Resistant starch of intestinal targeted slow-release polyphenol as well as preparation method and application of resistant starch

Abstract

The invention belongs to the technical field of drug delivery and food medicine, and particularly relates to resistant starch of intestinal targeted slow-release polyphenol and a preparation method and application of the resistant starch of the intestinal targeted slow-release polyphenol. Performing annealing recrystallization on the heat-dispersed high-amylose corn starch in an ethanol solution to obtain single-spiral structure resistant corn starch; and self-assembling the resistant corn starch with the single-spiral structure and the quinoa polyphenol extract in an alcohol-water system to obtain the intestinal targeted slow-release polyphenol resistant starch. According to the resistant starch of the intestinal targeted slow-release polyphenol prepared by the method disclosed by the invention, the specific starch spiral structure can realize the intestinal targeted controlled release of the polyphenol contained in the intestinal targeted slow-release polyphenol, so that the loss of phenolic compounds in the gastrointestinal digestion process is reduced, and the accurate and continuous release of the polyphenol compounds in the intestinal digestion stage is realized; the residence time of the polyphenol in the intestinal tract is prolonged, so that the bioavailability of the polyphenol is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of drug delivery and food medicine, and specifically relates to an intestinal tract-targeted polyphenol-slow-release resistant starch and a preparation method and application thereof. Background Art

[0002] Polyphenols, as secondary metabolites in plants, are a general term for a large class of heterogeneous phytochemicals and an important component of the human diet. They have outstanding biological activities, such as anti-inflammatory, anti-obesity, and improved postprandial blood sugar. However, after ingestion, polyphenols undergo extensive chemical degradation and modification in the gastrointestinal tract, which reduces the final concentration of intact polyphenols accumulated in the small intestine and colon, and has low bioavailability, which cannot fully exert its benefits to human health. Therefore, it is crucial to improve the stability of polyphenols in the digestive tract to promote their biological properties in the digestive tract. Quinoa is a pseudo-cereal rich in polyphenols. It is more beneficial to human health than other plant foods because of its balanced nutritional composition. Studies have shown that quinoa polyphenols have outstanding effects in lowering blood sugar levels and anti-diabetes, especially the activity of colored quinoa.

[0003] The prior art shows that the use of food-grade encapsulation carriers can improve the biological efficacy of polyphenols, and food-grade biopolymers such as corn starch can be used as an external matrix for encapsulating polyphenols. Corn starch is composed of amylose and amylopectin molecules and is one of the most abundant natural polysaccharides. It has the characteristics of safety, low price, good biocompatibility and biodegradability. However, natural corn starch has high digestibility and is easily hydrolyzed by digestive enzymes. After consumption, it is easy to cause high blood sugar response values, and further induce obesity and metabolic syndrome, which limits its application in the field of functional ingredient encapsulation. At the same time, since the internal pore size of natural corn starch is much larger than the hydrodynamic volume of polyphenol compounds, there is often a burst release when polyphenol compounds are released through the corn starch encapsulation system. Too fast and premature release will lead to excessive accumulation of polyphenol compounds in a short period of time, which will lead to a series of side reactions and affect its final efficacy. Based on the structural characteristics of natural corn starch, the corn starch-polyphenol complex prepared by the prior art still has the disadvantage of burst release.

[0004] Therefore, there is an urgent need to provide a method for preparing a sustained-release polyphenol corn starch. Summary of the invention

[0005] The purpose of the present invention is to provide an intestinal targeted slow-release polyphenol resistant starch and a preparation method and application thereof, which solves the problem of burst release when polyphenol compounds are released through a corn starch encapsulation system in the prior art.

[0006] The technical solution adopted by the present invention is: the present invention provides a method for preparing intestinal targeted sustained-release polyphenol resistant starch, comprising the following steps: Quinoa polyphenol extract is prepared; heat-dispersed high-amylose corn starch is annealed and recrystallized in an ethanol solution to obtain single-helix resistant corn starch; single-helix resistant corn starch and quinoa polyphenol extract are self-assembled in an alcohol-water system to obtain intestinal targeted sustained-release polyphenol resistant starch.

[0007] Preferably, the quinoa polyphenol extract is one or more of white quinoa polyphenol extract, red quinoa polyphenol extract and black quinoa polyphenol extract; the quinoa polyphenol extract is obtained by extracting white quinoa seeds, red quinoa seeds or black quinoa seeds using a methanol solution containing formic acid.

[0008] Preferably, the content of amylose in the high-amylose corn starch is 65%.

[0009] Preferably, the process of annealing and recrystallizing the heat-dispersed high-amylose corn starch in an ethanol solution is as follows: dissolving the high-amylose corn starch in a DMSO aqueous solution in a boiling water bath to obtain a first heat-dispersed system, washing the first heat-dispersed system cooled to room temperature with anhydrous ethanol, collecting a first precipitate, drying the first precipitate, crushing and sieving, dissolving the crushed and sieved first precipitate in an ethanol aqueous solution to obtain a second dispersion system, washing the second dispersion system, collecting a second precipitate, drying the second precipitate, and crushing and sieving.

