CN113615841A - Application of soluble dietary fiber in kelp in preparation of medicine and functional food for improving constipation - Google Patents
Application of soluble dietary fiber in kelp in preparation of medicine and functional food for improving constipation Download PDFInfo
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- CN113615841A CN113615841A CN202110850167.7A CN202110850167A CN113615841A CN 113615841 A CN113615841 A CN 113615841A CN 202110850167 A CN202110850167 A CN 202110850167A CN 113615841 A CN113615841 A CN 113615841A
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- dietary fiber
- soluble dietary
- intestinal
- kelp
- constipation
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Abstract
The invention discloses an application of soluble dietary fiber in kelp in preparation of a medicine and a functional food for improving constipation. According to the invention, the soluble dietary fiber is extracted from the kelp residues, a constipation mouse model is constructed, and the soluble dietary fiber in the kelp can promote defecation of a mouse, increase the moisture content of intestinal tracts and improve the intestinal tract transportation rate are verified for the first time; up-regulating the level of antioxidant enzyme; balancing intestinal neurotransmitter levels; increasing the abundance of firmicutes and actinomycetes, inhibiting the abundance of bacteroidetes, and increasing the production of short chain fatty acids. The soluble dietary fiber in the kelp is safe and nontoxic, avoids the occurrence of toxic and side effects of medicines, can be metabolized and fermented by intestinal flora and further mediates the level of antioxidant enzymes and the expression of intestinal neurotransmitter of organisms, improves symptoms related to constipation, and has good market application prospect.
Description
Technical Field
The invention belongs to the field of food health care, and particularly relates to application of soluble dietary fiber in kelp in preparation of a medicine and a functional food for improving constipation.
Background
The pathogenesis of constipation is mainly related to genetic susceptibility, low fiber and fluid intake, lack of activity, hormonal imbalance, drug side effects. Among the most common causes include lack of fiber and insufficient intake of drinking water or liquids, and thus constipation can be controlled and treated by dietary intervention to alter nutritional intake and lifestyle habits. Studies have shown that a high fiber diet can increase stool weight, promote intestinal motility, reduce colonic transit time, increase stool frequency and speed, while a low fiber diet can cause constipation.
Traditional Dietary Fiber (DF) products are mainly derived from some vegetarian materials such as grains, vegetables, fruits and the like, and related alga DF laxative products are few in the market. Kelp is a large edible algae and an industrial algae, and leftovers of the kelp contain abundant substances such as fucoidin, cellulose, hemicellulose, vitamins, mineral substances and the like, and are a high-quality source for producing DF.
Disclosure of Invention
The invention aims to provide application of soluble dietary fiber in kelp in preparation of medicines and functional foods for improving constipation.
In order to achieve the purpose, the invention adopts the technical scheme that:
use of soluble dietary fiber of herba Zosterae Marinae in preparing medicine and functional food for relieving constipation, wherein monosaccharide composition in the soluble dietary fiber comprises fucose, galactose, glucose, mannose, xylose, glucuronic acid, rhamnose, galacturonic acid, arabinose, and ribose.
Further, the extraction method of the soluble dietary fiber comprises the following steps:
(1) adding 4-6% NaCl solution into the residue at a ratio of 1:20-40, heating at 80-100 deg.C for 6-8 hr for decalcification, centrifuging, collecting precipitate, adding pure water, heating and boiling for 30 min;
(2) adding 5mol/L NaOH solution into the precipitate obtained in the step (1) according to the feed-liquid ratio of 1:20-40, adjusting the pH value to be alkaline, heating at 80-100 ℃ for 6-8 h, centrifuging and collecting the precipitate;
(3) and (3) adjusting the pH of the precipitate obtained in the step (2) to be neutral by using HCl, concentrating, dialyzing, standing at ultralow temperature, and freeze-drying to obtain the soluble dietary fiber.
Further, the pH value in the step (2) is adjusted to 13; heating at 90 deg.C for 6 hr, centrifuging to separate precipitate, and collecting precipitate to obtain soluble dietary fiber.
