress state and / or relieving intestinal inflammation.

Description

Technical Field

[0001] The present invention belongs to the field of food processing technology, and relates to a method for preparing small molecule hyaluronic acid by using an enzyme membrane reactor and its application, and specifically relates to a method for preparing small molecule hyaluronic acid by using an enzyme membrane reactor and its application in regulating intestinal health, and especially relates to a method for preparing small molecule hyaluronic acid by using enzyme membrane coupling technology, and the application of hyaluronic acids of different molecular weights in regulating intestinal health and / or with ulcerative colitis. Background Art

[0002] Hyaluronic acid (HA), as the main component of the extracellular matrix, is widely distributed in human tissues and body fluids. It exhibits unique biological activity and has functions such as antioxidant and immune regulation.

[0003] HA can be divided into three categories according to molecular weight differences: high molecular weight hyaluronic acid (HMWHA, Mw ≥ 100kDa), low molecular weight hyaluronic acid (LMWHA, Mw is 10-100kDa) and hyaluronic acid oligosaccharides (O-HA, Mw < 10kDa). HA of different molecular weights will show completely different biological activities. Enzymatic hydrolysis is an efficient method for preparing small molecule HA with biological activity, because hyaluronidase has strong specificity and only breaks glycosidic bonds to produce small molecule HA without destroying the monosaccharide structure of HA. It has the advantages of mild reaction and controllable degradation.

[0004] Enzymatic Membrane Reactor (EMR) combines bioreactor with membrane separation technology and is often used in the production of proteins, peptides, and oligosaccharides. It can be divided into intermittent EMR and continuous EMR according to its operation mode. At present, the industrial preparation of small molecule HA is mainly through intermittent enzymatic production, which uses the traditional model of enzymatic hydrolysis and separation and purification processes in batches. It is difficult to take into account the preparation of HA of different molecular weights at the same time, which brings difficulties to the industrial production of HA.

[0005] Currently, HA has been approved by the National Health Commission as a new type of food raw material and is allowed to be added to ordinary foods. In recent years, most HA-related research has focused on the field of skin beauty. As the first barrier exposed to the external bacterial environment, the anti-inflammatory and barrier repair effects of exogenous HA on the skin barrier are widely recognized. Similarly, the intestine is also an important tissue for the human body to contact the outside world. The digestive tract and gastrointestinal mucosal barrier are also in contact with external microorganisms for a long time, and are an important entrance to resist foreign bodies from invading the human body. As a type of food-borne polysaccharide, exogenous HA is orally ingested and digested and absorbed in the intestine, which can increase the precursors of HA synthesis in the body and increase the content of endogenous HA. In addition, HA of different molecular weights interact with intestinal cells through different receptors, participate in inflammation regulation or regulate cell functions between cells and ECM to regulate intestinal homeostasis and health.

[0006] Through the search found:

[0007] 1. Patent Publication No. CN 111893151 A provides a method and application for continuous production of low molecular weight hyaluronic acid. By coupling an enzyme catalytic reactor with an ultrafiltration membrane device, a method for continuous production of low molecular weight hyaluronic acid is realized, and the production cost of the method is low and the molecular weight distribution of the product is narrow. The method provides a new method for preparing small molecule hyaluronic acid by enzyme membrane coupling technology, but the method can only prepare hyaluronic acid with a molecular weight distribution, and does not explore the application of hyaluronic acid in the food field.

[0008] 2. Tangyou Mao et al. explored the mechanism of 35kDa HA in the gastrointestinal epithelial barrier. By constructing a Citrobacter rodentium infection and DSS-induced colitis model, the experimental results showed that 35kDa HA can increase the colonization of beneficial bacteria such as Eckermannia, regulate the abundance and diversity of intestinal colonization flora, and improve intestinal inflammation caused by pathogen infection or chemical induction. However, this document only focuses on the effect of hyaluronic acid with a molecular weight distribution of 35kDa on improving intestinal inflammation, and does not explore the difference in the repair effect of hyaluronic acid with other molecular weight distributions compared with 35kDa low molecular weight hyaluronic acid on intestinal inflammation.

[0009] 3. Patent Publication No. CN 114191447A provides a use of hyaluronic acid for improving intestinal flora imbalance. The patent selects 25kDa hyaluronic acid from low molecular weight hyaluronic acid and high molecular weight hyaluronic acid to effectively regulate the abundance of beneficial bacteria such as Eckermanella and Bacteroides in the intestine, plays a prebiotic role, and has the effect of improving intestinal inflammation. However, this method does not cover the effect of ultra-small molecular weight hyaluronic acid such as hyaluronic acid oligosaccharides on intestinal inflammation.

[0010] By comparison, the patent application of the present invention is essentially different from the above-mentioned published patents and documents. Summary of the invention

[0011] The purpose of the present invention is to solve the above problems and provide a method for preparing small molecule hyaluronic acid by using an enzyme membrane reactor and its use. This method can be used in the enzymatic hydrolysis process of high molecular weight hyaluronic acid to obtain small molecule HA products of different molecular weights, including low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides, which can better improve the commercial value of HA.