[0010] Preferably, the process of self-assembly of the single helical structure resistant corn starch and quinoa polyphenol extract in the alcohol-water system is: mixing the single helical structure resistant corn starch with the ethanol-water solution, adding the quinoa polyphenol extract, mixing well to obtain a third dispersed system, washing the third dispersed system cooled to room temperature, collecting the third precipitate, drying the third precipitate, and crushing and sieving.

[0011] Preferably, the ethanol aqueous solution is 40% ethanol by volume.

[0012] The present invention also provides an intestinal targeted sustained-release polyphenol resistant starch prepared by the preparation method.

[0013] Preferably, the intestinal targeted slow-release polyphenol resistant starch is used to prepare food or medicine for preventing and / or treating obesity.

[0014] Preferably, the intestinal targeted slow-release polyphenol resistant starch is used for preparing food or medicine for lowering blood sugar.

[0015] Preferably, the intestinal targeted slow-release polyphenol resistant starch is used to prepare food or medicine for protecting the intestinal barrier.

[0016] Preferably, it is characterized in that the intestinal targeted slow-release polyphenol resistant starch is used to prepare food or medicine for adjusting intestinal symbiotic flora.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing intestinal targeted slow-release polyphenol resistant starch, the steps comprising: Quinoa polyphenol extract is prepared; heat-dispersed high-amylose corn starch is annealed and recrystallized in an ethanol solution to obtain single-helix resistant corn starch; single-helix resistant corn starch and quinoa polyphenol extract are self-assembled in an alcohol-water system to obtain intestinal targeted sustained-release polyphenol resistant starch.

[0018] The intestinal targeted sustained release polyphenol resistant starch prepared by the present invention has a specific starch spiral structure that can achieve intestinal targeted controlled release of polyphenols, reduce the loss of phenolic compounds during gastrointestinal digestion, achieve accurate and sustained release of polyphenols during intestinal digestion, and prolong the retention time of polyphenols in the intestine, thereby improving its bioavailability. At the same time, the formed complex can be used as a prebiotic to promote the colon Lactobacillus , Bifidobacterium and Akkermansia The abundance of beneficial microorganisms increases, the production of short-chain fatty acids such as acetate and butyrate increases, and the Desulfovibrionaceae The relative abundance of harmful bacteria such as chlorella and broccoli has good probiotic properties.

[0019] The present invention forms a single helical structure resistant corn starch-quinoa polyphenol complex system in an alcohol-water system, which is used to regulate the diameter of the starch single helical pores and to interact with different small molecule polyphenol compounds, thereby regulating the release rate of the polyphenol compounds. Specifically, the starch single helical structure can form multiple interactions such as π-π interactions, hydrogen bonds, and electrostatic interactions with the polyphenol compounds, thereby achieving effective loading of the polyphenol compounds. This single helical structure resistant corn starch-quinoa polyphenol complex system is low in cost and has no toxic side effects.

[0020] The multi-scale single helical pore structure formed in the self-assembly system of the single helical resistant corn starch-quinoa polyphenol complex of the present invention can greatly increase the content of the ordered crystalline structure of starch, thereby prolonging the time required for starch to be hydrolyzed by digestive enzymes. The loading and encapsulation of polyphenol compounds by the single helical structure effectively avoids the degradation of polyphenol compounds by gastrointestinal digestion, thereby improving their bioavailability.

[0021] The present invention is based on the single helical structure of resistant corn starch-quinoa polyphenol complex can be partially or completely degraded by the intestinal microbiota in the colon, thereby triggering the release of polyphenol compounds in the target site. In addition, resistant starch can be used as a prebiotic to bring additional health benefits such as regulating the composition of intestinal flora and increasing the production of short-chain fatty acids to achieve better practical utility.

[0022] The single-helix structure resistant corn starch-quinoa polyphenol complex sustained-release system prepared by the present invention can completely avoid the burst release of drugs during gastric digestion, and the average daily cumulative release of drugs is less than 10%.

[0023] The preparation method of the present invention is simple and is conducive to low-cost industrial production. The materials used are natural bio-based materials with high safety. The obtained single-helix structure resistant corn starch-quinoa polyphenol complex can maintain structural stability during gastrointestinal digestion and is suitable for the field of polyphenol compound delivery.

[0024] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The natural corn starch prepared in Comparative Example 1 and the single helical resistant corn starch prepared in S2 in Example 1 13 C CP / MAS NMR spectrum.

[0026] Figure 2 It is the encapsulation efficiency of the corn starch-quinoa polyphenol complex prepared in Comparative Example 5 and the single helical structure resistant corn starch-quinoa polyphenol complex prepared in Example 4.

