Oat resistant starch as well as preparation method and application thereof

Abstract

The invention relates to oat resistant starch as well as a preparation method and application thereof. The preparation method comprises the following steps: uniformly mixing oat powder with water, adjusting the pH value, carrying out alkali extraction, sieving, centrifuging, collecting precipitate, scraping a brown upper layer, washing a white part, drying, crushing and sieving to obtain oat starch; water is added into the obtained oat starch to prepare starch paste, the starch paste is cooled after gelatinization, the pH is adjusted, pullulanase is used for conducting enzymolysis on the oat starch, meanwhile, ultrasonic treatment is conducted, enzyme deactivation is conducted after enzymolysis is finished, then aging is conducted, drying, smashing and sieving are conducted, and the oat resistant starch is obtained. According to the method disclosed by the invention, an ultrasonic wave combined enzyme hydrolysis technology is used, and the enzymolysis of pullulanase is promoted by ultrasonic waves, so that the problems of low enzymolysis reaction efficiency, long enzymolysis time and the like in the process of preparing the resistant starch by an enzyme method are solved, and the content of the resistant starch in the oat starch is effectively increased from 6.12% to 36.15%. The oat resistant starch ORSU disclosed by the invention is beneficial to improving the renal function, reducing the generation of uremic toxins and delaying the CKD process.

Description

Technical Field

[0001] The invention belongs to the technical field of functional foods, and in particular relates to oat resistant starch and a preparation method and application thereof. Background Art

[0002] Oats are rich in starch, protein, fat, dietary fiber, polyphenols and trace elements, and have many health benefits. They are functional grains widely consumed around the world. The starch content in oats is relatively high, about 60%, of which the amylose chain length is short and the crystallinity is high, making it suitable for the preparation of resistant starch. Although my country has abundant starch resources and a full range of varieties, domestic resistant starch mainly studies common varieties such as corn starch and potato starch, and there is less research on oat starch. At present, oats in my country are mainly used to produce oatmeal, oat flour and oat rice, etc., and their processing level is not high, and the added value of products is low. Nowadays, more and more scientific studies have revealed the functional properties of oats. Oats have become a recognized health food in the world. The market demand for oats and oat products is growing. Therefore, it is necessary to strengthen the full utilization of oat resources, research and develop products related to oat deep processing, and increase the added value of its products.

[0003] Chronic kidney disease (CKD) refers to a disease in which kidney damage (abnormal kidney structure and function) caused by various reasons lasts for more than 3 months. CKD is a chronic progressive disease. The occurrence of chronic diseases is closely related to the patient's diet. The intake of protein, fat, dietary fiber and other ingredients in food will affect it. Intestinal flora and uremic toxins play an important role in the progression of CKD. Streptococci in the intestinal flora contain tryptophanase and tyrosinase, which can metabolize tryptophan and tyrosine to produce uremic toxin precursors indole and p-cresol, respectively, which are further converted into uremic toxins indoxyl sulfate and p-cresol sulfate, accelerating the progression of CKD. Dietary nutritional intervention, as one of the auxiliary measures to improve CKD, can delay the progression of CKD and improve the quality of life of CKD patients. In their daily diet, CKD patients need to control the intake of sodium, potassium, phosphorus and protein. However, fruits and vegetables with high dietary fiber content are less consumed due to their high potassium content, which affects the dietary fiber intake of patients. Dietary fiber mainly comes from natural plants and compounds with health benefits processed from them. It is not easily digested and absorbed in the small intestine, and is partially or completely fermented by colonic bacteria in the colon to produce beneficial metabolites such as short-chain fatty acids. Due to the special structure and physiological characteristics of dietary fiber, it can maintain the balance of dietary structure and also play an auxiliary role in the treatment and prevention of some chronic diseases. For example, the short-chain fatty acids produced by the fermentation of dietary fiber by some intestinal flora help to improve the intestinal environment, regulate intestinal flora, and reduce the production of uremic toxins. Ordinary dietary fiber has the characteristics of poor taste and flavor when added to food, resulting in low market acceptance. Resistant starch is a new type of dietary fiber, which not only improves some of the shortcomings of ordinary dietary fiber, but also has similar physiological functions to dietary fiber, becoming a research hotspot in the health care product processing industry.

[0004] Liu Min et al. used microwave-moist heat method to prepare potato resistant starch with a resistant starch content of 9.77% by changing the mass fraction of potato starch milk, microwave power, time and other factors (Liu Min, Han Yumei, He Jun, Huang Huan, Wang Shaofan. Optimization of microwave-moist heat preparation process and structural characterization of potato resistant starch [J]. Food Industry Science and Technology, 2018, 39(15): 176-180). Wei Qiuyu et al. used cassava starch as raw material, starch milk concentration of 20%, high temperature gelatinization, and added pullulanase to obtain cassava resistant starch with a resistant starch content of 13.36% (Wei Qiuyu. Enzymatic preparation and mechanism study of cassava resistant starch [D]. Guangxi University, 2007). Related studies have shown that the yield and efficiency of resistant starch prepared by physical (such as autoclave, wet heat, etc.) or biochemical methods alone are low, and there are also problems such as long operation time, high energy consumption and safety. Summary of the invention

[0005] Based on the problem of low yield and efficiency of resistant starch prepared by physical (such as autoclave, wet heat, etc.) or biochemical methods alone in the prior art, the present invention provides oat resistant starch and a preparation method and application thereof.