[0012] The objective of the present invention is achieved through the following technical solutions:

[0013] The present invention provides a method for preparing and producing small molecule hyaluronic acid using an enzyme membrane reactor, the method comprising the following steps:

[0014] S1. Dissolution of high molecular weight hyaluronic acid: dissolve food grade high molecular weight hyaluronic acid in ultrapure water (on a magnetic stirrer) and swell evenly;

[0015] S2, hydrolysis of hyaluronic acid in enzyme membrane reactor: adding the high molecular weight hyaluronic acid solution obtained in step S1 into the enzyme membrane bioreactor device, and after the temperature in the tank body reaches a constant, adding hyaluronidase to carry out enzymolysis reaction; during the enzymolysis process, the hyaluronic acid oligosaccharide solution is filtered through an ultrafiltration membrane separation device, and the molecular weight of the high molecular weight hyaluronic acid in the reaction tank gradually decreases to a low molecular weight hyaluronic acid solution as the enzymolysis time increases (that is, the hyaluronic acid oligosaccharides produced by enzymolysis pass through the ultrafiltration membrane of the ultrafiltration membrane separation device to obtain a hyaluronic acid oligosaccharide filtrate; the hyaluronic acid aqueous solution retained by the ultrafiltration membrane separation device is refluxed into the reaction tank for continuous enzymolysis, and as the enzymolysis time increases, the molecular weight gradually decreases to obtain a low molecular weight hyaluronic acid retained solution); realizing a one-step method for preparing two small molecule hyaluronic acid solutions with different molecular weight distributions;

[0016] S3, drying of small molecule hyaluronic acid solution: the small molecule hyaluronic acid retentate and hyaluronic acid oligosaccharide filtrate obtained in step S2 are dehydrated and dried respectively to obtain low molecular weight hyaluronic acid and hyaluronic acid oligosaccharide. It can be seen that the present invention collects two parts of polysaccharides at the filtering end and the retentate end, which are hyaluronic acid oligosaccharide and low molecular weight hyaluronic acid.

[0017] Preferably, the concentration of the high molecular weight hyaluronic acid solution obtained in step S1 is 1-5 g / L.

[0018] Preferably, in step S2, the amount of hyaluronidase added is 1.0% to 5.0% of the mass of the high molecular weight hyaluronic acid in the reaction system.

[0019] Preferably, in step S1, the molecular weight of the high molecular weight hyaluronic acid is ≥120 kDa.

[0020] Preferably, in step S2, the shear rate of the enzyme membrane bioreactor on the substrate liquid is 200-600 rpm.

[0021] Preferably, in step S2, the pore size of the ultrafiltration membrane is 20-100 kDa.

[0022] Preferably, in step S2, the temperature during the enzymatic hydrolysis reaction is 25-50° C., and the pH value is 5.0-7.0.

[0023] Preferably, in step S2, the time during the enzymatic hydrolysis reaction is 1.0 h to 4.0 h.

[0024] Preferably, after the enzymatic hydrolysis reaction starts (hyaluronidase is added), the ultrafiltration membrane separation device is turned on to filter through the ultrafiltration membrane; after the filtration starts, ultrapure water is added to the reaction system every 15 minutes according to the volume of the filtered solution.

[0025] Preferably, the transmembrane pressure of the ultrafiltration membrane separation process is 0.05 to 0.20 mPa.

[0026] Preferably, in step S3, the dehydration and drying includes spray drying and freeze drying.

[0027] The present invention also provides an enzyme-membrane coupling device for preparing and separating small molecule hyaluronic acid used in the above method, comprising an enzyme-catalyzed bioreactor and a flat-plate polyethersulfone ultrafiltration membrane separation device, wherein the enzyme-catalyzed bioreactor and the ultrafiltration membrane separation device are connected by a pipeline.

[0028] The present invention also provides a small molecule hyaluronic acid obtained by the above method, comprising low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides, wherein the average molecular weight of the low molecular weight hyaluronic acid is between 1 and 20 kDa, and the average molecular weight of the hyaluronic acid oligosaccharides is less than 3 kDa. The embodiment shows that 10-50 kDa has the ability to significantly improve the activity of three antioxidant enzymes, among which the hyaluronic acid oligosaccharides less than 3 kDa have an effect second only to low molecular weight hyaluronic acid, and the repair effect is good compared with high molecular weight hyaluronic acid.

[0029] The present invention also provides an application of the small molecule hyaluronic acid obtained by the above method in preparing food for removing human free radicals. Compared with high molecular weight hyaluronic acid, the small molecule hyaluronic acid prepared by the present invention has significantly increased the ability to remove DPPH free radicals and hydroxyl free radicals (p < 0.05), indicating that the small molecule hyaluronic acid has certain antioxidant properties.

[0030] The present invention also provides an application of the small molecule hyaluronic acid obtained by the above method in preparing a food for improving intestinal oxidative stress and / or relieving intestinal inflammation. The present invention has found that hyaluronic acid with different molecular weights can be used for the application of functional food for improving the intestinal inflammatory state of patients.

