Preparation method and application of high-resistant starch content oat starch for relieving chronic kidney diseases

Abstract

The invention relates to a preparation method and application of high-resistant starch content oat starch for relieving chronic kidney diseases. The preparation method comprises the following steps: (1) preparing oat starch by a dilute alkali method; (2) preparing oat starch with high resistant starch content by an autoclaving method: mixing the oat starch with water, carrying out autoclaving and gelatinization treatment, cooling, and standing at low temperature, so that the gelatinized oat starch is aged; the pressure heat cooling treatment is repeated; and then drying, crushing and sieving to obtain the oat starch with high resistant starch content. The obtained oat starch is subjected to anaerobic fermentation by using intestinal flora of a CKD patient in an anaerobic incubator, and the results show that the renal function indexes such as creatinine, urea nitrogen, indole and p-cresol content, the tryptophanase activity and the tyrosinase activity are significantly reduced, and the beneficial metabolite short-chain fatty acid content is significantly increased. Compared with the prior art, the technology adopts an autoclaving cooling circulation method to prepare the oat starch with high resistant starch content of the oat dietary fiber, and is beneficial to relieving CKD symptoms.

Description

Technical Field

[0001] The invention belongs to the field of health care products, and specifically relates to a preparation method and application of oat starch with high resistant starch content for alleviating chronic kidney disease. Background Art

[0002] Oats are one of the important specialty crops in my country. They are rich in starch, protein, soluble dietary fiber, unsaturated fatty acids, phenolic substances and various trace elements. They are a high-quality healthy grain. The starch content in oats is relatively high, about 60%, of which the amylose chain length is short and the crystallinity is high, which is suitable for the preparation of resistant starch. Dietary fiber is recognized as the "seventh nutrient" in addition to the six major nutrients. Due to its special structure and physiological characteristics, it helps to treat and prevent some chronic diseases. Ordinary dietary fiber has the characteristics of poor taste and flavor when added to food. Resistant starch not only improves some of the shortcomings of ordinary dietary fiber, but also has physiological functions similar to dietary fiber. It is called a new dietary fiber and has become a research hotspot in the functional food processing industry. The new oat dietary fiber has broad application prospects in the development of functional foods or health products to prevent and treat obesity, diabetes, inflammation, cardiovascular disease and chronic kidney disease (CKD). For example, patent CN118452247A provides a gluten-free high-fiber cookie based on insoluble dietary fiber of oat bran and a preparation method thereof. The insoluble dietary fiber of oat bran effectively improves the antioxidant, texture and flavor of gluten-free high-fiber cookies. Patent CN118058476A provides a preparation method of oat dietary fiber with prebiotic function. The oat dietary fiber has the effect of regulating intestinal flora and promoting intestinal peristalsis, and has good prebiotic properties. Patent CN105341950B provides a hypoglycemic oat dietary fiber and a preparation method thereof. The obtained oat dietary fiber has no toxic side effects, high β-glucan content, and can effectively lower blood sugar.

[0003] Chronic kidney disease (CKD) refers to a disease in which kidney damage lasts for more than 3 months due to various reasons, characterized by a continuous and irreversible decline in renal function. As CKD progresses, it eventually turns into end-stage renal disease. It is reported that the number of CKD patients worldwide has reached nearly 700 million, accounting for 9.1% of the global population, and the prevalence of CKD is still rising, becoming an important public health issue. As an important part of human health, the intestinal flora provides beneficial effects to the host by regulating the immune system. There is a bidirectional relationship between the host and the intestinal flora. Intestinal microecological dysbiosis indicates an imbalance in the composition and metabolic capacity of the intestinal flora, which may promote the development of chronic diseases including kidney disease. The interaction between the intestine and the kidney forms the gut-kidney axis, of which the gut flora is an important part. The intestine and the kidney influence each other through multiple connections such as energy metabolism, immune inflammation, and intestinal flora. Intestinal flora dysbiosis is closely related to the pathogenesis of CKD, and its potential mechanism may involve intestinal barrier, systemic immunity, and metabolic disorders. During the progression of CKD, the composition of the intestinal flora and the intestinal environment are imbalanced, which is manifested in many aspects, including increased protein fermentation in the intestine, increased uremic toxins derived from the intestinal flora, reduced carbohydrate fermentation, and reduced production of beneficial metabolites. In addition, intestinal flora imbalance can destroy the integrity of the intestinal epithelium, enhance inflammation and immune processes, and induce endotoxemia caused by enterogenic uremic toxins and acidosis. Patent CN114191457A provides an application of a hibiscus flower extract in regulating intestinal microorganisms associated with diabetic nephropathy. The hibiscus flower extract can increase the relative abundance of different beneficial bacteria in the duodenum, ileum, and colon of the small intestine to varying degrees, reduce the relative abundance of harmful bacteria that can cause inflammatory reactions or damage the intestinal barrier, effectively improve and / or regulate the intestinal flora, and then effectively prevent or treat diseases such as chronic kidney disease and diabetic nephropathy through the effect on the intestinal flora.