[0006] The present invention combines the ultrasonic method with the enzymatic method, thereby effectively reducing the enzymatic hydrolysis time, increasing the yield of resistant starch, and effectively improving the reaction efficiency. In addition, the present application further studies the effect of oat resistant starch on the progression of chronic kidney disease, and further provides the use of oat resistant starch in the preparation of functional foods, health products or drugs for improving renal function.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention first provides a method for preparing oat resistant starch, which comprises the following steps:

[0009] (1) Preparation of oat starch

[0010] Take oatmeal powder and water and mix them evenly, adjust the pH, perform alkali extraction, sieve and centrifuge to collect the precipitate, scrape off the brown upper layer, wash the white part, and then dry, crush and sieve to obtain oatmeal starch;

[0011] (2) Preparation of oat resistant starch

[0012] The oat starch obtained in step (1) is added with water to prepare starch paste, cooled after gelatinization, and the pH is adjusted. The oat starch is enzymatically hydrolyzed with pullulanase and ultrasonically treated. After the enzymatic hydrolysis is completed, the enzyme is inactivated, and the oat starch is aged, and then dried, crushed and sieved to obtain oat resistant starch (ORSU).

[0013] In one embodiment of the present invention, in step (1), the ratio of oatmeal powder to water is 1:6-1:10 (w / v), and the water is distilled water.

[0014] Preferably, the ratio of oat flour to water is 1:8 (w / v).

[0015] In one embodiment of the present invention, in step (1), NaOH is used to adjust the pH of the oatmeal solution to 8.0-12.0, preferably 10.0.

[0016] Preferably, the concentration of NaOH is 2 mol / L.

[0017] In one embodiment of the present invention, in step (1), alkali extraction is performed in a water bath at 30-50° C. for 2.0-5.0 h.

[0018] Preferably, the alkali extraction is carried out in a 40°C water bath for 3.0 h.

[0019] In one embodiment of the present invention, in step (1), the two screenings are both through a 100-mesh sieve.

[0020] In one embodiment of the present invention, in step (1), the filtrate obtained by sieving is centrifuged at a rate of 3000-5000 r / min (preferably 4000 r / min) for 5-20 min, preferably 10 min, after which the supernatant is poured out, the brown material on the surface of the precipitate is scraped off, and the crude starch in the lower layer is retained.

[0021] In one embodiment of the present invention, in step (1), drying is performed in a blast drying oven, the drying temperature is 40-50° C., preferably 40° C., and the drying time is 24.0-36.0 h.

[0022] In one embodiment of the present invention, in step (1), the total starch content of the oat starch obtained is higher than 80%.

[0023] In one embodiment of the present invention, in step (2), the ratio of oat starch to water is 1:10-1:30 (w / v), preferably 1:20 (w / v).

[0024] In one embodiment of the present invention, in step (2), gelatinization is carried out in a boiling water bath for 20-30 minutes.

[0025] In one embodiment of the present invention, in the step (2), the gelatinization is followed by cooling to 40-60°C, preferably 50°C.

[0026] In one embodiment of the present invention, in step (2), HCl is used to adjust the pH to 4.0-6.0, preferably 5.0.

[0027] Preferably, the concentration of HCl is 0.05 mol / L.

[0028] In one embodiment of the present invention, in step (2), the enzyme activity of pullulanase is ≥ 1000 npun / g. Preferably, the commercial product produced by Sigma is selected.

[0029] In one embodiment of the present invention, in the step (2), the amount of pullulanase added is 20-40 npun / g, preferably 27.5 npun / g starch (the starch refers to the oat starch obtained in step (1)). The amount of enzyme used is related to the activity of the enzyme, and the activity of enzymes produced by different manufacturers may vary. However, in general, the present invention does have the advantage of requiring less enzyme.

[0030] In one embodiment of the present invention, in step (2), the enzymolysis temperature is 40-60°C and the enzymolysis time is 10.0-14.0h. The main function of pullulanase is to hydrolyze the α-1,6 glycosidic bonds on the amylopectin molecules to produce a large number of amylose molecules. The higher the content of amylose in starch, the more chances there are for crystallization between amylose molecules when the starch paste ages, and the yield of resistant starch increases accordingly. The enzymolysis time in some documents reaches 24.0h or even 36.0h, while the enzymolysis time in the present invention is only 10.0-14.0h, which is much shorter than the time reported in the literature. This effect is obtained by carrying out ultrasound and enzymolysis reaction simultaneously. Ultrasound is conducive to the diffusion of enzymes, increases the contact between enzymes and starch granules, and improves the efficiency of enzyme reactions.

[0031] Preferably, the enzymolysis temperature is 50°C and the enzymolysis time is 12.0 h.

[0032] In one embodiment of the present invention, in step (2), the power of ultrasonic treatment is 200-300w, the time of ultrasonic treatment is 5-20min, the temperature of ultrasonic treatment is 40-60°C, ultrasonic treatment and enzymatic hydrolysis are carried out simultaneously, and enzymatic hydrolysis is continued after the ultrasonic treatment ends.

[0033] Preferably, the power of ultrasonic treatment is 240w, the time of ultrasonic treatment is 10min, and the temperature of ultrasonic treatment is 50°C.

[0034] In one embodiment of the present invention, in step (2), after the enzymatic hydrolysis is completed, the enzyme is inactivated in a boiling water bath for 5-20 minutes, preferably 10 minutes.

[0035] In one embodiment of the present invention, in step (2), the aging process is specifically: aging at 2-6°C for 20.0-30.0h.

[0036] Preferably, the aging process is specifically: aging for 24.0 hours at 4° C. Starch aging is to rearrange and recombine the starch molecules that are dissolved and swollen during the gelatinization process to form a new dense and crystallized structure, making the starch molecules difficult to digest and forming resistant starch.