[0027] Figure 3 are X-ray diffraction patterns, A is the X-ray diffraction pattern of the natural corn starch prepared in Comparative Example 1 and the single helical resistant corn starch prepared by S2 in Example 1; B is the X-ray diffraction pattern of the single helical resistant corn starch-quinoa complex prepared in Examples 1 to 3.

[0028] Figure 4 It is the final accumulated quinoa polyphenol release efficiency of the corn starch-quinoa polyphenol complex prepared in Comparative Example 5 and the single helical structure resistant corn starch-quinoa polyphenol complex prepared in Example 4.

[0029] Figure 5 The polyphenol concentrations in serum at different times after intragastric administration of the corn starch-quinoa polyphenol complex prepared in Comparative Example 5 and the single helical resistant corn starch-quinoa polyphenol complex prepared in Example 4.

[0030] Figure 6 Changes in mice in each treatment group: A: changes in fasting blood glucose; B: changes in body weight.

[0031] Figure 7 These are the results of 16S rRNA sequencing of mouse fecal flora. A and B are the relative abundance of intestinal flora of each group of mice at the phylum level and genus level, respectively.

[0032] Figure 8 is the content of short-chain fatty acids SCFAs in the colon contents of mice, A~F: acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid. DETAILED DESCRIPTION

[0033] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0034] The inventive concept of the present invention is as follows: Existing technology shows that natural corn starch, as the external matrix for encapsulating polyphenols, is highly digestible and easily hydrolyzed by digestive enzymes. In addition, its internal pore size is much larger than the fluid dynamic volume of polyphenol compounds. Therefore, when polyphenol compounds are released through the natural corn starch encapsulation system, there is often an explosive release. Too fast and too early release will lead to excessive accumulation of polyphenol compounds in a short period of time, which will lead to a series of side reactions.

[0035] Based on this, the present invention proposes a method for preparing intestinal targeted sustained-release polyphenol resistant starch, the steps comprising: preparing quinoa polyphenol extract; annealing and recrystallizing heat-dispersed high-amylose corn starch in an ethanol solution to obtain single-helix structure resistant corn starch; self-assembling the single-helix structure resistant corn starch and quinoa polyphenol extract in an alcohol-water system to obtain intestinal targeted sustained-release polyphenol resistant starch.

[0036] The present invention is based on the non-covalent interaction between polyphenol compounds and the single helical structure of corn starch during the self-assembly process. By adjusting the multi-scale structure of corn starch, a specific starch helical structure can control the release of polyphenols in the inclusions, thereby reducing the loss of active substances in gastrointestinal digestion. At the same time, polyphenol compounds can promote the dense stacking of the single helical structure of corn starch, induce starch to form an ordered crystalline structure to resist hydrolysis by digestive enzymes, and form resistant starch resistant to enzymatic hydrolysis, which is indigestible in the upper intestine, but can be partially or completely degraded by intestinal microorganisms in the colon, exerting prebiotic properties and further realizing the fixed-point release of polyphenols. There may be a synergistic relationship between polyphenol compounds and starch, which jointly play a beneficial role in regulating the host. Therefore, the sustained and stable release of polyphenol compounds in the intestinal tract by constructing a new type of universal resistant starch single helical delivery system has considerable application prospects.

[0037] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0038] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0039] Example 1 A method for preparing intestinal targeted sustained-release polyphenol resistant starch, comprising the following steps: S1: preparing quinoa polyphenol extract, the method is as follows: After washing, vacuum drying and grinding into 60 mesh powder, the white quinoa seeds were ultrasonically extracted with 80% methanol at room temperature for 40 minutes, and the methanol also contained 0.1% formic acid. The mass volume ratio of white quinoa seeds to 80% methanol was 1:25. The white quinoa seeds were extracted twice according to this method, and the supernatants were combined and rotary evaporated at 45°C. The concentrated solution was freeze-dried, i.e., the white quinoa polyphenol extract, and stored at -20°C.

[0040] S2: The heat-dispersed high-amylose corn starch is annealed and recrystallized in an ethanol solution to obtain a single-helical resistant corn starch, as follows: High amylose corn starch with an amylose content of 65% was dissolved in a 95% DMSO aqueous solution under boiling water bath conditions, and stirred continuously in the boiling water bath for 1.5 hours to obtain a thermal dispersion system, and the mass volume ratio of high amylose corn starch to DMSO aqueous solution was 1:25. After cooling to room temperature, the thermal dispersion system was evenly mixed with 2.5 times the volume of anhydrous ethanol, and then the mixture was centrifuged at 5000g for 10 minutes, the supernatant was discarded, the precipitate was washed twice with anhydrous ethanol, dried overnight at 37°C in a vacuum drying oven at 5Pa, and crushed to pass through a 200-mesh sieve. Subsequently, the starch powder was bathed in a 40% ethanol aqueous solution at 70°C for 0.5 hours, washed and dried with anhydrous ethanol, and ground to 200 mesh to obtain a single helical resistant corn starch, recorded as HAMS, and stored in a drying dish.