[0031] In summary, the present invention provides a method for preparing small molecule hyaluronic acid by enzyme-membrane coupling technology, comprising the following steps: adding ultrapure water to high molecular weight hyaluronic acid to uniformly swell overnight to obtain a high molecular weight hyaluronic acid aqueous solution; adding the hyaluronic acid solution to an enzyme membrane reactor to continuously perform enzymatic hydrolysis reaction, and separating hyaluronic acid oligosaccharides and low molecular weight hyaluronic acid with different molecular weights through a membrane separation device; the pore size of the ultrafiltration membrane is any one between 20-100 kDa; the small molecule hyaluronic acid filtrate and the retentate are dehydrated and dried to obtain two small molecule hyaluronic acids with different molecular weight distributions.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The present invention Hyaluronidase is sheep testis hyaluronidase extracted from animal tissue , the reaction conditions are mild and the specificity is strong. It can specifically cut the glycosidic bond to produce small molecule HA without destroying the monosaccharide unit structure of HA, thus maintaining the structural integrity of small molecule hyaluronic acid;

[0034] 2) The present invention is realized Simultaneous enzymatic hydrolysis and separation of degradation products , hyaluronidase and high molecular weight hyaluronic acid (HA) substrate are retained in the reactor Continuous enzymatic reaction , and the low molecular weight (LMW-HA) produced by enzymatic hydrolysis is separated from the oligosaccharide product (O-HA) in real time through a membrane filtration device, thereby obtaining a one-step process for preparing hyaluronic acid with different molecular weight distributions. The preparation conditions are mild, the reaction is controllable, and it is suitable for industrial production.

[0035] 3) The results of the embodiment show that the main functional groups of the small molecule hyaluronic acid provided by the present invention are consistent with those of the high molecular weight hyaluronic acid before enzymatic hydrolysis in the infrared spectrum, indicating that the disaccharide unit structure of the small molecule hyaluronic acid obtained by the present invention is complete and no other branches are produced; the low molecular weight hyaluronic acid obtained by the present invention can be obtained by high performance liquid chromatography (equipped with a diode array and a differential detector) with an average molecular weight distribution between 10-50 kDa, which is a biologically active hyaluronic acid; the hyaluronic acid oligosaccharide obtained by the present invention can be obtained by matrix-assisted laser desorption tandem time-of-flight mass spectrometry with an average molecular weight distribution of less than 3 kDa, which is an oligosaccharide fragment with a very small molecular weight distribution, and is an oligosaccharide mixture of hyaluronic acid combinations of disaccharides to decasaccharides.

[0036] 4) The test results show that hyaluronic acid is an important component of the body's extracellular matrix, belongs to food-derived polysaccharides, has biocompatibility, and is therefore highly safe for consumption. The present invention compares three types of hyaluronic acid with different molecular weight distributions, which are administered orally to DSS-induced ulcerative colitis model mice, and after a period of drug administration intervention, the intestinal inflammation model is improved. The experimental results show that 10-50kDa hyaluronic acid can significantly enhance the antioxidant capacity of colon tissue. It can inhibit the activity of enzymes and effectively reduce the changes in intestinal permeability, thereby improving the function of intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0038] Figure 1 This is a schematic diagram of a device for preparing small molecule hyaluronic acid by using an enzyme membrane bioreactor in the present invention;

[0039] Figure 2 The infrared spectra of three different molecular weight hyaluronic acids before and after enzymatic hydrolysis in Example 1;

[0040] Figure 3 The infrared spectra of three different molecular weight hyaluronic acids before and after enzymatic hydrolysis in Example 2;

[0041] Figure 4 The infrared spectra of three different molecular weight hyaluronic acids before and after enzymatic hydrolysis in Example 3;

[0042] Figure 5 This is the GPC spectrum of the low molecular weight hyaluronic acid retained by the enzyme membrane bioreactor after enzymatic hydrolysis in Example 1;

[0043] Figure 6 This is the GPC spectrum of hyaluronic acid oligosaccharides filtered through the enzyme membrane bioreactor after enzymatic hydrolysis in Example 1;

[0044] Figure 7 This is the GPC spectrum of the low molecular weight hyaluronic acid retained by the enzyme membrane bioreactor after enzymatic hydrolysis in Example 2;

[0045] Figure 8 This is the GPC spectrum of hyaluronic acid oligosaccharides filtered through the enzyme membrane bioreactor after enzymatic hydrolysis in Example 2;

[0046] Fig. 9 This is the GPC spectrum of the low molecular weight hyaluronic acid retained by the enzyme membrane bioreactor after enzymatic hydrolysis in Example 3;

[0047] Fig.10 This is the GPC spectrum of hyaluronic acid oligosaccharides filtered through the enzyme membrane bioreactor after enzymatic hydrolysis in Example 3;

[0048] Fig.11The change of the scavenging ability of hyaluronic acid with different molecular weights on DPPH free radicals with the change of concentration in Test Example 1;

[0049] Fig.12 The change of the scavenging ability of hyaluronic acid with different molecular weights on hydroxyl free radicals with the change of concentration in Test Example 1;

[0050] Fig.13 The effect of different molecular weight hyaluronic acid on the disease activity index of enteritis mice in Test Example 2;

[0051] Fig.14 To test the effect of different molecular weight hyaluronic acid on the pathological results of enteritis mice in Example 2;

[0052] Fig.15 The effect of different molecular weight hyaluronic acid on the FITC-dextran content in the serum of enteritis mice in Test Example 2;

[0053] Fig.16 The effect of different molecular weight hyaluronic acid on GPx enzyme activity in the colon tissue of enteritis mice in test example 2;

[0054] Fig.17 The effect of different molecular weight hyaluronic acid on T-SOD enzyme activity in the colon tissue of enteritis mice in test example 2;

[0055] Fig.18 This is the effect of hyaluronic acid of different molecular weights in Test Example 2 on CAT enzyme activity in the colon tissue of enteritis mice. DETAILED DESCRIPTION

[0056] The present invention provides a method for preparing small molecule hyaluronic acid by using an enzyme membrane bioreactor, characterized in that it comprises the following steps:

[0057] 1) Dissolve food grade hyaluronic acid in ultrapure water and swell evenly on a magnetic stirrer to a concentration of 1-5 g / L.