[0037] In one embodiment of the present invention, in step (2), drying is performed in a forced air drying oven, the drying temperature is 40-50° C., preferably 40° C., and the drying time is 24.0-36.0 h.

[0038] In one embodiment of the present invention, in step (2), the sieving is through a 100-mesh sieve.

[0039] In one embodiment of the present invention, in step (2), the content of resistant starch in oat resistant starch (ORSU) is 36.15%.

[0040] The second object of the present invention is to provide oat resistant starch prepared based on the above preparation method. Compared with conventional oat starch, ultrasound combined with enzymatic hydrolysis treatment changes the starch crystal type from type A to type B + C, and the crystallinity is significantly increased. The crystal type of oat resistant starch prepared by conventional physical processing methods such as autoclave cooling cycle may be type B or type B + V, and the crystallinity is lower than that of oat resistant starch prepared by ultrasound combined with enzyme hydrolysis. In addition, the volume average particle size and surface area average particle size of oat resistant starch prepared by ultrasound combined with enzyme hydrolysis are both smaller than those prepared by autoclave cooling cycle. In terms of apparent morphological characteristics, the surface of oat resistant starch prepared by ultrasound combined with enzyme hydrolysis has more groove-like stripes, which may be due to the addition of ultrasonic treatment during the enzymatic hydrolysis process. When the cavitation bubbles burst under the action of ultrasound, a higher pressure gradient and local velocity are formed near the bubbles, generating shear force, damaging starch granules, and cutting long chains. The cavitation effect of ultrasound acts on the enzymatic hydrolysis system, destroying the surface shape of starch granules, generating some cracks, and further promoting enzymatic hydrolysis.

[0041] In one embodiment of the present invention, the content of resistant starch in the oat resistant starch (ORSU) is 36.15%.

[0042] The third object of the present invention is to provide the use of oat resistant starch prepared based on the above preparation method in the preparation of functional foods, health products or medicines.

[0043] The functional food or health product refers to a drug that has the effect of improving kidney function and / or delaying the progression of CKD. The functional food or health product mainly improves CKD by a dietary nutritional intervention. The drug refers to a drug that has the effect of improving kidney function and / or delaying the progression of CKD.

[0044] In some embodiments of the present invention, oat resistant starch is used in the preparation of functional foods, health products or medicines that target streptococci to improve renal function.

[0045] In some embodiments of the present invention, the application includes the use of ingestion of oat resistant starch ORSU to help improve the body weight, kidney index, renal function indicators serum creatinine, urea nitrogen, uric acid, lipopolysaccharide, TNF-α, uremic toxin levels and targeted regulation of streptococci in different CKD model mice, but does not involve a method of treating the disease.

[0046] The present application discloses that oat resistant starch can be prepared into functional food, health product or medicine having the effect of improving kidney function.

[0047] This application applies oat resistant starch ORSU to different CKD model mice to explore its effect on mouse renal function. Specifically, this application constructs a humanized CKD mouse model of intestinal flora and intervenes in CKD mice with oat resistant starch to study the effect of ORSU on renal function, uremic toxins, and intestinal flora of CKD mice; and screens out the key characteristic bacteria that promote the CKD process-Streptococcus Paris (S. lutetiensis) and ORSU for in vitro co-culture, and constructs a pseudo-sterile CKD mouse model colonized with S. lutetiensis to study the effect of ORSU on S. lutetiensis regulating uremic toxins.

[0048] The most commonly used enzyme debranching is pullulanase. The working principle of the pullulanase debranching method is: under the action of enzymes, the α-1,6 glucosidic bonds in the amylopectin in starch are hydrolyzed, so that the hydrolyzate contains more free straight-chain molecules, and the specific parts of the straight-chain starch are intertwined and folded to form a stable double helix structure. The double helix can be further polymerized through hydrogen bonds and van der Chem forces to form ordered crystals that are extremely resistant to amylase. Therefore, resistant starch cannot be digested, decomposed and absorbed in the human gastrointestinal tract. In the process of enzyme reaction, the enzyme concentration and enzymatic hydrolysis time have a great influence on the final yield of resistant starch.

[0049] When ultrasound is used as a physical method in conjunction with the enzymatic method to prepare resistant starch, it is beneficial to improve the yield. Ultrasonic waves have high frequency and high energy, produce acoustic effects, increase mass transfer rates, accelerate enzymatic reactions, increase the yield of reaction products, reduce the amount of enzymes used, and save operating costs to a certain extent. As a relatively new food processing technology, ultrasound has not been widely used in the industrial production of resistant starch, and its application in the preparation of oat resistant starch has not yet been reported.

[0050] Oat resistant starch is fermented by the flora in the colon to produce short-chain fatty acids, of which acetic acid, propionic acid, and butyric acid are the main components. Among them, acetic acid is an important source of energy for the body, propionic acid helps inhibit kidney inflammation and fibrosis, and butyric acid is the main source of energy for intestinal epithelial cells. Short-chain fatty acids are beneficial for maintaining a stable intestinal environment, strengthening the intestinal barrier, regulating intestinal flora, and also participating in the regulation of immunity, apoptosis, inflammation, and lipid metabolism. The physiological role of short-chain fatty acids in slowing the progression of CKD has also been continuously proven, including inhibiting inflammatory responses, inhibiting oxidative stress, regulating autophagy, and improving energy metabolism and immune pathways.