Further, after the precipitate which is adjusted to be neutral in pH in the step (3) is concentrated by rotary evaporation, the precipitate is dialyzed in a 1500 Da dialysis bag for 10 to 12 hours, and the precipitate is frozen and dried for 36 hours after being placed in a refrigerator at the temperature of minus 80 ℃ to obtain the dried soluble dietary fiber.
Further, the unit of the feed-liquid ratio is mg/mL.
Further, the effective dosage of the soluble dietary fiber for improving constipation is 0.5 g/kg-3.5 g/kg.
Further, the most effective dosage of the soluble dietary fiber for improving constipation is 3.5 g/kg.
Furthermore, the soluble dietary fiber can increase the quantity, the weight and the water content of excrement of a constipated mouse, so that the defecation function of the mouse is enhanced.
Further, the soluble dietary fiber can promote the intestinal peristalsis and improve the intestinal transport function by increasing the water content of the intestinal tract of the constipation mice and increasing the intestinal transport rate.
Further, the soluble dietary fiber can increase the content of superoxide dismutase SOD and glutathione peroxidase GSH-Px in serum and increase the content of SCFAs, thereby inhibiting the oxidative stress level disorder caused by constipation.
Further, the SCFAs include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, isocaproic acid.
Further, the soluble dietary fiber can down-regulate the expression level of inducible nitric oxide synthase iNOS and up-regulate the expression levels of intestinal neurotransmitter acetylcholinesterase AchE, endothelial nitric oxide synthase eNOS, glial cell line-derived neurotrophic factor GDNF and brain-derived neurotrophic factor BDNF.
Further, the soluble dietary fiber can adjust the structure of intestinal flora of constipation mice by reducing the abundance of bacteroidetes phyla in intestinal tracts of the mice and increasing the abundance of firmicutes and actinomycetes.
Further, the soluble dietary fiber can increase the ratio of Murebaudiaceae to Lachnospiraceae at the family level and decrease the ratio of Lactobacillaceae to Mariniferae to Helicobacteraceae to norak _ o __ Clostridium _ UCG-014.
Compared with the prior art, the invention has the beneficial effects and advantages that:
the raw material of the soluble dietary fiber extracted by the invention is the kelp residue after the algin is separated, and the source of the kelp residue is rich and the use is safe. The soluble dietary fiber extracted by the invention belongs to natural active substances, contains various monosaccharide types, and is proved to have uneven surface and increased folds, so that the water absorption swelling function can be promoted, the capability of the dietary fiber for absorbing glucose, cholesterol and harmful substances in gastrointestinal tracts can be increased, the high additional utilization value of kelp is improved, and the soluble dietary fiber is high in safety and low in price.
Animal model experiments prove that the extracted soluble dietary fiber can promote intestinal movement by regulating the expression of genes related to intestinal neurotransmitters, and further has remarkable effects on improving constipation symptoms of mice and regulating intestinal flora and metabolites thereof, so that the soluble dietary fiber can be used as a prebiotic component for improving constipation by targeting regulation of the intestinal flora and the metabolites thereof, and has great potential in being a functional food for improving constipation.
Drawings
FIG. 1 is an electron microscope scanning image of soluble dietary fiber of kelp;
FIG. 2 is an infrared spectrum of soluble dietary fiber of kelp;
FIG. 3 is an index plot of constipation mouse feces;
FIG. 4 is a graph of intestinal transit rates in constipated mice;
FIG. 5 is a graph showing the water content in the intestinal tract of a constipated mouse;
FIG. 6 is a graph of serum SOD and GSH-Px in constipated mice;
FIG. 7 is a graph showing the relative expression amount of colonic intestinal neurotransmitter mRNA in constipated mice;
FIG. 8 is a graph showing the relative expression amounts of colonic intestinal neurotransmitter proteins in constipated mice;
FIG. 9 is a distribution diagram of intestinal flora OUT of constipation-treated mice;
FIG. 10 is a bar graph of the composition of the intestinal flora and phylum horizontal flora of constipated mice;
FIG. 11 is a graph showing the content of Short Chain Fatty Acids (SCFAs) in feces of constipated mice.