[0058] 2) adding a high molecular weight hyaluronic acid solution to an enzyme membrane bioreactor device, setting a constant temperature, and after the temperature in the tank reaches a constant, adding a certain mass of hyaluronidase for enzymatic hydrolysis, filtering the hyaluronic acid oligosaccharide filtrate obtained in the enzymatic hydrolysis process through an ultrafiltration membrane separation device, and the high molecular weight hyaluronic acid in the reaction tank gradually decreases to a low molecular weight hyaluronic acid retentate as the enzymatic hydrolysis time increases, and two small molecular weight hyaluronic acid solutions with different molecular weight distributions can be prepared in one step; the pore size of the ultrafiltration membrane is any one between 20-100 kDa;

[0059] 3) Dehydrating and drying the small molecular weight hyaluronic acid retentate to obtain low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides.

[0060] The hyaluronidase used in the present invention is sheep testicular hyaluronidase extracted from animal tissue, and its suitable temperature is 25-50°C, and its suitable pH value is 5.0-7.0. The enzyme activity of the hyaluronidase used in the present invention is preferably 300-500U / mg, and more preferably 400U / mg. The hyaluronidase reaction conditions in the present invention are mild, and the specificity is good. It can specifically hydrolyze the glycosidic bonds in hyaluronic acid. The product contains a reducing end, which makes the small molecule hyaluronic acid have a certain antioxidant property, and can improve the body's anti-inflammatory effect.

[0061] The molecular weight of the food-grade sodium hyaluronate selected in the present invention is preferably ≥1.2×10 6 Da is more preferably 2.0×10 6 The hyaluronic acid with this molecular weight selected by the present invention has stable quality and is easy to store for a long time, and the degradation of high molecular hyaluronic acid by this method can control the trend of molecular weight decrease by controlling the enzymatic hydrolysis time.

[0062] The present invention dissolves high molecular weight food grade hyaluronic acid in ultrapure water, swells to a transparent uniform 0.2% aqueous solution on a magnetic stirrer overnight, adds 0.2% (m / v) of hyaluronic acid aqueous solution to an enzyme membrane reactor device, performs enzymatic action under the action of the hyaluronidase, and the hyaluronic acid oligosaccharides produced under enzymolysis pass through an ultrafiltration membrane to obtain a hyaluronic acid oligosaccharide filtrate, and the hyaluronic acid aqueous solution is retained by an ultrafiltration membrane separation device, and the molecular weight is gradually reduced under the ongoing enzymolysis to obtain a low molecular weight hyaluronic acid retentate; A one-step method is implemented to prepare two small molecule hyaluronic acid solutions with different molecular weight distributions. In the present invention, the ultrafiltration membrane pore size used is any one between 20-100kDa, more preferably 50kDa, and the present invention selects the hyaluronic acid of the molecular weight to be able to maintain the enzymatic reaction of HA in the connected operation of the enzyme membrane reactor, and the hyaluronic acid oligosaccharides are filtered out of the enzyme membrane reactor under continuous enzymolysis, and the low molecular weight hyaluronic acid is refluxed in the reaction tank for continuous enzymolysis. In the present invention, the amount of hyaluronidase added is preferably 2.0%-8.0% of the mass volume of high molecular weight hyaluronic acid, and more preferably 5.0%. In the present invention, the temperature of the enzymolysis is preferably 25-50°C, and more preferably 37°C; the pH value of the enzymolysis is preferably 5.0-7.0°C, and more preferably 6.0. In the present invention, the enzymolysis time is preferably 1.0h-4.0h, and preferably 2.0h.

[0063] The enzymolysis conditions of the present invention are mild. By screening the enzymolysis conditions, the continuous degradation of the molecular weight of the feed liquid retained by the ultrafiltration membrane separation device during the enzymolysis process can be controlled and regulated until the molecular weight is reduced to between 10-50kDa, reaching the molecular weight interval of low molecular weight hyaluronic acid, which is the molecular weight distribution of biologically active hyaluronic acid. Among the parameters of the enzyme membrane reactor in the present invention, the transmembrane pressure is preferably 0.05-2.0mPa, more preferably 0.15mPa; the shear rate is preferably 200-800rpm, more preferably 200rpm.

[0064] The parameter optimization of the enzyme membrane reactor of the present invention is suitable for the separation of hyaluronic acid oligosaccharides, so that the hyaluronic acid oligosaccharides produced by enzymolysis are filtered out of the enzymolysis reaction tank under a certain pressure, thereby improving the utilization rate of hyaluronic acid oligosaccharides. The present invention prepares small-molecule hyaluronic acid by coupling an enzymolysis bioreactor and an ultrafiltration membrane separation device, realizes the separation of low-molecular-weight hyaluronic acid and hyaluronic acid oligosaccharides during the enzymolysis reaction, prepares two small-molecule hyaluronic acids with different molecular weight distributions by a one-step method, and the preparation process is simple, the structure of the small-molecule hyaluronic acid obtained by membrane separation is complete, and the structure is consistent with that of high-molecular-weight hyaluronic acid, which greatly reduces the production cost of small-molecule hyaluronic acid.