[0051] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0052] (1) The present invention uses ultrasound combined with enzyme hydrolysis technology in the process of preparing oat resistant starch ORSU, and ultrasound promotes the enzymatic hydrolysis of pullulanase, in order to solve the problems of low enzymatic hydrolysis reaction efficiency and long enzymatic hydrolysis time in the process of preparing resistant starch by enzymatic method, and effectively increases the content of resistant starch in oat starch from 6.12% to 36.15%.

[0053] (2) The present invention uses oat starch as a raw material to prepare oat resistant starch with a high resistant starch content. Since resistant starch has multiple physiological functions and good processing characteristics, it is beneficial to the comprehensive utilization of oats and improves the added value of its products.

[0054] (3) The oat resistant starch ORSU prepared by ultrasound combined with enzyme hydrolysis of the present invention helps to improve the renal function of different CKD models, reduce the generation of uremic toxins, and delay the progression of CKD. Compared with the model group, the mice of different CKD models intervened by ORSU had increased weight, decreased renal index, significantly decreased renal function-related indicators serum creatinine, urea nitrogen and uric acid, significantly decreased inflammatory factors serum lipopolysaccharide and TNF-α, decreased indole, tryptophanase activity, indoxyl sulfate, p-cresol, tyrosinase activity, and p-cresol sulfate levels, decreased streptococcus abundance, and significantly increased short-chain fatty acid content.

[0055] (4) The present invention broadens the application scope of oat resistant starch, provides an important theoretical basis for the deep processing of oats, provides a theoretical basis for the application of ultrasound combined with enzyme hydrolysis technology in the preparation of resistant starch, and provides theoretical guidance for the application of oat resistant starch in foods for improving renal function, and has important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Design diagram for the CKD mouse experiment;

[0057] Figure 2 is the weight change of CKD mice in each group;

[0058] Figure 3 is the final body weight of CKD mice in each group;

[0059] Figure 4 is the renal index of CKD mice in each group;

[0060] Figure 5 is the serum creatinine of CKD mice in each group;

[0061] Figure 6 is the serum urea nitrogen of CKD mice in each group;

[0062] Figure 7 is the serum uric acid of CKD mice in each group;

[0063] Figure 8is the serum lipopolysaccharide of CKD mice in each group;

[0064] Fig. 9 is the serum TNF-α of CKD mice in each group;

[0065] Fig.10 Indole, tryptophanase activity, indoxyl sulfate, p-cresol, tyrosinase activity, and p-cresol sulfate levels in each group of CKD mice;

[0066] Fig.11 is the relative abundance of Streptococcus in each group of CKD mice;

[0067] Fig.12 The growth curves, indole content, tryptophanase activity, p-cresol content, and tyrosinase activity of each group of S. lutetiensis in vitro fermentation were shown;

[0068] Fig.13 Figure 1 shows the experimental design, weight changes, final weight, renal index, serum creatinine, serum urea nitrogen, serum uric acid, serum lipopolysaccharide, and serum TNF-α of each group of mice in the Ade combined with S. lutetiensis transplantation CKD mouse model;

[0069] Fig.14 The levels of indole, p-cresol, p-cresol, indoxyl sulfate, and p-cresol sulfate in each group of mice in the Ade combined with S. lutetiensis transplantation CKD mouse model. DETAILED DESCRIPTION

[0070] First, a method for preparing oat resistant starch is provided, which specifically comprises the following steps:

[0071] (1) Preparation of oat starch:

[0072] Take oatmeal powder and water and mix them evenly, the ratio of oatmeal powder to water is 1:6-1:10 (w / v), and adjust the pH to 8.0-12.0, extract them in a 30-50℃ water bath for 2.0-5.0h, centrifuge them through a 100-mesh sieve and collect the precipitate, scrape off the brown upper layer, wash the white part, repeat several times to obtain the lower starch precipitate. Then dry, crush and sieve to obtain oatmeal starch, and dry them in a blast drying oven, the drying temperature is 40-50℃, and the drying time is 24.0-36.0h.

[0073] (2) Preparation of oat resistant starch

[0074] The oat starch obtained in step (1) is added with water to prepare starch paste, wherein the ratio of oat starch to water is 1:10-1:30 (w / v), and gelatinization is carried out in a boiling water bath for 20-30 min. After gelatinization, the starch paste is cooled to 40-60° C. and the pH is adjusted to 4.0-6.0. The oat starch is enzymolyzed with pullulanase, wherein the enzyme activity of the pullulanase is ≥1000 npun / g, the amount of the pullulanase added is 20-40 npun / g starch, the enzymolysis temperature is 40-60° C., the enzymolysis time is 10.0-14.0 h, and ultrasonic treatment is applied during the enzymolysis. After the enzymolysis is completed, the starch paste is inactivated in a boiling water bath for 5-20 min, and the starch paste is aged at 2-6° C. for 20.0-30.0 h, and then dried, crushed and sieved to obtain high-amylose mung bean resistant starch. The power of ultrasonic treatment is 200-300w, the time of ultrasonic treatment is 5-20min, the temperature of ultrasonic treatment is 40-60℃, a circulating water bath is used to control the temperature during the ultrasonic process, drying is carried out in a blast drying oven, the drying temperature is 40-50℃, the drying time is 24.0-36.0h, and after crushing, it is sieved through a 100-mesh sieve to obtain oat resistant starch ORSU.

[0075] The present invention will be described in detail below in conjunction with specific drawings and specific embodiments, but this should not be understood as limiting the scope of the present invention to the following embodiments.