[0004] Autoclave is a process of gelatinizing starch under high temperature and high pressure. During the autoclave process, the crystal structure and amorphous lumps of starch are destroyed, and the extract contains more amylose molecules, which are then retrograded to form resistant starch. Kang Lingtao et al. prepared sweet potato resistant starch and studied the effects of factors such as starch milk mass fraction, autoclave time, autoclave temperature, refrigeration time, and pH on the yield of resistant starch. Finally, sweet potato resistant starch with a content of 9.41% was obtained (Kang Lingtao, Song Ying, Liu Sihan, Cui Guijuan, Li Gaoyang. Process optimization of preparing sweet potato resistant starch by autoclave [J]. Food Industry Science and Technology, 2019). Patent CN105001343B provides a product and preparation method thereof that uses autoclave treatment to prepare high resistant starch (RS3) content in combination with enzymatic debranching. Starch is used as raw material and a method combining autoclave treatment and enzymatic debranching is adopted. The resistant starch (RS3) content in the obtained product reaches more than 40%, but the autoclave treatment time is long and only treated once. In addition, the enzymolysis time reaches 12 hours, and a hazardous chemical reagent (2M hydrochloric acid) is added to the starch paste to adjust the pH. Patent CN115947874B provides a method for preparing composite radish RS3 resistant starch, which increases the yield of resistant starch to 62.24% ± 0.21%, but the method first uses an electron beam irradiation device for electron beam irradiation treatment, which increases the production cost. In addition, the method also uses a hazardous chemical reagent (hydrochloric acid) to adjust the pH of the starch solution. In summary, the advantage of autoclave treatment is that the product is highly safe, and the autoclave cooling cycle treatment also belongs to the physical method. The treatment process experienced by the starch solution can ensure the safety of the final product.

[0005] Currently, there are few studies on the preparation of oat starch with high resistant starch content using the autoclave cooling cycle process, and there is no research on the effects of oat starch with high resistant starch content on alleviating CKD. Summary of the invention

[0006] Based on the lack of research in the prior art on the use of autoclave cooling cycle treatment in the preparation of oat starch with a high resistant starch content, and the lack of research on the use of oat starch with a high resistant starch content in alleviating chronic kidney disease (CKD), the purpose of the present invention is to provide an oat starch with a high resistant starch content for alleviating chronic kidney disease, and its preparation method and application. Based on the preparation of oat starch with a high resistant starch content (herein, the resistant starch special oat resistant starch, i.e., ORSA) by autoclave cooling cycle treatment, the present application further studies the effect of oat starch with a high resistant starch content on CKD-related indicators by in vitro fermentation of the intestinal flora of CKD patients using oat starch with a high resistant starch content.

[0007] Oat starch with high resistant starch content can be fermented in the colon, promote intestinal health, and prevent chronic diseases; it is beneficial to host health through multiple mechanisms, such as promoting fecal excretion, regulating lipid and bile acid metabolism, reducing the risk of cancer and cardiovascular disease, and regulating the composition of intestinal flora and the production of beneficial metabolites. Supplementation of oat starch with high resistant starch content is beneficial to regulate CKD-related microecological imbalance, promote the growth of beneficial intestinal bacteria, reduce intestinal uremic toxins, inflammation and oxidative stress, and improve metabolic status, thereby delaying the development of CKD and its complications.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] One of the technical solutions of the present invention provides an oat starch with a high resistant starch content for alleviating chronic kidney disease, wherein the total starch content in the oat starch is 82-85%, and the total starch contains amylopectin and amylose; the total starch includes more than 11.01% oat resistant starch (ORSA).