[0065] The present invention provides a device for preparing small molecule hyaluronic acid using an enzyme membrane reactor, comprising an enzyme bioreactor, an ultrafiltration membrane, and a flat membrane separation device connected in sequence; the enzyme bioreactor and the flat membrane separation device are connected by a pipeline, and the pipeline is provided with a feed pipe and a reflux pipe; the ultrafiltration membrane is fixed by two flat membrane separation devices through a knob, and ultrapure water is used to connect and run for 30 minutes under preferred pressure conditions before each enzymatic hydrolysis reaction before enzyme membrane coupling production is performed. The schematic diagram of the enzyme membrane reactor of the present invention is shown in Figure 1 The bioreactor is connected to the membrane separation device through a PU hose. The enzymatic hydrolysis reaction process ensures constant reaction conditions by controlling the transmembrane pressure, stirring rate, pH, and temperature. Low molecular weight hyaluronic acid larger than the pore size of the ultrafiltration membrane will flow back into the reaction tank to continue the enzymatic hydrolysis reaction. At the same time, the hyaluronic acid oligosaccharides produced by the enzymatic hydrolysis will be filtered and separated from the reaction system during the reaction.

[0066] The method of the above scheme provided by the present invention obtains low molecular weight hyaluronic acid retentate and hyaluronic acid oligosaccharide filtrate through a membrane separation device and spray-dries them through a spray dryer device. The device parameter setting temperature of the spray dryer is preferably 160-180°C, and more preferably 170°C; the peristaltic pump speed is preferably 60-80%, and more preferably 70%. The parameter setting of the spray dryer device in the present invention is suitable for the drying of small molecule hyaluronic acid, and can realize the dehydration and drying of small molecule hyaluronic acid filtrate with low concentration to form dry powder, which can more quickly improve the preparation process efficiency of small molecule hyaluronic acid and is more suitable for industrial production.

[0067] The present invention provides the application of the small molecule hyaluronic acid obtained by the above technical solution in food processing technology.

[0068] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0069] <Preparation Example>: Preparation of small molecule hyaluronic acid

[0070] Example 1

[0071] 0.2% (m / v) hyaluronic acid aqueous solution (Mw>1.2×10 6 Da), add 1L HA aqueous solution to a 1.5L enzyme catalytic reaction tank, adjust the enzyme membrane reactor equipment to control the temperature at 37°C, the pH value at 6.0±0.1, and the stirring speed at 200rpm to make the substrate solution uniform, then add hyaluronidase in an amount of 5% by weight of high molecular weight hyaluronic acid, open the membrane separation device at the beginning of enzymolysis, set the transmembrane pressure to 0.15mPa, and during the enzymolysis for 1h, hyaluronic acid oligosaccharides with a molecular weight of less than 3kDa can be obtained by ultrafiltration membrane separation. After enzymolysis for 1h, the molecular weight in the reaction tank is reduced to 100-150kDa, and then the filtrates obtained from the two parts are passed through a spray dryer to obtain small molecular weight hyaluronic acid dry powders with two molecular weight distributions.

[0072] The high molecular weight hyaluronic acid before and after enzymatic hydrolysis and the small molecular weight hyaluronic acid after enzymatic hydrolysis were scanned and analyzed by infrared spectrometer. The results showed that Figure 2 It can be seen that the main functional groups of the hyaluronic acid molecular structure before and after enzymatic hydrolysis are consistent, indicating that the monosaccharide units of the hyaluronic acid obtained before and after enzymatic hydrolysis remain unchanged.

[0073] The experimental results show that ( Figure 5 , 6 ) According to the log, the average molecular weight of low molecular weight hyaluronic acid is 130.0 kDa, and the PDI is 1.35, which belongs to the molecular weight distribution range of low molecular weight hyaluronic acid. The average molecular weight of hyaluronic acid oligosaccharide pair is 1.6 kDa, and the PDI is 1.10, which belongs to the hyaluronic acid oligosaccharide mixture.

[0074] Example 2

[0075] A 0.2% (m / v) hyaluronic acid aqueous solution is swollen on a magnetic stirrer, and 1L HA aqueous solution is added to a 1.5L enzyme catalytic reaction tank. The enzyme membrane reactor equipment is adjusted to control the temperature at 37°C, the pH value at 6.0±0.1, and the stirring speed is 200rpm to make the substrate solution uniform. Then, 5% by weight of hyaluronidase is added, and the membrane separation device is turned on at the beginning of enzymolysis, and the transmembrane pressure is set to 0.15mPa. During the 2h enzymolysis process, hyaluronic acid oligosaccharides with a molecular weight of less than 3kDa can be obtained by ultrafiltration membrane separation. After 2h of enzymolysis, the molecular weight in the reaction tank is reduced to about 10-50kDa. The filtrates obtained from the two parts are then passed through a spray dryer to obtain small molecule hyaluronic acid dry powders with two molecular weight distributions.

[0076] The high molecular weight hyaluronic acid before and after enzymatic hydrolysis and the small molecular weight hyaluronic acid after enzymatic hydrolysis were scanned and analyzed by infrared spectrometer. The results showed that Figure 3 It can be seen that the main functional groups of the hyaluronic acid molecular structure before and after enzymatic hydrolysis are consistent, indicating that the monosaccharide units of the hyaluronic acid obtained before and after enzymatic hydrolysis remain unchanged.

[0077] The experimental results show that ( Figure 7 , 8 ) shows that the average molecular weight of low molecular weight hyaluronic acid is 36.7 kDa, and PDI is 2.12, which belongs to the molecular weight distribution range of biologically active hyaluronic acid. The average molecular weight of hyaluronic acid oligosaccharide pair is 1.7 kDa, and PDI is 1.14, which belongs to hyaluronic acid oligosaccharide mixture.