[0076] Example 1

[0077] A method for preparing oat resistant starch targeting streptococci to improve renal function, the specific steps are as follows:

[0078] (1) Preparation of oat starch

[0079] Take 1kg of oatmeal powder and mix it evenly with water, the ratio of oatmeal powder to water is 1:8 (w / v), adjust the pH of the oatmeal powder solution to 10.0 with 2mol / L NaOH, and extract it in a 40℃ water bath for 3.0h. Then pass it through a 100-mesh sieve, centrifuge the filtrate at 4000r / min for 10min, collect the precipitate, scrape off the brown upper layer, wash the white part, and repeat several times to obtain the lower starch precipitate. Then dry, crush and sieve to obtain oat starch, which is dried in a blast drying oven at a drying temperature of 40℃ and a drying time of 36.0h.

[0080] The above steps obtained about 600 g of oat starch. The total starch content of the oat starch was measured by using a Megazyme total starch assay kit and a resistant starch assay kit, which was 83.26% and 6.12% respectively.

[0081] The formula used in the above numerical calculation is:

[0082] Total starch content % = ΔA × F × EV × D × 0.9 / w × 100%;

[0083] 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.

[0084] Resistant starch content% = ΔE × F / W × 90 × 100%;

[0085] 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.

[0086] (2) Preparation of oat resistant starch ORSU

[0087] Take 50g of the oat starch obtained in step (1), add 1000ml of distilled water and mix well. After gelatinization in a boiling water bath for 20min, cool it to 50°C, adjust the pH to 5.0 with HCl (0.05mol / L), and enzymolyze the oat starch with pullulanase (the addition amount of pullulanase is 27.5npun / g starch, the enzyme activity of the pullulanase is greater than 1000npun / g, produced by Sigma). The enzymolysis temperature is 50°C and the enzymolysis time is 12.0h. While enzymolyzing the oat starch, a certain amount of ultrasonic treatment is applied. A circulating water bath is used to control the temperature during the ultrasonic process. The power of the ultrasonic treatment is 240w, the time of the ultrasonic treatment is 10min, and the temperature of the ultrasonic treatment is 50°C. After the enzymolysis is completed, the enzyme is inactivated in a boiling water bath for 10min, aged at 4°C for 24.0h, dried at 40°C in a forced air drying oven, and then crushed through a 100-mesh sieve to obtain oat resistant starch ORSU.

[0088] The total weight of the oat resistant starch ORSU obtained in the above steps is 46 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 81.57%, and the resistant starch content increases to 36.15%.

[0089] Comparative Example 1

[0090] An oat beta-glucan (OBGM), the specific information is as follows:

[0091] 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.

[0092] Comparative Example 2

[0093] Fructooligosaccharides (FOS), fructooligosaccharide content greater than 90.0%, glucose + sucrose + fructose: ≤ 10%, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0094] The embodiments of the present invention also provide a study on improving the renal function of CKD mice using oat resistant starch ORSU, and the specific method is as follows:

[0095] (1) Preparation of oat resistant starch ORSU: The preparation was carried out according to Example 1, wherein the resistant starch content of ORSU was 36.15%.

[0096] (2) Mouse breeding and establishment and grouping of CKD model

[0097] According to the guidelines of the "Chinese Laboratory Animal Standards", animal experiments were conducted in accordance with internationally valid guidelines and protocols and approved by the Animal Ethics Committee. Male C57BL / 6J mice (6-8 weeks old) were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. Mice were raised under standard conditions (12h light / dark cycle) and had free access to food and water. After one week of adaptive feeding, the experimental process was entered.

[0098] The mice were randomly divided into 5 groups (8 mice in each group): Con group, CKD group (Ade mice), CKD-FMT group (Ade-FMT-CKD mice), CKD-FMT-ORSU group (Ade-FMT-CKD mice + ORSU), CKD-FMT-OBGM group (Ade-FMT-CKD mice + OBGM). Except for the Con group, the other 3 groups of mice were given adenine Ade 250 mg / kg mouse body weight in the first week, gavage once every 2 days; Ade 200 mg / kg was given in the 2nd-3rd week, gavage once every 4 days, a total of 7 times. The amount of Ade suspension gavaged into the mice each time was 0.1 mL / 10 g to create a CKD model. According to the settings of different groups, Ade, CKD bacterial solution and ORSU were gavaged at the same time. The Con group was gavaged with PBS in the same way. The samples for FMT were prepared from fecal samples of non-dialysis CKD patients in the Nephrology Department of the 905th Hospital of the Chinese People’s Liberation Army Navy. The concentration of CKD bacterial solution was about 1.2×10 9 CFU / mL, the amount of oral administration to mice was 0.1mL / 10g. The oral administration of ORSU and OBGM was 1g / kg, once a day, for a total of 51 days. At the end of the animal experiment, blood was collected from the mouse eyeballs, and the mice were quickly killed by cervical dislocation. Blood and feces were collected and stored at -80℃ until further use.

[0099] (3) S. lutetiensis in vitro fermentation

[0100] Culture and measure OD according to the conditions in the S. lutetiensis strain instructions 600 The growth curve was determined based on the value of. FOS, ORSU and OBGM (1% w / v) were added to 20 mL of culture medium and fermented with S. lutetiensis for 24 h. They were set as Con group, S. lutetiensis group, S. lutetiensis-FOS group, S. lutetiensis-ORSU group and S. lutetiensis-OBGM group.

[0101] (4) Establishment and grouping of CKD mouse model with Ade combined with S. lutetiensis transplantation

[0102] According to the guidelines of the Chinese Laboratory Animal Standards, animal experiments were performed in accordance with internationally valid guidelines and protocols and approved by the Animal Ethics Committee. Male C57BL / 6J mice (6-8 weeks old) were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. Mice were housed under standard conditions (12 h light / dark cycle) with free access to food and water, and were adaptively fed for one week.