[0041] S3: The single helical resistant corn starch and quinoa polyphenol extract are self-assembled in an alcohol-water system to obtain a single helical resistant corn starch-quinoa polyphenol complex, as follows: The single helical corn starch prepared by S2 was mixed with a 40% ethanol solution at a mass volume ratio of 1:100, and the quinoa polyphenol extract prepared by S1 was added, and the amount of quinoa polyphenol extract added was 10% of the weight of the single helical resistant corn starch. Stirring was continued for 0.5 h in a 70°C water bath, and after cooling to room temperature, it was mixed with 2.5 times the volume of anhydrous ethanol, and then the mixture was centrifuged at 5000g for 10 min, the supernatant was discarded, and the precipitate was washed twice with anhydrous ethanol, dried overnight at 37°C in a vacuum drying oven, and crushed through a 200-mesh sieve to obtain a single helical resistant corn starch-quinoa polyphenol complex, which was recorded as HAMS-WQP, i.e., intestinal targeted sustained-release polyphenol resistant starch, and stored in a drying dish for subsequent experimental determination.

[0042] Example 2 A method for preparing intestinal targeted sustained-release polyphenol resistant starch, comprising the following steps: Red quinoa seeds were used to prepare red quinoa polyphenol extract. When preparing the single helical resistant corn starch-quinoa polyphenol complex, the red quinoa polyphenol extract was used. The other processes were exactly the same as in Example 1. The obtained single helical resistant corn starch-quinoa polyphenol complex was recorded as HAMS-RQP, i.e., intestinal targeted sustained-release polyphenol resistant starch.

[0043] Example 3 A method for preparing intestinal targeted sustained-release polyphenol resistant starch, comprising the following steps: Black quinoa seeds were used to prepare black quinoa polyphenol extract. When preparing the single helical resistant corn starch-quinoa polyphenol complex, the black quinoa polyphenol extract was used. The other processes were exactly the same as in Example 1. The obtained single helical resistant corn starch-quinoa polyphenol complex was recorded as HAMS-BQP, i.e., intestinal targeted sustained-release polyphenol resistant starch.

[0044] Example 4 A method for preparing intestinal targeted sustained-release polyphenol resistant starch, comprising the following steps: According to S1 of Examples 1 to 3, white quinoa polyphenol extract, red quinoa polyphenol extract and black quinoa polyphenol extract were prepared. The white quinoa polyphenol extract, the red quinoa polyphenol extract and the black quinoa polyphenol extract were mixed in a weight ratio of 1:1:1 to prepare a mixed quinoa polyphenol extract, and the obtained single helical resistant corn starch-quinoa polyphenol complex was recorded as HAMS-QP. When preparing the single helical resistant corn starch-quinoa polyphenol complex, the mixed quinoa polyphenol extract was used, and the other processes were exactly the same as in Example 1.

[0045] In order to further illustrate the characteristics of the intestinal targeted sustained-release polyphenol resistant starch prepared by the method of the present invention, the present invention provides comparative examples 1 to 5.

[0046] Comparative Example 1 A method for preparing corn starch, comprising the following steps: The natural corn starch was used as a control sample. The natural corn starch was dried in a vacuum drying oven overnight, ground to 200 mesh, recorded as NHAMS, and stored in a drying dish.

[0047] Comparative Example 2 A method for preparing a corn starch-quinoa polyphenol complex comprises the following steps: S1: Prepare quinoa polyphenol extract using the same method as in Example 1.

[0048] S2: Prepare corn starch-quinoa polyphenol complex as follows: The natural corn starch was mixed with a 40% ethanol solution at a mass volume ratio of 1:100, and the quinoa polyphenol extract prepared by S1 was added, and the amount of quinoa polyphenol extract added was 10% of the weight of the natural corn starch. Stirring was continued for 0.5 h in a 70°C water bath, and after cooling to room temperature, it was mixed with 2.5 times the volume of anhydrous ethanol, and then the mixture was centrifuged at 5000g for 10 min, the supernatant was discarded, and the precipitate was washed twice with anhydrous ethanol, dried in a vacuum drying oven at 37°C overnight, and crushed through a 200-mesh sieve to obtain a corn starch-quinoa polyphenol complex, recorded as NHAMS-WQP, and stored in a drying dish for subsequent experimental determination.

[0049] Comparative Example 3 A method for preparing a corn starch-quinoa polyphenol complex comprises the following steps: S1: Prepare red quinoa polyphenol extract using the same method as in Example 2.

[0050] S2: Prepare corn starch-quinoa polyphenol complex as follows: The quinoa polyphenol added was red quinoa polyphenol extract, and the rest of the process was exactly the same as that of Comparative Example 2, to obtain a corn starch-quinoa polyphenol complex, which was recorded as NHAMS-RQP.

[0051] Comparative Example 4 A method for preparing a corn starch-quinoa polyphenol complex comprises the following steps: S1: Prepare black quinoa polyphenol extract using the same method as in Example 3.