Detailed Description
The technical solutions of the present invention will be further described in detail with reference to the drawings and the detailed description, but the scope of the present invention is not limited to the scope described by the examples.
Example 1
The extraction method of the soluble dietary fiber comprises the following steps:
(1) adding 5% NaCl solution into the kelp residue according to the ratio of 1:30, and heating at 90 ℃ for 7 h for decalcification;
(2) centrifuging to remove supernatant, adding appropriate amount of pure water into the precipitate, heating and boiling for 30 min;
(3) adding 5mol/L NaOH solution into the precipitate obtained in the step (2) according to the ratio of the material to the liquid of 1:30, adjusting the pH value to 13, and heating for 6 h at 90 ℃;
(4) centrifuging, and collecting precipitate to obtain SDF;
(5) adjusting pH of SDF to neutral with HCl, rotary steaming, concentrating, dialyzing in 1500 Da dialysis bag for 10-12 h, placing in refrigerator at-80 deg.C for 12h, and freeze drying for 36 h to obtain dried soluble dietary fiber.
Example 2: identification of basic structure of Soluble Dietary Fiber (SDF) of kelp
1. Molecular weight distribution and monosaccharide composition of soluble kelp dietary fiber
The molecular weight distribution and monosaccharide composition of the soluble dietary fiber of kelp prepared in example 1 were investigated using GPC and HPLC, respectively.
Complete acid hydrolysis: an appropriate amount of sample was put into a hydrolysis tube, 1mL of 4mol/L TFA (trifluoroacetic acid) was added, and the mixture was hydrolyzed in an oven at 120 ℃ for 2 hours. Taking out, and drying by nitrogen; and (3) derivatization reaction: adding 1mL of 0.5mol/L PMP-methanol solution and 0.5 mL of 0.3 mol/L NaOH solution into the dried sample, carrying out water bath at 70 ℃ for 60 min, cooling, adding 0.5 mL of 0.3 mol/L HCl solution, adding 0.5 mL of chloroform, shaking uniformly, standing for 20 min, discarding the lower layer, extracting for three times, taking the water layer, and passing through a membrane machine.
The instrument method comprises the following steps: a chromatographic column: SHISEIDO C18 column (4.6 mm. times.250 mm, 5 μm), mobile phase: 0.1 mol/L0.1M KH2PO4 (pH 6.8), B: acetonitrile, A: b = 82: 18, flow rate: 1.0 mL/min, a column temperature of 25 ℃, a sample injection amount of 10 mu L and a wavelength of 245 nm.
Calculating the formula: x = V × N × (Cx-Co)/m;
in the formula, V-constant volume, N-dilution times, Cx-detection concentration, Co-blank, m-sample weighing and X-sample concentration.
The results are shown in table 1, where 10 monosaccharide compositions of the soluble diet of kelp were: fucose (39.495%), galactose (17.572%), glucose (11.897%), mannose (10.088%), xylose (5.700%), glucuronic acid (8.892%), rhamnose (5.780%), galacturonic acid (0.575%), arabinose (< 0.001%), ribose (< 0.001%).
Table 1: molecular weight distribution of soluble kelp diet
2. Electron microscope scanning of soluble kelp dietary fiber
Uniformly placing a proper amount of dried soluble kelp dietary fiber sample on an objective table with double-sided adhesive tape, performing surface gold plating by using an ion sputtering coating method, taking out, placing under a scanning electron microscope, and performing scanning observation, wherein the voltage is set to be 5Kv, and the magnification is 150X.