[0010] The second technical solution of the present invention provides a method for preparing the above-mentioned oat starch with high resistant starch content for alleviating chronic kidney disease, comprising the following steps:

[0011] (1) Preparation of oat starch by dilute alkali method

[0012] Mix oatmeal powder with water to obtain an oatmeal powder solution, adjust the pH and then react;

[0013] The oatmeal powder solution after the reaction was sieved and then centrifuged to collect the precipitate;

[0014] The upper brown precipitate is removed, and the lower white precipitate is collected. The obtained white precipitate is dried, crushed and sieved to obtain oat starch;

[0015] (2) Preparation of oat starch with high resistant starch content by autoclave cooling cycle method

[0016] Mixing the oat starch obtained in step (1) with water to obtain an oat starch solution;

[0017] The obtained oat starch solution is subjected to autoclave gelatinization treatment to gelatinize the oat starch; the gelatinized oat starch is cooled and allowed to stand at low temperature to age the gelatinized oat starch;

[0018] Repeat the above autoclave gelatinization + cooling aging operation to obtain a mixture;

[0019] The mixture is dried, crushed and sieved to obtain oat starch with high resistant starch content prepared by an autoclave cooling cycle method.

[0020] Furthermore, the mixing ratio of the oatmeal powder and water in step (1) is 1:6 to 1:10 (w / v); preferably, the ratio of the oatmeal powder and water is 1:8 (w / v).

[0021] Furthermore, in step (1), the pH of the oatmeal powder solution is adjusted to 8.0-12.0, preferably 10.0; the reagent used to adjust the pH is any one of NaOH and KOH, and the concentration of the reagent is preferably 1-3 mol / L.

[0022] Furthermore, in step (1), after adjusting the pH, the reaction is carried out at 30 to 50° C. for 2 to 6 hours; preferably, the reaction is carried out in a 40° C. water bath for 3 hours.

[0023] Furthermore, in the sieving operation in step (1) or (2), the sieve used is a 100-mesh sieve.

[0024] Furthermore, in step (1), the filtrate obtained by sieving is centrifuged at a rate of 3000 to 5000 r / min for 5 to 20 min, preferably at a rate of 4000 r / min for 10 min to obtain a precipitate.

[0025] Furthermore, in step (1), when the white precipitate is dried, the drying temperature is 40 to 50° C. and the drying time is 24 to 48 hours.

[0026] Furthermore, in step (1), the total starch (amylose + amylopectin) content of the oat starch obtained is measured to be higher than 80%.

[0027] Furthermore, in step (2), the ratio of oat starch to water is 1:1 to 1:10 (w / v), preferably 1:5 (w / v).

[0028] Furthermore, in step (2), the pressure heat cooling cycle method refers to multiple cycles of pressure heat gelatinization treatment + cooling low-temperature static aging treatment; the pressure heat gelatinization treatment is carried out in a high-pressure sterilization pot, the treatment time is 20 to 40 minutes, and the reaction temperature is 115 to 125°C; preferably, the reaction is carried out at 121°C for 30 minutes; the low-temperature static temperature is 0 to 8°C, the time is 12 to 36 hours, preferably 24 hours at 4°C.

[0029] The process of low-temperature standing is the process of starch aging. Starch aging is the process of rearranging and combining the starch molecules that have dissolved and expanded during the gelatinization process to form a new dense, crystalline structure, which makes the starch molecules difficult to digest and forms resistant starch.

[0030] Furthermore, in step (2), the number of autoclave cooling cycles is at least 1, thereby increasing the production of resistant starch by increasing the number of starch gelatinization and aging.

[0031] Furthermore, in step (2), the drying is carried out in a blast drying oven, the drying temperature is 40 to 50° C., and the drying time is 24.0 to 36.0 hours.

[0032] The third technical solution of the present invention provides an application of the oat starch with a high resistant starch content for alleviating chronic kidney disease, wherein the application is to prepare the oat starch with a high resistant starch content for alleviating chronic kidney disease into a health product to alleviate CKD symptoms.

[0033] In some specific embodiments of the present invention, the CKD symptoms include high levels of creatinine, urea nitrogen, indole and p-cresol, high activities of tryptophanase and tyrosinase, and low levels of short-chain fatty acids.