[0103] The mice were randomly divided into 4 groups (8 mice in each group), namely Con group, S.lutetiensis group (Ade+S.lutetiensis), S.lutetiensis-ORSU group (Ade+S.lutetiensis+ORSU), S.lutetiensis-OBGM group (Ade+S.lutetiensis+OBGM). Except for the Con group, the other 3 groups of mice drank water containing antibiotics for 2 weeks to eliminate the intestinal flora of the mice and construct a pseudo-sterile mouse model. At the same time, adenine (Ade) was gavaged. In the first week, Ade 250 mg / kg of mouse body weight was administered, once every 2 days; in the second to third weeks, Ade 200 mg / kg was administered, once every 4 days; each gavage of Ade suspension 0.1 mL / 10 g was used to construct the Ade-S.lutetiensis-CKD mouse model. After 2 weeks of antibiotic treatment, gavage of S.lutetiensis, ORSU and OBGM was started. The concentration of S.lutetiensis bacterial solution is about 1*10 9 CFU / mL, oral administration volume was 10mL / kg mouse body weight, PBS 1mL / kg, ORSU and OBGM 1g / kg, once a day. After the experiment, mice were killed by cervical dislocation. Blood was collected and stored at -80℃ for future use.

[0104] (5) Measurement of mouse body weight and kidney weight

[0105] The body weight and kidney weight of each group of mice were measured using an analytical balance.

[0106] (6) Determination of renal function indexes in mice

[0107] The renal function indices of the mice in each group, serum creatinine, urea nitrogen, and uric acid, were measured using enzymatic kits according to the manufacturer's instructions.

[0108] (7) Determination of inflammatory indicators in mice

[0109] The levels of lipopolysaccharide and inflammatory factor TNF-α in the serum of mice in each group were detected using ELISA kits according to the manufacturer's instructions.

[0110] (8) Determination of uremic toxin precursors and their generating enzyme activities and uremic toxin levels in mice

[0111] The levels of indole and p-cresol were detected according to the following method: the feces of each group of mice were mixed with normal saline at a ratio of 1:5 (w / v) and centrifuged at 10000r / min for 3min. 100μL of supernatant was taken, and 180μL of ether and 90μL of ethyl acetate were added. Vortex, then centrifuge at 12000r / min for 10min, and 200μL of supernatant was taken. The ether and ethyl acetate were removed by nitrogen blowing, and 200μL of acetonitrile was added. After centrifugation again, the supernatant was taken for testing. HPLC detection conditions: mobile phase A was ammonium acetate buffer (0.2M, pH 4.5); mobile phase B was acetonitrile. 60% B phase was eluted isocratically, injection time was 12min, injection volume was 10μL, total flow rate was 0.7mL / min, column temperature was 35℃, and detection wavelength was 270nm.

[0112] The activity detection of indole-generating enzyme tryptophanase and p-cresol-generating enzyme tyrosinase was carried out according to the instructions of the kit (purchased from Shanghai ELISA Biotechnology Co., Ltd.).

[0113] The levels of uremic toxins indoxyl sulfate and p-cresol sulfate in the serum of mice in each group were detected according to the instructions of the kit (purchased from Shanghai ELISA Biotechnology Co., Ltd.).

[0114] (9) Determination of relative abundance of Streptococcus Parisi in mouse intestinal flora

[0115] Intestinal flora analysis: DNA was extracted from the cecal contents of each group of mice using a fecal genomic DNA kit according to the instructions. After confirming the quality of the DNA sample by agarose gel electrophoresis, the primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') 806R

[0116] (5'-GGACTACHVGGGTWTCTAAT-3') The V3-V4 region of the 16S rRNA gene was amplified from the purified DNA by polymerase chain reaction (PCR). After the amplicon was purified and quantified, a paired-end library was constructed according to the Illumina genomic DNA library preparation procedure ( Rapid DNA-Seq Kit, Bioo Scientific, USA), and high-throughput sequencing was performed on the MiSeqIllumina sequencing platform (PE300) (MiSeq Reagent Kit v3, Illumina, USA). The 16S rRNA sequencing data were analyzed using QIIME2V.2018.2, and the classification was assigned according to the Greengenes database. The sequences were clustered into OTUs based on a similarity of 97%. The RDP classifier software was used to annotate the species classification of each sequence, and the Silva 16S rRNA database was compared with the alignment threshold of 70%.

[0117] (10) Determination of short-chain fatty acids in mouse feces

[0118] Mix the feces and saline at a ratio of 1:5 (w / v) and centrifuge at 10000r / min for 10min. Take 2mL of supernatant, add 0.1mL HCl and 2mL anhydrous ether, mix and let stand for 20min. Centrifuge at 4000r / min for 5min, take the supernatant, add 0.5mL 1M NaOH and let stand for 20min, then centrifuge again. Collect the lower aqueous phase, add 0.1mL HCl and mix, and the obtained sample is then tested by liquid phase.

[0119] HPLC detection conditions: mobile phase A 0.025% phosphoric acid solution (pH 2.8), mobile phase B 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.