The electron microscope scanning result of the soluble kelp dietary fiber is shown in fig. 1, the left column is the soluble kelp dietary fiber, the right column is the raw material kelp residue, and the graph from 150 times to 500 times shows that the surface of the soluble kelp dietary fiber is uneven, the wrinkles are increased, and the granularity is obviously lower than that of the kelp residue. The particle size of the dietary fiber determines the specific surface area, the specific surface area greatly increases the affinity with water, the water absorption swelling function is promoted, and the uneven surface increases the capacity of the dietary fiber for absorbing glucose, cholesterol and harmful substances in the gastrointestinal tract.
3. Infrared spectrum of soluble kelp dietary fiber
Taking 2 mg of dry sample and 100 mg of KBr (spectral grade), grinding, pressing into a transparent sheet after grinding, using blank KBr as background, scanning times of 20 times and resolution of 4 cm-1Scanning range 4000--1。
As shown in FIG. 2, a strong peak at 3431.09 in the IR spectrum, indicating stretching vibration with O-H bonds, and more rounded indicating the presence of intermolecular and intramolecular hydrogen bonding, a strong peak at 1631.62 indicating stretching vibration with C = O, peaks at 2920.12 and 2850.67 indicating stretching vibration with C-H groups with methyl groups, and a peak at 1261.56 indicating angular motion with C-H groups, are typical characteristic absorption peaks of carbohydrates.
Example 3
After adaptive feeding for 7 days, 72 male ICR mice were randomly divided into 9 groups according to body weight, and 12 mice in each group were selected from normal group (NC), model group (MC), cisapride group (PC), SDF low, medium and high dose groups (L-SDF, M-SDF, H-SDF). After 7 d of adaptive feeding, the test substance gavage intervention experiment is started, and the intervention scheme is shown in table 2. Starting from 8 d, mice in each of the other groups except the NC group were gavaged with 3 mg/kg Lop at 9 am daily, and after 1 h with different subjects for 7 d. The state of the mouse feces is observed every day, including the indexes of the quantity of the feces, the wet weight of the feces, the dry weight of the feces and the like. After the last feeding, the mice are fasted and are killed by taking blood from eye sockets and then removing cervical vertebrae in the next day without water supply overnight.
TABLE 2 Constipation model Experimental intervention protocol
| Time/packet | 1234567 (Tian) |
| NC group | Gavage 0.1 mL/10 g normal saline |
| MC group | Gavage 3 mg/kg Lop +1 h later, gavage normal saline |
| PC group | Gavage is carried out for 3 mg/kg Lop +1 h, and then gavage is carried out for 0.25 g/kg cisapride |
| L-SDF group | Gavage is carried out for 3 mg/kg Lop +1 h, and then gavage is carried out for 0.5 g/kg SDF |
| M-SDF group | Gavage after 3 mg/kg Lop +1 h for 1.5 g/kg SDF |
| Group of H-SDF | Gavage 3 mg/kg Lop +1 h later, gavage 3.5 g/kg SDF |
The results of the experiment are shown in FIG. 3: the SDF promotes the defecation quantity, the excrement weight and the excrement water content of a constipation mouse for 1 h, and further enhances the defecation function of the mouse.
Example 4
The experimental groups and gavage dose were the same as in example 3. The mice were sacrificed 30 min after the administration of 5% charcoal powder-10% acacia gum semisolid paste (1 mL/100 g body weight) 1 h before anesthesia, the abdominal cavity was dissected open, and blood samples were left for serological examination. The distance from the pylorus to the rectum and the distance from the front end of the carbon tip to the pylorus were measured, and the percent advancement of the carbon tip was calculated using the following formula:
carbon powder advancing rate (%) = distance from front edge of carbon powder to pylorus/distance from pylorus to rectum x 100%
The results of the experiment are shown in FIG. 4: SDF can improve intestinal transport function and promote intestinal transport rate.