[0078] Example 3

[0079] A 0.2% (m / v) hyaluronic acid aqueous solution is swollen on a magnetic stirrer, and 1L HA aqueous solution is added to a 1.5L enzyme catalytic reaction tank. The enzyme membrane reactor equipment is adjusted to control the temperature at 37°C, the pH value at 6.0±0.1, and the stirring speed is 200rpm to make the substrate solution uniform. Then, 5% by weight of hyaluronidase is added, and the membrane separation device is turned on at the beginning of enzymolysis, and the transmembrane pressure is set to 0.15mPa. During the 4h enzymolysis process, hyaluronic acid oligosaccharides with a molecular weight of less than 3kDa can be obtained by ultrafiltration membrane separation. After 4h of enzymolysis, the molecular weight in the reaction tank is reduced to about 10-50kDa. The filtrates obtained from the two parts are then passed through a spray dryer to obtain small molecule hyaluronic acid dry powders with two molecular weight distributions.

[0080] The high molecular weight hyaluronic acid before and after enzymatic hydrolysis and the small molecular weight hyaluronic acid after enzymatic hydrolysis were analyzed by infrared scanning. The results showed that Figure 4 It can be seen that the main functional groups of the hyaluronic acid molecular structure before and after enzymatic hydrolysis are consistent, indicating that the monosaccharide units of the hyaluronic acid obtained before and after enzymatic hydrolysis remain unchanged.

[0081] The experimental results show that ( Fig. 9 , 10 ) shows that the average molecular weight of low molecular weight hyaluronic acid is 18.5 kDa, and PDI is 1.71, which belongs to the molecular weight distribution range of biologically active hyaluronic acid. The average molecular weight of hyaluronic acid oligosaccharide pair is 2.0 kDa, and PDI is 1.20, which belongs to hyaluronic acid oligosaccharide mixture.

[0082] <Test example>

[0083] Test Example 1: Study on the Antioxidant Activity of HA with Different Molecular Weights

[0084] The following experiments were conducted using "2000kDa food-grade hyaluronic acid raw material, 35kDa low molecular weight hyaluronic acid, and 3kDa hyaluronic acid oligosaccharide":

[0085] 1.1 Changes in scavenging rate of HA with different molecular weights on DPPH free radicals with concentration

[0086] Experimental design: 0.5 mL of HA solution of different concentrations, 0.1 mL (400 μmol / L) DPPH solution, and 1.0 mL of distilled water were added to the EP tube, mixed, and placed in a dark place at room temperature to react for 30 minutes. The absorbance was measured at 517 nm using a UV spectrophotometer.

[0087] Calculation formula: DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100%. Wherein, A0 is the absorbance when water replaces the sample, A1 is the absorbance of the sample experiment, and A2 is the absorbance of the sample blank experiment.

[0088] Experimental analysis: attached Fig.11 It can be seen that the two small molecule hyaluronic acids with different molecular weight distributions prepared by the enzyme membrane reactor showed a concentration-dependent effect on the scavenging rate of DPPH. When the HA concentration reached 1.8 mg / mL, the scavenging rates of LMW-HA (48.88%) and O-HA (46.07%) for nitrogen free radicals (DPPH) were significantly higher than those of HMW-HA (18.91%) (p < 0.05), but the DPPH free radical scavenging ability of the three different molecular weight hyaluronic acids was significantly lower than that of the positive ascorbic acid solution (p < 0.05), indicating that small molecule hyaluronic acid has a certain ability to scavenge DPPH free radicals compared with high molecular weight hyaluronic acid.

[0089] 1.2 Changes in the scavenging rate of hydroxyl radicals by HA of different molecular weights with concentration

[0090] Experimental design: 300 μL o-phenanthroline ethanol solution, 300 μL sodium phosphate buffer, and 200 μL FeSO4 solution were added to the EP tube, and 200 μL of sample solution of different concentrations were added after thorough mixing. At the same time, negative control tubes and normal tubes were set up, without adding sample solution, and 200 μL H2O2 solution was added to each tube. H2O2 solution was not added to the normal tube, and the volume was supplemented with an equal volume of distilled water, and reacted in a 37°C water bath for 30 minutes, and the absorbance at 536 nm was measured by a UV spectrophotometer.

[0091] Calculation formula: Hydroxyl free radical scavenging rate (%) = [(A2-A1) / (A0-A1)] × 100%. Where A0 is the absorbance value of the normal tube; A1 is the absorbance value of the negative control tube, and A2 is the absorbance value of the sample tube.

[0092] Experimental analysis: Through the Fig.12 It can be seen that the scavenging rate of hydroxyl radicals by HA at lower concentrations did not change significantly, showing a slowly rising trend. As the concentration increased to 1.2 mg / mL, the scavenging rate of free radicals by small molecule HA increased significantly, and then gradually leveled off when the concentration was greater than 1.8-2.1 mg / mL. The scavenging rates of hydroxyl radicals by LMW-HA and O-HA were significantly higher than those by HMW-HA (p < 0.05). However, the three HAs of different molecular weights still had significant differences compared with ascorbic acid (p < 0.05), indicating that small molecule hyaluronic acid has a certain ability to scavenge hydroxyl radicals compared with high molecular weight hyaluronic acid.

[0093] Summary: Small molecule hyaluronic acid of different molecular weights can significantly enhance the ability to scavenge free radicals in the human body and possess a certain degree of antioxidant properties.