[0034] In some specific embodiments of the present invention, the novel oat dietary fiber prepared in step (2) of the above technical scheme, i.e., oat starch with a high resistant starch content, is fermented in vitro by the intestinal flora of CKD patients to explore its effect on CKD-related indicators. The results show that in vitro fermentation of ORSA helps to reduce the levels of creatinine, urea nitrogen, indole and p-cresol in the fermentation broth, as well as the activities of tryptophanase and tyrosinase, and increase the content of short-chain fatty acids, but does not involve a method for treating the disease.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention uses a pressure heat cooling cycle technology in the process of preparing a new type of oat dietary fiber - oat starch with a high resistant starch content, in order to solve the problems of low resistant starch content in oat starch and improve its product safety, and effectively increase the resistant starch content in oat starch from 5.31% to 11.01%. The high temperature and high pressure conditions in the autoclave cause the oat starch to gelatinize. The crystal structure and amorphous agglomerates of the starch are destroyed during the pressure heat process. The extract contains more straight-chain starch molecules, which are then regenerated and polymerized to form resistant starch after low-temperature aging. The surface of the resistant starch treated by pressure heat is broken, and a large number of wrinkles appear, and the distance between the starch particles is reduced to form a dense structure. This structure is conducive to increasing its anti-digestion performance and freeze-thaw stability, as well as improving the physical and chemical properties of the original starch such as gel texture characteristics and thermal stability. The pressure heat recrystallization treatment causes the starch crystal form to change from type A to type B or type B+V crystals, and the crystallinity is significantly increased. In addition, the pressure heat cooling cycle treatment is a physical processing process, and the product safety is high.

[0037] (2) The present invention uses oat starch as a raw material to prepare oat starch with a high resistant starch content and high product safety. Since resistant starch has multiple physiological functions, it is beneficial to increase the added value of oat-related products.

[0038] (3) The novel oat dietary fiber prepared by the autoclave cooling cycle method of the present invention, oat starch ORSA with high resistant starch content, helps alleviate CKD and delay the progression of CKD. In the fermentation liquid of stool samples of CKD patients treated with ORSA, the renal function indicators creatinine, urea nitrogen, uremic toxin precursors indole and p-cresol, and the activities of their production enzymes tryptophanase and tyrosinase were significantly reduced.

[0039] (4) The present invention broadens the application scope of oat starch with high resistant starch content, provides an important theoretical basis for the deep processing of oats, and provides a theoretical basis for the application of autoclave cooling cycle technology in the preparation of resistant starch with high product safety. It provides theoretical guidance for the application of new oat dietary fiber in health products with the effect of alleviating CKD, and has important academic value and application prospects.

[0040] (5) The novel oat dietary fiber provided by the present invention, oat starch with a high resistant starch content, produces beneficial metabolites, short-chain fatty acids (SCFAs), during fermentation by intestinal flora. Studies have shown that SCFAs have significant physiological regulatory functions, such as providing part of the energy required by the human body and regulating electrolyte balance, protecting the intestinal mucosal barrier, regulating lipid metabolism, regulating the pH value in the intestine, inhibiting intestinal inflammation, as well as anti-tumor effects and regulating immune responses. Supplementing dietary fiber is beneficial for regulating CKD-related intestinal microecological imbalance, promoting the growth of beneficial intestinal bacteria, reducing intestinal-derived uremic toxins, inflammation and oxidative stress, and improving metabolic status. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the creatinine content in the fermentation broth of each group;

[0042] Figure 2 is the urea nitrogen content in the fermentation broth of each group;

[0043] Figure 3 is the indole content in the fermentation broth of each group;

[0044] Figure 4 is the p-cresol content in the fermentation broth of each group;

[0045] Figure 5 is the tryptophanase activity in the fermentation broth of each group;

[0046] Figure 6 is the tyrosinase activity in the fermentation broth of each group. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations 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.

[0048] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0049] Example 1: Preparation of oat starch with high resistant starch content for alleviating chronic kidney disease, the specific steps are as follows:

[0050] (I) Preparation of oat starch

[0051] Take 500g oatmeal powder, add 4L water and mix well, adjust the pH of the oatmeal powder solution to 10 with 2mol / L NaOH, and react at 40℃ for 3h. After the reaction is completed, pass through a 100-mesh sieve, centrifuge the filtrate at 4000r / min for 10min, collect the precipitate, scrape off the brown upper layer, and obtain the white lower layer starch; dry, crush and sieve the obtained lower layer white starch (100-mesh sieve), and dry it in a blast drying oven at a drying temperature of 40℃ and a drying time of 36h. The final product is oatmeal starch.

[0052] The total weight of the oat starch obtained in the above steps is 310 g; the total starch content (amylose + amylopectin) of the oat starch measured by Megazyme total starch assay kit and resistant starch assay kit is 83.26%, and the resistant starch content is 5.31%.

[0053] The formula used in the above numerical calculation is:

[0054] Total starch content % = ΔA × F × EV × D × 0.9 / w × 100%;

[0055] Where: ΔA is the absorbance value of the sample solution after deducting the reagent blank; F is the factor, 100 divided by (the average absorbance value corresponding to 4 portions of D-Glucose Standard after incubation); EV is the fixed volume, 100mL; D is the dilution factor 1; w is the sample weight, mg.