Example 5
The experimental groups and gavage dose were the same as in example 3. After 7 d of experiment, the final fasting body weight and the length of the mice (from the tip of the nose to the length of the anus) were measured and recorded after the mice had fasted without water deprivation for 12 h. The small intestine (jejunum, ileum and colon) of the constipated mice was isolated and a portion of the intestinal system was weighed, dried at 40 ℃ for 5 h, weighed again and the water content of the intestine was calculated using the following formula:
intestinal tract water content (%) = (intestinal tract wet weight-intestinal tract dry weight)/intestinal tract wet weight × 100%
The results of the experiment are shown in FIG. 5: SDF can increase water content of intestinal tract and promote intestinal peristalsis.
Example 6
The experimental groups and gavage dose were the same as in example 3. And (3) after 7 d intervention, fasting for 12h, performing ether anesthesia, then taking blood from an orbit, standing the whole blood at room temperature for 30 min, centrifuging for 15 min at the temperature of 4 ℃ and at the speed of 7500 rpm, separating serum, and detecting the content of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) according to the instruction of a kit.
The results of the experiment are shown in FIG. 6: SDF can enhance the antioxidant capacity of organism and inhibit oxidative stress level disorder caused by constipation by enhancing the activities of SOD and GSH-Px.
Example 7
The experimental groups and gavage dose were the same as in example 3. After 7 days of experiment, 100 mg of small intestine tissue was separately extracted for total RNA according to the instructions of Transzol, and the total RNA was reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit for real-time fluorescent quantitative PCR detection of key genes of intestinal neurotransmitter: expression levels of mRNA of AchE, iNOS, eNOS, BDNF, and GDNF.
The results of the experiment are shown in FIG. 7: SDF down-regulates intestinal neurotransmitter iNOS mRNA expression levels, up-regulates AchE, eNOS, BDNF, and GDNF mRNA expression levels.
Example 8
The experimental groups and gavage dose were the same as in example 3. After the experiment is finished, 0.1 g of colon tissue extract protein is respectively taken out, and the protein expression levels of inflammatory factors AchE, iNOS, eNOS, BDNF and GDNF are detected by a protein immunoblotting method.
The results of the experiment are shown in FIG. 8: SDF down-regulates the intestinal neurotransmitter iNOS protein expression level, up-regulates AchE, eNOS, BDNF, and GDNF protein expression levels.
Example 9
The experimental groups and gavage dose were the same as in example 3. One day before the experiment, mice were placed in clean and sterile mouse cages, and mouse feces were collected. 16S rRNA V3-V4 region amplification is carried out through the processes of DNA extraction, primer joint design and synthesis, PCR amplification and product purification, PCR product quantification and homogenization, PE library construction and the like, and a sequencing platform is Illumina. OTU clustering is carried out on the non-repetitive sequences according to 97% similarity, and species composition analysis is carried out on OTU representative sequences with 97% similarity level by adopting RDP classifier Bayesian algorithm.
The results of the experiment are shown in figures 9 and 10: SDF can adjust OUT distribution of intestinal flora of constipation mice, reduce bacteroidetes abundance, increase abundances of firmicutes and actinomycetes, mainly increase ratio of Murivulariaceae to Lachnospiraceae, and reduce ratio of Lactobacillus, Marinifilacea, Helicobacteraceae to norak _ o __ Clostridium _ UCG-014.
Example 10
The experimental grouping and gavage dose were the same as in example 3, and the mice were placed in clean and sterile mouse cages one day before the experiment was completed, and the mouse feces were collected. Firstly, accurately weighing a certain mass of sample, and adding an extracting solution to perform metabolite extraction treatment in a low-temperature environment. Preparing standard solutions with different concentrations, performing GC-MS on-machine detection on the standard solutions and a sample to be detected under the same condition, calculating the concentration of a target substance of the detected sample according to a standard curve, and finally converting to obtain the concentration of the sample.
The results of the experiment are shown in FIG. 11: SDF increases short-chain fatty acid levels, such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, isocaproic acid, and is involved in the regulation of the mechanisms of constipation.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. An application of soluble dietary fiber in kelp in preparing medicines and functional foods for improving constipation is characterized in that: the monosaccharide composition in the soluble dietary fiber comprises fucose, galactose, glucose, mannose, xylose, glucuronic acid, rhamnose, galacturonic acid, arabinose and ribose.