[0094] Test Example 2: Effects of Hyaluronic Acids of Different Molecular Weights on Intestinal Inflammation

[0095] The following experiments were conducted using "2000kDa food-grade hyaluronic acid raw material, 35kDa low molecular weight hyaluronic acid, and 3kDa hyaluronic acid oligosaccharide":

[0096] Experimental design: 40 6-8 week old ICR mice were fed adaptively in a sterile clean environment for 7 days in the previous week. They were randomly divided into 5 groups, 8 mice in each group. The specific treatment plan is shown in Table 1:

[0097] Table 1 Dosage design table

[0098]

[0099] During the experiment, the weight of mice was measured at fixed points every day, the fecal status of mice was observed on the same day, and the disease activity index (DAI) of mice was scored according to Table 2 below. On the 14th day, the mice were deprived of food and water for 4 hours before euthanasia. FITC-dextran (80 mg / kg) was given to each group of mice. Blood was collected through the eye sockets before dissection, and the changes in intestinal barrier permeability were evaluated by the fluorescence intensity in the serum. Euthanasia was performed after approval by the Experimental Animal Ethics and Use Committee of Shanghai Jiao Tong University. After sacrifice, colon tissue was obtained, fixed with 10% formaldehyde solution, embedded in paraffin, and stained with conventional hematoxylin-eosin for colon tissue pathology analysis.

[0100] Table 2 DAI scoring criteria

[0101] score Weight loss rate (%) Stool characteristics Blood in stool 0 ＜1 Molded particles No bleeding 1 1-5 2 6-10 Semi-loose stools that do not stick to the anus Occult blood in stool 3 11-15 4 ＞15 Loose stools, sticking to the anus Naked bloody stool

[0102] 2.1 Effects of different molecular weight hyaluronic acid on the disease activity index of enteritis mouse model

[0103] During the DSS-induced colitis model in mice, mice will have clinical signs of colitis such as blood in stool, loose stools, and weight loss. Therefore, the DAI score can be used to assess the severity of the mouse UC model. The daily weight changes and fecal status of each group of mice were recorded. The DAI score results are shown in Figure 2. Fig.13 shown. Fig.13 AThe results showed that compared with the control group, the symptoms of intestinal inflammation in the model group on the 7th day were significantly improved, with a DAI score of 2.42, and varying degrees of loose stools and bloody stools, indicating that the model of colitis disease in this experiment was successfully established. Fig.13 B The results showed that compared with the model group, the intervention of hyaluronic acid with different molecular weights showed different degrees of recovery of intestinal inflammation symptoms, among which the low molecular weight hyaluronic acid of 10-50kDa had the best recovery effect, significantly reducing the DAI score of colitis mice (p < 0.05). The above results proved that hyaluronic acid with different molecular weights can alleviate the symptoms of mouse colitis model induced by DSS, but low molecular weight hyaluronic acid has the best effect in relieving intestinal inflammation in colitis mice, and can effectively relieve symptoms such as diarrhea and bloody stools in mice, and promote the recovery of intestinal health.

[0104] 2.2 Effects of different molecular weight hyaluronic acid on colon histopathological findings in enteritis mouse model

[0105] The pathological symptoms of colon tissue can directly analyze the severity of colitis mouse model, so the improvement effect of each sample on UC can be evaluated by pathological analysis of HE staining. Fig.14As shown, the epithelial mucosal layer structure of the blank group mice was intact, the villi and crypts were clear, regular and with complete striated borders, and the goblet cell content was high. However, the muscular layer of the UC mice induced by DSS showed obvious basal inflammatory cell infiltration, and the muscular layer of the mucosa was ulcerated, the crypt structure was changed, and the surface was irregular and branched. After intervention with three molecular weight hyaluronic acid, the lesions and damage of the colon tissue of each group of mice recovered to varying degrees, the colon muscle layer and mucosal structure were restored, the gland and crypt structure were relatively complete, the number of goblet cells increased, and the inflammatory cell infiltration in the muscle layer and submucosal layer was reduced. Among them, the recovery of the villi and crypt structure of the colon tissue after treatment with low molecular weight hyaluronic acid of 10-50kDa was significantly improved, which can be seen that the colon damage of DSS-induced colitis mice was significantly improved, and the colon tissue was promoted to restore the integrity of the intestinal barrier.

[0106] 2.3 Effects of different molecular weight hyaluronic acid on intestinal permeability in enteritis mouse model

[0107] FITC-Dextran (FD4) is a common fluorescently labeled polysaccharide, which is often used to evaluate changes in intestinal permeability in experimental animals by oral gavage. Fig.15 As shown in the figure, compared with the blank group, the serum FD4 content of the model group increased to 3.78ug / mL, indicating that the intestinal barrier of the DSS-induced colitis model was damaged and the intestinal permeability was significantly increased. The FD4 content of each sample showed a decreasing trend to varying degrees, and all showed a significant decreasing trend compared with the model group. The FD4 content of O-HA and LMW-HA was 1.88 and 2.49ug / mL, respectively, which had no significant difference compared with the blank group (p>0.05). The results showed that small molecule hyaluronic acid can effectively inhibit the damage of intestinal mucosa caused by DSS, reduce intestinal permeability, and help enhance the recovery of intestinal epithelial barrier.