[0056] Resistant starch content% = ΔE × F / W × 90 × 100%;

[0057] Where: ΔE is the absorbance value of the control reagent blank reading; F is the conversion factor of absorbance value to micrograms, 100 divided by the GOPOD absorbance value of 100 micrograms of D-glucose; W is the dry weight of the analyzed sample.

[0058] (II) Preparation of oat starch with high resistant starch content by autoclave cooling cycle method

[0059] Take 100 g of the oat starch obtained in step (1), add 500 mL of distilled water and mix well, put it into a high pressure sterilizer and react at 121° C. for 30 min to gelatinize the starch and destroy the starch molecular structure; after the reaction is completed, cool it to room temperature and then place it at 4° C. for 24 h to allow the starch to age and the starch molecules to rearrange to form resistant starch.

[0060] The above autoclave cooling cycle is repeated once to obtain a product containing resistant starch. The obtained product is dried, crushed and sieved, and the drying is carried out in a forced air drying oven at a drying temperature of 40° C. for 36 hours to finally obtain oat starch with a high resistant starch content.

[0061] The total weight of the oat starch with high resistant starch content obtained in the above steps is about 90 g; the total starch content of the oat starch is measured by using a Megazyme total starch assay kit and a resistant starch assay kit, which is 83.61%, and the resistant starch content increases to 11.01%.

[0062] The present application provides oat starch with a high resistant starch content. Since the processing method is purely physical processing, the increase in the resistant starch content is limited, but the product is highly safe.

[0063] Comparative Example 1

[0064] An oat beta-glucan (OBGM), the specific information is as follows:

[0065] The molecular weight of oat β-glucan (OBGM) is 5.04×10 4 Da (70%, HPLC) was purchased from Guangzhou Beta Health Biotechnology Co., Ltd.

[0066] Comparative Example 2

[0067] An oat beta-glucan (OBGH), the specific information is as follows:

[0068] The molecular weight of oat β-glucan (OBGH) is 1.77×10 5 Da (70%, HPLC) was purchased from Guangzhou Beta Health Biotechnology Co., Ltd.

[0069] Example 2: In vitro fermentation of oat starch with high resistant starch content by intestinal flora of CKD patients and preparation of fermentation broth

[0070] This example is based on the oat starch and oat starch with a high resistant starch content prepared in Example 1, and studies the in vitro fermentation of oat starch with a high resistant starch content by the intestinal flora of CKD patients to explore the effect of oat starch with a high resistant starch content on the intestinal flora.

[0071] 1. Experimental materials and experimental groups:

[0072] Example 1 prepared oat starch, oat starch with high resistant starch content ORSA, wherein the resistant starch content of ORSA was 11.01%, and a bacterial solution prepared from fecal samples of CKD patients (in accordance with the relevant requirements of the Medical Ethics Committee of the 905th Hospital of the Chinese People's Liberation Army Navy, fecal samples of non-dialysis CKD patients in the Nephrology Department were obtained) with a concentration of about 1.2×10 9 CFU / mL, the bacterial solution prepared from the stool samples of CKD patients' family members and hospital nurses has a concentration close to that of the bacterial solution prepared from the patient samples, which is about 1.2×10 9 CFU / mL;

[0073] The experimental groups are:

[0074] HC group: 20 mL of bacterial solution prepared from fecal samples of healthy people + 1% (w / v) oat starch;

[0075] BC group: 20 mL of bacterial solution prepared from stool samples of CKD patients + 1% (w / v) oat starch;

[0076] ORSA group: 20 mL of bacterial solution prepared from stool samples of CKD patients + 1% (w / v) oat starch ORSA with high resistant starch content;

[0077] OBGM group: 20 mL of bacterial solution prepared from stool samples of CKD patients + 1% (w / v) oat β-glucan (OBGM);

[0078] OBGH group: 20 mL of bacterial solution prepared from stool samples of CKD patients + 1% (w / v) oat β-glucan (OBGH).

[0079] (II) Fermentation experiment:

[0080] The bacterial liquids of the above different groups were placed in a 280L anaerobic incubator. After fermentation for 24 hours, the fermentation liquids were collected for further use.

[0081] Example 3: Effect of high resistant starch content oat starch ORSA on creatinine in fermentation broth

[0082] The fermentation broth obtained in Example 2 was used to further explore the effect of high resistant starch content oat starch ORSA on creatinine in the fermentation broth. The creatinine content in the fermentation broth was determined using an enzymatic kit according to the instructions provided by the kit. The results are as follows: Figure 1 shown.