2. Use according to claim 1, characterized in that: the extraction method of the soluble dietary fiber comprises the following steps:
(1) adding 4-6% NaCl solution into the residue at a ratio of 1:20-40, heating at 80-100 deg.C for 6-8 hr for decalcification, centrifuging, collecting precipitate, adding pure water, heating and boiling for 30 min;
(2) adding 5mol/L NaOH solution into the precipitate obtained in the step (1) according to the feed-liquid ratio of 1:20-40, adjusting the pH value to be alkaline, heating at 80-100 ℃ for 6-8 h, centrifuging and collecting the precipitate;
(3) and (3) adjusting the pH of the precipitate obtained in the step (2) to be neutral by using HCl, concentrating, dialyzing, standing at ultralow temperature, and freeze-drying to obtain the soluble dietary fiber.
3. Use according to claim 2, characterized in that: the pH value is adjusted to 13 in the step (2); heating at 90 deg.C for 6 hr, centrifuging, and precipitating to obtain soluble dietary fiber.
4. Use according to claim 2, characterized in that: and (3) after the precipitate with the pH adjusted to be neutral is subjected to rotary evaporation and concentration, dialyzing in a 1500 Da dialysis bag for 10-12 h, placing in a refrigerator at the temperature of-80 ℃ for 12h, and freeze-drying for 36 h to obtain the dried soluble dietary fiber.
5. Use according to claim 1, characterized in that: the effective dosage of the soluble dietary fiber for improving constipation is 0.5 g/kg-3.5 g/kg.
6. Use according to claim 1, characterized in that: the soluble dietary fiber can increase the quantity, the weight and the water content of excrement of a constipated mouse, so that the defecation function of the mouse is enhanced.
7. Use according to claim 1, characterized in that: the soluble dietary fiber can promote intestinal peristalsis and improve intestinal transport function by increasing the water content of intestinal tracts of constipated mice and increasing the intestinal transport rate.
8. Use according to claim 1, characterized in that: the soluble dietary fiber can increase the content of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum and increase the content of SCFAs, thereby inhibiting oxidative stress level disorder caused by constipation.
9. Use according to claim 1, characterized in that: the soluble dietary fiber can reduce the expression level of inducible nitric oxide synthase iNOS and can up-regulate the expression levels of intestinal neurotransmitter acetylcholinesterase AchE, endothelial nitric oxide synthase eNOS, glial cell-derived neurotrophic factor GDNF and brain-derived neurotrophic factor BDNF.
10. Use according to claim 1, characterized in that: the soluble dietary fiber can adjust the structure of intestinal flora of constipation mice by reducing the abundance of bacteroidetes in intestinal tracts of the mice and increasing the abundance of firmicutes and actinomycetes.
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| CN103211230A (en) * | 2005-10-24 | 2013-07-24 | 雀巢技术公司 | Dietary fiber formulation and method of administration |
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| WO2021014469A1 (en) * | 2019-07-25 | 2021-01-28 | Chitra Vasant Savangikar | Dietary fiber composition |
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| CN103211230A (en) * | 2005-10-24 | 2013-07-24 | 雀巢技术公司 | Dietary fiber formulation and method of administration |
| CN103168993A (en) * | 2013-03-28 | 2013-06-26 | 中国人民解放军第三军医大学第三附属医院 | Fiber nutritional preparation for preventing and treating constipation and being used as assembly meal of dietary fiber |
| CN109717484A (en) * | 2019-03-15 | 2019-05-07 | 南京中医药大学 | A kind of preparation method with the anti-dietary fiber to treat constipation |
| WO2021014469A1 (en) * | 2019-07-25 | 2021-01-28 | Chitra Vasant Savangikar | Dietary fiber composition |
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Application publication date: 20211109 |