[0052] S2: Prepare corn starch-quinoa polyphenol complex as follows: The quinoa polyphenol added was black quinoa polyphenol extract, and the rest of the process was exactly the same as that of Comparative Example 2, to obtain a corn starch-quinoa polyphenol complex, which was recorded as NHAMS-BQP.

[0053] Comparative Example 5 A method for preparing a corn starch-quinoa polyphenol complex comprises the following steps: S1: preparing a mixed quinoa polyphenol extract of white quinoa polyphenol extract, red quinoa polyphenol extract and black quinoa polyphenol extract, the method is exactly the same as that of Example 4.

[0054] S2: Prepare corn starch-quinoa polyphenol complex as follows: The quinoa polyphenol added was a mixed quinoa polyphenol extract, and the rest of the process was exactly the same as that of Comparative Example 2, to obtain a corn starch-quinoa polyphenol complex, which was recorded as NHAMS-QP.

[0055] The single helical structure resistant corn starch-quinoa polyphenol complex prepared in Examples 1 to 4, the corn starch prepared in Comparative Example 1, and the corn starch-quinoa polyphenol complex prepared in Comparative Examples 2 to 5 were subjected to starch multi-scale structure test.

[0056] (1) Helical structure test of native corn starch and single helical resistant corn starch.

[0057] The natural corn starch prepared in Comparative Example 1 and the single helical resistant corn starch prepared in S2 in Example 1 were collected using a solid-state nuclear magnetic spectrometer equipped with a 4-mm MAS probe. The operating conditions were set as follows: resonance frequency 188.6 MHz, rotor speed 10 kHz, contact time 1 ms, delay time 5 s, cumulative scanning times more than 2400 times, and all experiments were carried out at 25 ° C.

[0058] The results are as follows Figure 1 As shown, compared with the natural corn starch sample, the single-helix structure resistant corn starch HAMS sample presents a tiny single peak at 94ppm~105ppm, which is characterized by a typical empty helical polymorph, which may be caused by the formed left-handed single-chain helical structure, proving the successful preparation of single-helix structure resistant corn starch.

[0059] (2) Encapsulation efficiency test of corn starch-quinoa polyphenol complex and single helical structure resistant corn starch-quinoa polyphenol complex.

[0060] The single helical resistant corn starch-quinoa polyphenol complex prepared in Example 4 and the corn starch-quinoa polyphenol complex prepared in Comparative Example 5 were subjected to the encapsulation efficiency test. 200 mg of the sample was mixed with 10 mL of ethanol to remove free polyphenol compounds, centrifuged at 5000 g for 10 min, and the supernatant was collected and the polyphenol content was determined by the Folin phenol method. The encapsulation efficiency was then calculated using the following equation: .

[0061] The results are as follows Figure 2 As shown, the single helical resistant corn starch-quinoa polyphenols complex exhibited a higher encapsulation rate than the corn starch-quinoa polyphenols complex, indicating the efficient loading and encapsulation of polyphenol compounds by the single helical resistant corn starch.

[0062] (3) Crystal structure test of native corn starch, single helical resistant corn starch and single helical resistant corn starch-quinoa polyphenol complex.

[0063] The single helical resistant corn starch-quinoa polyphenol composite prepared in Examples 1 to 3, the natural corn starch prepared in Comparative Example 1, and the single helical resistant corn starch prepared in S2 in Example 1 were subjected to X-ray diffraction measurement using an X-ray diffractometer at 40 kV and 40 mA, with Cu-Kα radiation as the X-ray source. The diffraction angle 2θ ranged from 5° to 50°, and the scanning speed was 10° / min.

[0064] The results are as follows Figure 3 As shown, compared with natural corn starch, single helix resistant corn starch exhibits typical V-shaped crystallization peaks at 7.4°, 13.3° and 20.3°, and has the potential to accommodate guest molecular phenolic compounds. Compared with single helix resistant corn starch, single helix resistant corn starch-quinoa polyphenol complex forms sharper diffraction peaks, indicating that a more ordered structure has been formed.

[0065] (4) Testing the release characteristics of phenolic compounds in single helical resistant corn starch-quinoa polyphenol complex during in vitro simulated gastrointestinal digestion.

[0066] The digestion release characteristics of the single helical resistant corn starch-quinoa polyphenol complex prepared in Example 4 and the corn starch-quinoa polyphenol complex prepared in Comparative Example 5 were tested. The digestive fluid was collected at 5min, 10min, 20min, 30min, 60min, 90min and 120min in the simulated gastric digestion stage and 5min, 10min, 20min, 30min, 60min, 90min, 120min, 240min, 360min, 480min and 600min in the simulated intestinal digestion stage, respectively, and the enzyme was inactivated and centrifuged at 5000g for 10min to take the supernatant, and the polyphenol content was determined by the Folin phenol method.