[0083] Creatinine content (μmol / L) = (ΔA determination - ΔA blank) / (ΔA standard - ΔA blank) × C standard;

[0084] Among them, ΔA determination, ΔA blank, and ΔA standard are the absorbance differences of the determination sample group, blank group, and standard product, respectively;

[0085] C standard: standard concentration, 442 μmol / L.

[0086] Depend on Figure 1 It can be seen that after 24 hours of in vitro fermentation, the creatinine content of the BC group increased significantly compared with the HC group. The creatinine content of the ORSA group decreased significantly compared with the BC group, OBGM group, and OBGH group, indicating that ORSA intervention helps to reduce the creatinine level in the fermentation liquid of CKD intestinal flora.

[0087] Example 4: Effect of oat starch with high resistant starch content on urea nitrogen in fermentation broth

[0088] The fermentation broth obtained in Example 2 was used to further explore the effect of oat starch ORSA with high resistant starch content on urea nitrogen in the fermentation broth. The urea nitrogen content in the fermentation broth was determined using an enzymatic kit according to the instructions provided by the kit. The results are as follows: Figure 2 shown.

[0089] Urea nitrogen content (mmol / L) = (A determination - A blank) / (A standard - A blank) × C standard × N;

[0090] Among them, A determination, A blank, and A standard are the absorbance values ​​of the determination sample group, blank group, and standard product, respectively;

[0091] C standard: standard concentration, 10mmol / L;

[0092] N: sample dilution multiple.

[0093] Depend on Figure 2 It can be seen that after 24 hours of in vitro fermentation, the urea nitrogen content of the BC group increased significantly compared with the HC group. Compared with the BC group, the OBGM group, and the OBGH group, the urea nitrogen content of the ORSA group decreased significantly, indicating that the intervention of ORSA helps to reduce the urea nitrogen level in the fermentation liquid of the CKD intestinal flora.

[0094] Example 5: Effect of oat starch with high resistant starch content on uremic toxin precursor indole in fermentation broth

[0095] The fermentation broth obtained in Example 2 was used to further explore the effect of oat starch ORSA with a high resistant starch content on indole, a precursor of uremic toxins, in the fermentation broth. The method for determining the indole content in the fermentation broth is as follows:

[0096] The fermentation broth was centrifuged at 10000r / min for 3min. 100mL of supernatant was taken and 180mL of ether and 90mL of ethyl acetate were added. Vortex, then centrifuged at 12000r / min for 10min, and 200mL of supernatant was taken. The ether and ethyl acetate were removed by nitrogen blowing, 200mL of acetonitrile was added, and the supernatant was taken after centrifugation again and tested by high performance liquid chromatography.

[0097] HPLC detection conditions: mobile phase A is ammonium acetate buffer (0.2M, pH 4.5); mobile phase B is acetonitrile. 60% B phase isocratic elution, injection time 12min, injection volume 10mL, total flow rate 0.7mL / min, column temperature 35℃, detection wavelength 270nm. The elution time is based on the elution time of the indole standard.

[0098] Depend on Figure 3 It can be seen that after 24 hours of in vitro fermentation, the indole content in the BC group increased significantly compared with the HC group. Compared with the BC group, the indole content in the ORSA group decreased significantly, and was also lower than that in the OBGM group and the OBGH group, indicating that the intervention of ORSA helps to reduce the level of indole, a precursor of uremic toxins, in the fermentation liquid of the CKD intestinal flora.

[0099] Example 6: Effect of oat starch with high resistant starch content on the activity of indole-generating enzyme tryptophanase in fermentation broth

[0100] The fermentation broth obtained in Example 2 was used to further explore the effect of high resistant starch content oat starch ORSA on the activity of indole-generating enzyme tryptophanase in the fermentation broth. The tryptophanase activity in the fermentation broth was determined using an ELISA kit according to the instructions provided by the kit. The results are as follows: Figure 4 shown.

[0101] Depend on Figure 4 It can be seen that after 24 hours of in vitro fermentation, the activity of indole-generating enzyme-tryptophanase in the BC group increased significantly compared with the HC group. Compared with the BC group, the activity of indole-generating enzyme-tryptophanase in the ORSA group decreased significantly, and was also lower than that in the OBGM group and the OBGH group, indicating that the intervention of ORSA helps to reduce the activity level of indole-generating enzyme-tryptophanase, a precursor of uremic toxins, in the fermentation liquid of the CKD intestinal flora.