[0108] 2.4 Effects of different molecular weight hyaluronic acid on the activity of antioxidant enzymes in colon tissue of enteritis mouse model

[0109] DSS-induced UC model will lead to an increase in free radical content in the body, resulting in an imbalance of reactive oxygen species in the body and inflammation. Antioxidant enzymes in the body are key substances for removing a large amount of ROS produced by oxidative stress. Glutathione peroxidase (GSH-Px) is an important enzyme in the body that catalyzes the decomposition of hydrogen peroxide. It can act as a proton donor to remove lipid hydroperoxides produced in tissue peroxidation. Total superoxide dismutase (T-SOD) can remove superoxide anion free radicals (O2 - ) protects tissues from peroxidation damage; catalase (CAT) can further decompose H202 produced by the metabolism of SOD enzyme into water. Therefore, the enzyme activity of these three types of antioxidant enzymes can be used to evaluate the efficacy of samples in improving mouse UC models. The experimental results are as follows Figure 16-18 As shown in the figure, compared with the blank group, the enzyme activities of the three antioxidant enzymes in the model group after DSS induction were significantly reduced (p < 0.05). After the intervention of HA with different molecular weights, the enzyme activities recovered to varying degrees compared with the model group. Among them, low molecular weight hyaluronic acid of 10-50kDa can significantly improve the activity of endogenous antioxidant enzymes. The enzyme activities of LMW-HA and O-HA are higher than those of HMW-HA, indicating that small molecular hyaluronic acid can exert antioxidant protection, remove excessive free radicals in colon tissue, reduce peroxidation damage to colon tissue, and achieve the effect of improving enteritis.

[0110] Based on the above experimental results, it can be preliminarily shown that hyaluronic acid has a certain degree of food safety, a certain antioxidant activity, can alleviate intestinal inflammatory damage caused by DSS, and effectively restore the healthy state of the intestine. Among the hyaluronic acids with different molecular weight distributions, low molecular weight hyaluronic acid of 10-50kDa is preferred (according to experimental data, the therapeutic effect on enteritis is LMW-HA>O-HA>HMW-HA), and its effect in repairing inflammatory damage is more significant.

[0111] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing small molecule hyaluronic acid using an enzyme membrane reactor, characterized in that: The method comprises the following steps: S1. Dissolution of high molecular weight hyaluronic acid: Add food grade high molecular weight hyaluronic acid into ultrapure water and swell evenly; S2, hydrolysis of hyaluronic acid in an enzyme membrane reactor: adding the high molecular weight hyaluronic acid solution obtained in step S1 to an enzyme membrane bioreactor device, and after the temperature in the tank reaches a constant, adding hyaluronidase to carry out enzymatic hydrolysis; the hyaluronic acid oligosaccharides produced by the enzymatic hydrolysis pass through the ultrafiltration membrane of the ultrafiltration membrane separation device to obtain a hyaluronic acid oligosaccharide filtrate; the hyaluronic acid aqueous solution retained by the ultrafiltration membrane separation device is refluxed into the reaction tank for continuous enzymatic hydrolysis, and as the enzymatic hydrolysis time increases, the molecular weight gradually decreases to obtain a low molecular weight hyaluronic acid retained solution; S3. Drying of the small molecule hyaluronic acid solution: The small molecule hyaluronic acid retentate and the hyaluronic acid oligosaccharide filtrate obtained in step S2 are dehydrated and dried respectively to obtain low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides.

2. The method according to claim 1, characterized in that The concentration of the high molecular weight hyaluronic acid solution obtained in step S1 is 1-5 g / L; the molecular weight of the high molecular weight hyaluronic acid is ≥120 kDa.

3. The method according to claim 1, characterized in that In step S2, the amount of hyaluronidase added is 1.0% to 5.0% of the mass of the high molecular weight hyaluronic acid in the reaction system.

4. The method according to claim 1, characterized in that: In step S2, the shear rate of the enzyme membrane bioreactor on the substrate liquid is 200-600 rpm; the pore size of the ultrafiltration membrane is 20-100 kDa.

5. The method according to claim 1, characterized in that In step S2, the temperature during the enzymatic hydrolysis reaction is 25-50°C, the pH value is 5.0-7.0; and the time during the enzymatic hydrolysis reaction is 1.0h-4.0h.

6. The method according to claim 1, characterized in that After the enzymatic hydrolysis reaction begins, the ultrafiltration membrane separation device is turned on to filter through the ultrafiltration membrane; after the filtration begins, ultrapure water is added to the reaction system every 15 minutes according to the volume of the filtered solution; the transmembrane pressure of the ultrafiltration membrane separation process is 0.05-0.20 mPa.

7. A device for preparing and separating small molecule hyaluronic acid by enzyme-membrane coupling according to the method of claim 1, comprising an enzyme-catalyzed bioreactor and a flat-plate polyethersulfone ultrafiltration membrane separation device, wherein the enzyme-catalyzed bioreactor and the ultrafiltration membrane separation device are connected by a pipeline.

8. A small molecule hyaluronic acid obtained according to the method of any one of claims 1-6, comprising low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides, wherein the average molecular weight of the low molecular weight hyaluronic acid is between 1 and 20 kDa, and the average molecular weight of the hyaluronic acid oligosaccharides is less than 3 kDa.

9. Use of the small molecule hyaluronic acid obtained by the method according to any one of claims 1 to 6 in preparing food for removing free radicals from the human body.

10. Use of the small molecule hyaluronic acid obtained by the method according to any one of claims 1 to 6 in preparing food for improving intestinal oxidative stress and / or relieving intestinal inflammation.

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