[0120] The following specific test examples are set up to verify the effects of oat resistant starch ORSU on renal function, uremic toxins, intestinal flora, etc. in CKD mice

[0121] Experimental Example 1: Effect of oat resistant starch ORSU on body weight of CKD mice

[0122] Depend on Figure 2 and Figure 3It can be seen that compared with the Con group, the weight of the four groups of mice gavaged with Ade in the first three weeks decreased significantly, and the weight increased after the gavage of Ade was stopped. This shows that Ade caused damage to the mice's body, causing the mice to lose weight significantly. After the gavage of Ade was stopped, the weight of the mice recovered. In addition, the intervention of ORSU helps to increase the weight of CKD mice.

[0123] Experimental Example 2: Effects of oat resistant starch ORSU on renal index in CKD mice

[0124] Depend on Figure 4 It can be seen that compared with the Con group, the renal index of the CKD and CKD-FMT groups increased significantly. However, compared with the CKD and CKD-FMT groups, the renal index of the CKD-FMT-ORSU group decreased significantly, indicating that the intervention of ORSU helps to reduce the kidney weight of CKD mice and alleviate the problem of renal edema.

[0125] Experimental Example 3: Effect of oat resistant starch ORSU on renal function in CKD mice

[0126] Depend on Figure 5-7 It can be seen that compared with the Con group, the three indicators of serum creatinine, urea nitrogen and uric acid in the CKD group and CKD-FMT group were significantly increased, and increased by about 2-3 times, indicating that the CKD mouse model was successfully established. Compared with the CKD-FMT group, the levels of serum creatinine, urea nitrogen and uric acid in the CKD-FMT-ORSU group were significantly reduced, indicating that supplementation with ORSU helps improve the renal function of CKD mice.

[0127] Experimental Example 4: Effect of oat resistant starch ORSU on inflammation in CKD mice

[0128] Depend on Figure 8 It can be seen that the lipopolysaccharide content in the CKD-FMT-ORSU group was significantly lower than that in the CKD group and the CKD-FMT group. Fig. 9 It can be seen that the TNF-α content in the CKD-FMT-ORSU group was lower than that in the CKD group, and was significantly lower than that in the CKD-FMT group, indicating that supplementation of ORSU helps to alleviate systemic inflammation in CKD mice.

[0129] Experimental Example 5: Effects of oat resistant starch ORSU on the activities of uremic toxin precursors and their generating enzymes, as well as the levels of uremic toxins in CKD mice

[0130] Depend on Fig.10 AC showed that the indole, tryptophanase activity, and indoxyl sulfate levels in the CKD-FMT-ORSU group were significantly lower than those in the CKD group and the CKD-FMT group. Fig.10DF showed that the levels of p-cresol, tyrosinase activity, and p-cresol sulfate in the CKD-FMT-ORSU group were significantly lower than those in the CKD-FMT group, indicating that supplementation with ORSU can help reduce the levels of uremic toxins and their precursors in CKD mice.

[0131] Experimental Example 6: Effect of oat resistant starch ORSU on the relative abundance of Streptococcus lutetiensis in CKD mice

[0132] Depend on Fig.11 It can be seen that the abundance of Streptococcus Paris was the highest in the CKD-FMT group, indicating that it may be a key characteristic bacterium that promotes the progression of CKD. Compared with the CKD-FMT group, the relative abundance of Streptococcus Paris in the CKD-FMT-ORSU group was significantly reduced, indicating that supplementation with ORSU helps to target Streptococcus Paris and improve the renal function of CKD mice.

[0133] Experimental Example 7: Effects of oat resistant starch ORSU on short-chain fatty acids (SCFAs) in CKD mice

[0134] As shown in Table 1, the SCFAs content in the Con group was the highest, the SCFAs content in the CKD and CKD-FMT groups was the lowest, and the SCFAs content in the CKD-FMT-ORSU group was higher than that in the CKD-FMT group, indicating that the intestinal flora of CKD mice fermented ORSU, increasing the level of SCFAs in CKD mice. Due to the increase in SCFAs, the intestinal barrier function and anti-inflammatory function of CKD mice were enhanced. Therefore, the intervention of ORSU helps to delay the disease progression of CKD mice.

[0135] Table 1 Contents of short-chain fatty acids in feces of Con, CKD, CKD-FMT, CKD-FMT-ORSU, and CKD-FMT-OBGM groups

[0136]

[0137]

[0138] Note: The results are expressed as mean ± standard deviation. The values ​​with different letters in the same row are significantly different (p<0.05)

[0139] Experimental Example 8: Effect of in vitro fermentation of oat resistant starch ORSU by Streptococcus lutetiensis

[0140] Depend on Fig.12 A shows that the intervention of ORSU helps to inhibit the growth of S.lutetiensis. Fig.12 B and C show that ORSU intervention helps inhibit tryptophanase activity and reduce the production of indole, a precursor of uremic toxins. Fig.12D and E show that the intervention of ORSU helps to inhibit tyrosinase activity and reduce the production of uremic toxin precursor p-cresol. This indicates that OSRU helps to reduce the activity of uremic toxin precursors and their production enzymes in the in vitro fermentation model of S. lutetiensis.

[0141] Experimental Example 9: Effects of ORSU on body weight and renal index of Ade-S.lutetiensis-CKD mice in each group

[0142] Depend on Fig.13 B and C show that compared with the Con group, the body weight of mice in the S. lutetiensis group decreased significantly, and the body weight of mice increased significantly after OSRU intervention. Fig.13 D shows that compared with the Con group, the renal index of mice in the S.lutetiensis group increased significantly, and the renal index of mice after OSRU intervention decreased significantly, with no significant difference compared with the Con group. This indicates that ORSU intervention can significantly improve the abnormalities of body weight and renal index in Ade-S.lutetiensis-CKD mice.