[0102] Example 7: Effect of oat starch with high resistant starch content on uremic toxin precursor substance - p-cresol in fermentation broth

[0103] The fermentation broth obtained in Example 2 was used to further explore the effect of oat starch ORSA with a high resistant starch content on p-cresol, a precursor of uremic toxins in the fermentation broth. The method for determining the p-cresol content in the fermentation broth is as follows:

[0104] The fermentation broth was centrifuged at 10000 r / min for 3 min. 100 mL of supernatant was taken and 180 mL of ether and 90 mL of ethyl acetate were added. Vortex, then centrifuged at 12000 r / min for 10 min, and 200 mL of supernatant was taken. The ether and ethyl acetate were removed by nitrogen blowing, 200 mL of acetonitrile was added, and the supernatant was taken after centrifugation again and detected by high performance liquid chromatography.

[0105] HPLC detection conditions: mobile phase A is ammonium acetate buffer (0.2M, pH 4.5); mobile phase B is acetonitrile. 60% B phase isocratic elution, injection time 12min, injection volume 10mL, total flow rate 0.7mL / min, column temperature 35℃, detection wavelength 270nm. The elution time is based on the p-cresol standard.

[0106] Depend on Figure 5 It can be seen that after 24 hours of in vitro fermentation, the p-cresol content in the BC group increased significantly compared with the HC group. Compared with the BC group, the p-cresol content in the ORSA group decreased significantly, and was also lower than that in the OBGM group and the OBGH group, indicating that the intervention of ORSA helps to reduce the level of p-cresol, a precursor of uremic toxins, in the fermentation liquid of the CKD intestinal flora.

[0107] Example 8: Effect of oat starch with high resistant starch content on the activity of tyrosinase, a p-cresol-generating enzyme, in fermentation broth

[0108] The fermentation broth obtained in Example 2 was used to further explore the effect of oat starch ORSA with a high resistant starch content on the activity of tyrosinase, an enzyme that generates p-cresol, in the fermentation broth. The tyrosinase activity in the fermentation broth was determined using an ELISA kit according to the instructions provided by the kit.

[0109] Depend on Figure 6 It can be seen that after 24 hours of in vitro fermentation, the activity of p-cresol-generating enzyme-tyrosinase in the BC group was significantly increased compared with the HC group. Compared with the BC group, the activity of p-cresol-generating enzyme-tyrosinase in the ORSA group was significantly reduced, and was also lower than that in the OBGM group and the OBGH group, indicating that the intervention of ORSA helps to reduce the level of activity of p-cresol-generating enzyme-tyrosinase, a precursor of uremic toxins, in the fermentation liquid of the CKD intestinal flora.

[0110] Example 9: Effect of oat starch with high resistant starch content on the content of short-chain fatty acids (SCFAs) in fermentation broth

[0111] The fermentation broth obtained in Example 2 was taken to further explore the effect of oat starch ORSA with high resistant starch content on the content of short-chain fatty acids SCFAs in the fermentation broth (including the content of each of acetic acid, propionic acid, butyric acid and the total content of the three). Short-chain fatty acids (SCFAs) can be made from all carbohydrates, but are mainly made from prebiotic dietary fiber, which is beneficial to bacterial activity. These organic compounds have many important functions in the gastrointestinal tract and in human health. The method for determining the content of SCFAs in the fermentation broth is as follows:

[0112] The fermentation broth was centrifuged at 10000r / min for 10min, 2mL of supernatant was taken, 0.1mL of HCl was added and the mixture was thoroughly shaken and mixed, then 2mL of anhydrous ether was added, the mixture was extracted for 20min, then centrifuged at 4000r / min for 5min, and the supernatant was taken. 0.5mL of 1M NaOH was added and extracted for 20min, then centrifuged at 4000r / min for 5min, the lower aqueous phase was collected, 0.1mL of HCl was added and mixed, and the obtained sample was filtered through a 0.22μm filter membrane and transferred to a liquid phase injection bottle for testing.

[0113] HPLC detection conditions: mobile phase A is 0.025% phosphoric acid solution (pH=2.8), mobile phase B is acetonitrile. 5% B phase isocratic elution, flow rate 1 mL / min, detection wavelength 210 nm, column temperature 30°C, injection volume 20 μL, injection time 15 min. The peak time is based on the SCFAs standard.

[0114] Table 1 Content of SCFAs in fermentation broth of each group

[0115]

[0116] Note: The results are expressed as mean ± standard deviation. The values ​​with different letters in the same row are significantly different (p<0.05).