[0067] The results are as follows Figure 4 As shown in the figure, during the gastric digestion stage, 4.5% of phenolic compounds were released from the digestion supernatant of corn starch-quinoa polyphenol complex, and 4.3% of phenolic compounds were released from the single helical resistant corn starch-quinoa polyphenol complex. After entering the intestinal digestion stage, quinoa polyphenols were rapidly released from the corn starch-quinoa polyphenol complex, which was caused by the digestive hydrolysis of starch by starch digestive enzymes. In contrast, after 2 hours of digestion, the sustained release of quinoa polyphenols from the single helical resistant corn starch-quinoa polyphenol complex was still observed. The ordered helical structure of HAMS delayed and protected the polyphenol compounds encapsulated in the cavity from degradation by digestive enzymes and digestive juices. This result shows the intestinal pH response and controlled release characteristics of HAMS.

[0068] (5) The single helical resistant corn starch-quinoa polyphenol complex prepared in Example 4 and the corn starch-quinoa polyphenol complex prepared in Comparative Example 5 were subjected to a pharmacokinetic test of phenolic substances in rats.

[0069] 6-week-old male Sprague-Dawley, SD rats were purchased from Hangzhou Medical College. The feeding conditions were 12h light cycle, 25°C, 60% relative humidity, and the animals had free access to rat feed and drinking water. All animal experiments were approved by the Animal Ethics Committee of Zhejiang Province and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals. After one week of adaptation, the rats were randomly divided into 3 groups, including a blank control group, a model group, and an experimental group, and fasted for 12h before the pharmacokinetic test of phenolic substances. According to body weight, the blank control group, model group, and experimental group rats were gavaged at a dose of 30mg / kg. The blank control group was gavaged with 1mM, pH7.4 phosphate buffer PBS for rats; the model group was gavaged with the corn starch-quinoa polyphenol complex prepared in Comparative Example 4 dissolved in 1mM, pH7.4 phosphate buffer PBS for rats; the experimental group was gavaged with the single helical resistant corn starch-quinoa polyphenol complex prepared in Example 3 dissolved in 1mM, pH7.4 phosphate buffer PBS for rats. At specific time points after intragastric administration, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 20 h, the rat orbital blood was collected and centrifuged at 3000 rpm for 10 min to collect serum. Then 200 μL of acetonitrile was added to 100 μL of serum to extract phenolic substances in the serum, and the mixture was centrifuged at 13000 rpm for 20 min. The supernatant was filtered with a 0.22 μm filter membrane and then analyzed by ultra-performance liquid chromatography UPLC.

[0070] The results are as follows Figure 5 As shown, rats were gavaged with PBS solutions containing equal amounts of phenolic compounds of corn starch-quinoa polyphenol complex and single helical resistant corn starch-quinoa polyphenol complex, and the concentrations of phenolic compounds in serum were quantified at different time points and the pharmacokinetic parameters were calculated. The maximum blood concentration of quinoa polyphenols in the single helical resistant corn starch-quinoa polyphenol complex group was C max appeared 60 minutes after oral administration, while the maximum blood concentration of corn starch-quinoa polyphenols complex was C max The single helical resistant corn starch can slowly release phenolic compounds and maintain a certain blood concentration range for a long time. These results are consistent with the results of phenolic substance release in simulated gastrointestinal digestion, confirming the regulatory ability of the single helical resistant corn starch-quinoa polyphenol complex to protect the release of phenolic compounds.

[0071] (6) In vivo experiments were conducted on mice using the single helical resistant corn starch-quinoa polyphenol complexes prepared in Examples 1 to 3 and the single helical resistant corn starch prepared in S2 in Example 1.

[0072] 6.1 The feeding and grouping of mice in animal experiments are as follows: Six-week-old male C57BL / 6J mice were purchased from Hangzhou Medical College. The animals were kept under the conditions of 12 h light cycle, 25 °C, and 60% relative humidity, and they had free access to mouse feed and drinking water. After 1 week of adaptation, 36 mice were randomly divided into 6 groups, 6 mice / group, including a normal control group, a model group, and 4 experimental groups. The normal control group was given a basic diet CD; the model group was given a high-fat diet HFD; the HFD HAMS 、HFD HAMS–WQP 、HFD HAMS–RQP and HFD HAMS–BQP The four dietary intervention experimental groups were fed with 20% HFD of carbohydrate replacement amount of single helical resistant corn starch prepared by S2 in Example 1 and single helical resistant corn starch-quinoa polyphenol complex prepared by Examples 1 to 3, that is, 20% of the mass of carbohydrates in the mouse feed ingredients were replaced by HAMS or HAMS-QP. Therefore, the mass of corn starch in carbohydrates in the model group was 72.8 gm; the mass of corn starch in carbohydrates in the dietary intervention group was 58.3 gm%, plus 14.5 gm% of single helical resistant corn starch or single helical resistant corn starch-polyphenol complex. All groups were fed for 8 weeks, and the specific feed formula is shown in Table 1.

[0073] Table 1 Mouse feed composition

[0074] Note: “-” means there is no such item.