[0143] Experimental Example 10: Effect of ORSU on renal function of Ade-S.lutetiensis-CKD mice in each group

[0144] Depend on Fig.13 EG showed that compared with the Con group, the three renal function indicators of the S.lutetiensis group mice, serum creatinine, urea nitrogen, and uric acid, were significantly increased, and increased by about 2-3 times, indicating that the Ade-S.lutetiensis-CKD mouse model was successfully established. Compared with the S.lutetiensis group, the levels of renal function indicators of the S.lutetiensis-ORSU group mice, serum creatinine, urea nitrogen, and uric acid, were significantly reduced after ORSU intervention, indicating that supplementation with ORSU helps improve the renal function of Ade-S.lutetiensis-CKD mice.

[0145] Experimental Example 11: Effects of ORSU on inflammation in each group of Ade-S.lutetiensis-CKD mice

[0146] Depend on Fig.13 H and I showed that compared with the Con group, the levels of serum lipopolysaccharide and TNF-α, the inflammatory indicators of mice in the S.lutetiensis group, were significantly increased. Compared with the S.lutetiensis group, the levels of serum lipopolysaccharide and TNF-α, the inflammatory indicators of mice in the S.lutetiensis-ORSU group, were significantly decreased after ORSU intervention, indicating that supplementation of ORSU helps to alleviate systemic inflammation in Ade-S.lutetiensis-CKD mice.

[0147] Experimental Example 12: Effects of ORSU on uremic toxin precursors and uremic toxin levels in Ade-S.lutetiensis-CKD mice

[0148] Depend on Fig.14 It can be seen that the levels of uremic toxins indoxyl sulfate, p-cresol sulfate and their precursors indole and p-cresol in the S.lutetiensis group were significantly higher than those in the Con group. Compared with the S.lutetiensis group, the levels of uremic toxins and their precursors in the S.lutetiensis-ORSU group mice decreased significantly after ORSU intervention, and the decrease was greater than that in the S.lutetiensis-OBGM group after OBGM intervention, indicating that ORSU helps to reduce the production of uremic toxins in Ade-S.lutetiensis-CKD mice.

[0149] In summary, oat resistant starch ORSU prepared by ultrasound combined with enzymatic hydrolysis can help target streptococci to reduce the production of uremic toxins, improve renal function, and delay the progression of CKD.

[0150] 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. A method for preparing oat resistant starch, characterized in that: The preparation method comprises the following steps: (1) Preparation of oat starch Take oatmeal powder and water and mix them evenly, adjust the pH, perform alkali extraction, sieve and centrifuge to collect the precipitate, scrape off the brown upper layer, wash the white part, and then dry, crush and sieve to obtain oatmeal starch; (2) Preparation of oat resistant starch The oat starch obtained in step (1) is added with water to prepare starch paste, cooled after gelatinization, and the pH is adjusted. The oat starch is enzymatically hydrolyzed with pullulanase and ultrasonically treated. After the enzymatic hydrolysis is completed, the enzyme is inactivated, and the oat starch is aged. After that, the oat starch is dried, crushed and sieved to obtain oat resistant starch.

2. The method for preparing oat resistant starch according to claim 1, characterized in that: Select one or a combination of the following conditions: In step (1), the ratio of oatmeal powder to water is 1:6-1:10 (w / v); In step (1), the pH of the oatmeal solution is adjusted to 8.0-12.0; In step (1), alkali extraction is performed in a water bath at 30-50° C. for 2.0-5.0 h; In step (1), the filtrate obtained by sieving is centrifuged at a rate of 3000-5000 r / min for 5-20 min; In step (1), the drying temperature is 40-50° C. and the drying time is 24.0-36.0 h.

3. The method for preparing oat resistant starch according to claim 1, characterized in that: In the step (1), the total starch content of the obtained oat starch is higher than 80%.

4. The method for preparing oat resistant starch according to claim 1, characterized in that: Select one or a combination of the following conditions: In step (2), the enzyme activity of pullulanase is ≥1000 npun / g; In step (2), the amount of pullulanase added is 20-40 npun / g; In step (2), the enzymolysis temperature is 40-60°C and the enzymolysis time is 10.0-14.0h; In step (2), the power of ultrasonic treatment is 200-300w, and the time of ultrasonic treatment is 5-20min.

5. The method for preparing oat resistant starch according to claim 1, characterized in that: Select one or a combination of the following conditions: In step (2), the ratio of oat starch to water is 1:10-1:30 (w / v); In step (2), gelatinization is carried out in a boiling water bath for 20-30 min; In step (2), after gelatinization, cooling to 40-60° C.; In step (2), the pH is adjusted to 4.0-6.0; In step (2), after the enzymatic hydrolysis is completed, the enzyme is inactivated in a boiling water bath for 5-20 minutes; In step (2), the aging process is specifically: aging at 2-6°C for 20.0-30.0h; In step (2), the drying temperature is 40-50° C. and the drying time is 24.0-36.0 h.

6. Oat resistant starch prepared by the preparation method according to any one of claims 1 to 5.

7. The oat resistant starch according to claim 6, characterized in that The content of resistant starch in oat resistant starch is 36.15%.

8. Use of the oat resistant starch according to claim 6 or 7 in the preparation of functional foods, health products or medicines.

9. The use according to claim 8, characterized in that: The functional food or health product refers to a food that has the effect of improving kidney function and / or delaying the progression of CKD; the drug refers to a drug that has the effect of improving kidney function and / or delaying the progression of CKD.

10. The use according to claim 8, characterized in that: Application of oat resistant starch in the preparation of functional foods, health products or medicines targeting streptococci to improve renal function.

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