[0117] As shown in Table 1, after 24 hours of in vitro fermentation, the short-chain fatty acid content in the ORSA group was significantly higher than that in other groups, indicating that ORSA intervention helps increase the content of short-chain fatty acids, a beneficial metabolite, in the fermentation liquid of the CKD intestinal flora, which is beneficial to the health of the host and delays the progression of CKD.

[0118] In summary, the new type of oat dietary fiber prepared by the autoclave cooling cycle treatment - oat starch ORSA with high resistant starch content helps to improve renal function, reduce the levels of renal function indicators such as creatinine, urea nitrogen, indole, and p-cresol, and reduce the activity of indole-producing enzyme - tryptophanase and p-cresol-producing enzyme - tyrosinase, increase the content of beneficial metabolites short-chain fatty acids, and relieve chronic kidney disease.

[0119] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An oat starch with a high resistant starch content for alleviating chronic kidney disease, characterized in that: The total starch content of the oat starch is 82-85%, the total starch contains amylopectin and straight-chain starch, and the total starch includes more than 11.01% of oat resistant starch ORSA.

2. The method for preparing oat starch with high resistant starch content for alleviating chronic kidney disease according to claim 1, characterized in that: The following steps are involved: (1) Preparation of oat starch by dilute alkali method Mix oatmeal powder with water to obtain an oatmeal powder solution, adjust the pH and then react; The oatmeal powder solution after the reaction was sieved and then centrifuged to collect the precipitate; The upper brown precipitate is removed, and the lower white precipitate is collected. The obtained white precipitate is dried, crushed and sieved to obtain oat starch; (2) Preparation of oat starch with high resistant starch content by autoclave cooling cycle method Mixing the oat starch obtained in step (1) with water to obtain an oat starch solution; The obtained oat starch solution is subjected to autoclave gelatinization treatment to gelatinize the oat starch; the gelatinized oat starch is cooled and allowed to stand at low temperature to age the gelatinized oat starch; Repeat the above autoclave gelatinization + cooling aging operation to obtain a mixture; The mixture is dried, crushed and sieved to obtain oat starch with high resistant starch content prepared by an autoclave cooling cycle method.

3. The method for preparing oat starch with high resistant starch content for alleviating chronic kidney disease according to claim 2, characterized in that: In step (1), the mixing ratio of the oatmeal powder and water is 1:6 to 1:10 (w / v); The pH of the oatmeal powder solution is adjusted to 8.0-12.0; the reagent used to adjust the pH is any one of NaOH and KOH.

4. The method for preparing oat starch with high resistant starch content for alleviating chronic kidney disease according to claim 2, characterized in that: In step (1), after adjusting the pH, the reaction is carried out at 30 to 50° C. for 2 to 6 hours.

5. The method for preparing oat starch with high resistant starch content for alleviating chronic kidney disease according to claim 2, characterized in that: In the sieving operation described in step (1) or (2), the sieve used is a 100-mesh sieve.

6. The method for preparing oat starch with high resistant starch content for alleviating chronic kidney disease according to claim 2, characterized in that: In step (1), the filtrate obtained by sieving is centrifuged at a rate of 3000 to 5000 r / min for 5 to 20 min to obtain a precipitate; When the white precipitate is dried, the drying temperature is 40 to 50°C and the drying time is 24 to 48 hours.

7. The method for preparing oat starch with high resistant starch content for alleviating chronic kidney disease according to claim 2, characterized in that: In step (2), the ratio of oat starch to water is 1:1 to 1:10 (w / v).

8. The method for preparing oat starch with high resistant starch content for alleviating chronic kidney disease according to claim 2, characterized in that: In step (2), the autoclave cooling cycle method refers to multiple cycles of autoclave gelatinization treatment + cooling low-temperature static aging treatment; The pressure heat gelatinization treatment time is 20 to 40 minutes, and the reaction temperature is 115 to 125°C; The low temperature standing temperature is 0 to 8°C and the time is 12 to 36 hours; The number of the autoclave cooling cycle is at least 1.

9. The method for preparing oat starch with high resistant starch content for alleviating chronic kidney disease according to claim 2, characterized in that: In step (2), the drying temperature is 40-50° C. and the drying time is 24.0-36.0 h.

10. The use of oat starch with high resistant starch content for alleviating chronic kidney disease as claimed in claim 1, characterized in that: The application is to prepare the oat starch with high resistant starch content for relieving chronic kidney disease into a health product for relieving CKD symptoms; the CKD symptoms include high levels of creatinine, urea nitrogen, indole and p-cresol, high activities of tryptophanase and tyrosinase, and low content of short-chain fatty acids.

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