[0075] 6.2 Mouse body weight and fasting blood glucose were measured as follows: The weight and fasting blood glucose levels of mice were measured every week. After fasting for 12 hours, the fasting blood glucose levels of the tail vein blood of mice were measured by a blood glucose meter.

[0076] like Figure 6 As shown in the figure, after 8 weeks of dietary intervention, the weight gain of mice in the HFD group was significantly higher, and the weight gain of HFD mice was more than 10% higher than that of mice in the CD group, indicating that the HFD-induced obesity model in mice was successfully established. HAMS-BQP The average weight of mice in the HFD group was much lower than that in the CD group. The fasting blood glucose of mice was measured to investigate the hypoglycemic effect of the single helical resistant corn starch-quinoa polyphenol complex on HFD-induced mice. Compared with the CD group, the fasting blood glucose level of the HFD group was significantly increased. HAMS and HFD HAMS-WQP 、HFD HAMS-RQP and HFD HAMS-BQP The increase in blood sugar in the dietary intervention group was significantly suppressed, and the HFD HAMS-RQP and HFD HAMS-BQPIn general, single helical resistant corn starch and single helical resistant corn starch-quinoa polyphenol complex have a certain anti-obesity effect on HFD-induced obese mice, and also reduce the blood glucose level of obese mice.

[0077] 6.3 16S rRNA sequencing of mouse fecal flora is as follows: For gut microbiome analysis, total DNA was extracted from mouse feces. After determining DNA concentration and integrity, the V3-V4 region was amplified and sequenced using the Illumina MiSeq platform. The obtained sequences were processed using MOTHUR for alignment and clustering, while chimera detection was performed using UPARSE software and UCHIME. The final reads were clustered into operational taxonomic units with a 97% similarity threshold. OTUs were further classified using the Ribosomal Database Project Classifier v.2.13 with a minimum confidence threshold of 0.6 and trained by QIIME v1.9.1. The linear discriminant analysis effect size method was applied to distinguish statistically significant and biologically relevant taxa. Significant species or functions were determined by R, v3.4.1 based on the Wilcox test.

[0078] To evaluate the effects of single-helix resistant corn starch and single-helix resistant corn starch-quinoa polyphenols complex on the gut microbial composition of HFD-induced obese mice, the relative abundance of taxa in the different groups was compared. Figure 7 , at the phylum level, Firmicutes Firmicutes and Bacteroidetes Bacteroidetes The relative abundance of Firmicutes and Bacteroidetes The ratio of the gate F / B value is considered to be highly correlated with the risk of obesity, and some studies have reported that an increase in the F / B value is also one of the inducing factors of inflammatory bowel disease. The F / B value of HFD-induced obese mice was significantly increased compared with the CD group, while the F / B value of mice in the single helical resistant corn starch-quinoa polyphenols complex dietary intervention group was reduced to varying degrees. In order to further analyze the intervention and regulatory effect of the single helical resistant corn starch-quinoa polyphenols complex on the intestinal flora of HFD-induced obese mice, the intestinal flora of mice was evaluated at the genus level. Akkermansia Probiotics such as Figure 7 As shown in B, after dietary intervention with single helical resistant corn starch-quinoa polyphenols complex, the beneficial Lactobacillus , Bifidobacterium and Akkermansia The relative abundance of microbiota including Bifidobacterium and LachnospiraceaeStudies have shown that the intake of dietary fiber such as resistant starch can effectively reduce Desulfovibrionaceae The relative abundance of bacterial genera, Desulfovibrionaceae As a sulfate-reducing bacterium, it is usually increased in ulcerative colitis hosts. The lipopolysaccharide it produces can damage the intestinal barrier and further cause metabolic disorders and inflammatory responses. The hydrogen sulfide product produced by reducing sulfate is a recognized epithelial cell toxic product, which can cause abnormal proliferation and metabolism of intestinal epithelial cells and thus damage the intestinal barrier function. After dietary intervention with single-helix resistant corn starch-quinoa polyphenol complex, the conditional pathogens of the genus Desulfovibrionacea The abundance of beneficial bacteria was significantly reduced. The above facts indicate that dietary intervention with single-helix resistant corn starch-quinoa polyphenol complex significantly increased the relative abundance of beneficial bacteria, which to a certain extent reflects the probiotic characteristics of the single-helix resistant corn starch-quinoa polyphenol complex.

[0079] 6.4 Determination of the content of short-chain fatty acids (SCFAs) in the intestinal contents of mice, as follows: Phosphate buffered saline (PBS) was added to freeze-dried mouse feces, and the sample was frozen and ground, ultrasonicated for 10 minutes, centrifuged, 13000g, 15min, 4°C. The supernatant was taken out and added to the n-butanol solvent for extraction, vortexed for 10s, low-temperature ultrasonicated for 10min, centrifuged, 13000g, 15min, and the supernatant was taken to the injection bottle for SCFAs quantification by gas chromatography-mass spectrometry. Chromatographic conditions: HP FFAP capillary column, 30m×0.25mm×0.25μm, carrier gas was high-purity helium, flow rate was 1.0mL / min, and injection port temperature was 180°C. Split injection, injection volume 1μL, split ratio 10:1, solvent extension 2.5min. Program temperature rise: the initial temperature of the column oven was 80°C, and the temperature was increased to 120°C at 20°C / min, and then increased to 160°C at 5°C / min, and then ran at 220°C for 3min. Mass spectrometry conditions: electron impact ion source EI, ion source temperature 230°C, quadrupole temperature 150°C, transfer line temperature 230°C, electron energy 70eV. The scanning mode is selected ion scanning mode SIM. The default parameters of Masshunter quantitative software are used to automatically identify and integrate the target SCFAs ion fragments, and manual inspection is assisted. The detection concentration of each sample is calculated by the standard curve, and the actual content of SCFAs in the sample, namely acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid, is converted.

[0080] To further verify the probiotic properties of the single helical resistant corn starch-quinoa polyphenol complex, SCFAs were identified and quantified in the intestinal contents of mice. Figure 8 As shown in Figure 2, acetate accounts for the largest proportion of SCFAs, followed by propionate and butyrate.HAMS-BQP The dietary intervention group significantly increased the SCFAs content in the intestinal contents of mice. Bifidobacterium The relative abundance of quinoa polyphenols increased significantly, which is potentially related to the production of SCFAs. It is inferred that the single-helix resistant corn starch-quinoa polyphenol complex increases the production of SCFAs by increasing the abundance of SCFAs-producing bacteria and promoting the growth of host-friendly intestinal microbiota, further exerting its anti-inflammatory physiological function in the intestine and alleviating intestinal and systemic circulatory inflammation. This result provides new evidence at the animal in vivo level to verify that the quinoa polyphenol-starch complex is a new type of resistant starch system, filling the gap in its in vivo evaluation.

[0081] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0082] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing intestinal targeted sustained-release polyphenol resistant starch, characterized in that: The following steps are involved: preparing quinoa polyphenol extract; The heat-dispersed high-amylose corn starch was annealed and recrystallized in an ethanol solution to obtain single-helical resistant corn starch; Single helical resistant corn starch and quinoa polyphenol extract were self-assembled in an alcohol-water system to obtain intestinal targeted sustained-release polyphenol resistant starch.

2. The preparation method according to claim 1, characterized in that: The quinoa polyphenol extract is one or more of white quinoa polyphenol extract, red quinoa polyphenol extract and black quinoa polyphenol extract; The quinoa polyphenol extract is obtained by extracting white quinoa seeds, red quinoa seeds or black quinoa seeds using a methanol solution containing formic acid.

3. The preparation method according to claim 1, characterized in that: The content of amylose in the high-amylose corn starch is 65%.

4. The preparation method according to claim 1, characterized in that: The process of annealing and recrystallizing the heat-dispersed high-amylose corn starch in an ethanol solution is as follows: Dissolve high-amylose corn starch in a DMSO aqueous solution in a boiling water bath to obtain a first thermal dispersion system, wash the first thermal dispersion system cooled to room temperature with anhydrous ethanol, collect a first precipitate, dry the first precipitate, crush and sieve, dissolve the crushed and sieved first precipitate in an ethanol aqueous solution to obtain a second dispersion system, wash the second dispersion system, collect a second precipitate, dry the second precipitate, and crush and sieve.

5. The preparation method according to claim 1, characterized in that: The process of self-assembly of the single helical resistant corn starch and quinoa polyphenol extract in an alcohol-water system is as follows: The single helical structure resistant corn starch is mixed with an ethanol aqueous solution, and quinoa polyphenol extract is added and mixed to obtain a third dispersion system. The third dispersion system cooled to room temperature is washed and a third precipitate is collected. The third precipitate is dried, crushed and sieved.

6. An intestinal targeted sustained-release polyphenol resistant starch prepared by the preparation method as claimed in claim 1.

7. The use of the intestinal targeted sustained-release polyphenol resistant starch according to claim 6, characterized in that: The intestinal targeted slow-release polyphenol resistant starch is used for preparing food or medicine for preventing and / or treating obesity.

8. The use of the intestinal targeted sustained-release polyphenol resistant starch according to claim 6, characterized in that: The intestinal targeted slow-release polyphenol resistant starch is used for preparing food or medicine for lowering blood sugar.

9. The use of the intestinal targeted sustained-release polyphenol resistant starch according to claim 6, characterized in that: The intestinal targeted slow-release polyphenol resistant starch is used for preparing food or medicine for protecting intestinal barrier.

10. The use of the intestinal targeted sustained-release polyphenol resistant starch according to claim 6, characterized in that: The intestinal targeted slow-release polyphenol resistant starch is used for preparing food or medicine for adjusting intestinal symbiotic flora.

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