CN111808910A - Method for evaluating activity of dietary polysaccharide - Google Patents
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Abstract
The invention relates to a method for evaluating the activity of dietary polysaccharide. The method comprises the steps of adding dietary polysaccharide into a microbial culture medium as a carbon source, culturing the dietary polysaccharide and fecal microorganisms at 37 ℃ for 24 hours under an in vitro anaerobic condition, centrifuging at 10000rmp, collecting fermentation liquor through a 0.22 mu m membrane, and detecting the metabolic products of the dietary polysaccharide by utilizing metabonomics. Caco-2 cells are planted in an upper chamber of a Transwell technology, cells for evaluating the activity of the dietary polysaccharides are planted in a lower chamber of the upper chamber of the Transwell technology, fermentation liquor is added into an upper chamber cell culture medium to be cultured for 24 hours, liquid phase, gas phase or metabonomics and other means are used for detecting metabolites which can pass through a Caco-2 monolayer cell model, and relevant indexes of lower chamber cells are measured. The method of the invention can systematically and scientifically evaluate the biological activity of the dietary polysaccharide.
Description
Technical Field
The invention relates to the field of food science and engineering, in particular to a method for evaluating the activity of dietary polysaccharide.
Background
Polysaccharides are natural high molecular compounds in the cell walls of higher plants, animal cell membranes and microorganisms, and are widely involved in various life phenomena of cells. Due to the diverse biological activities and the wide use in the fields of food health care and disease prevention, the development and utilization of polysaccharide resources are increasingly active, and the polysaccharide resources become hot spots for research of food science, natural medicines and biochemistry. More and more researches show that the dietary polysaccharide has the functions of resisting oxidation, inhibiting bacteria, resisting obesity, resisting inflammation, resisting diabetes, resisting tumor, protecting cardiovascular and cerebrovascular vessels and nervous system, and reducing the damage of ultraviolet rays to skinInjury, etc. At present, more than 300 polysaccharide compounds are separated from natural products, wherein water-soluble polysaccharide extracted from plants is the most important, and nearly hundreds of plant polysaccharides are widely applied to health-care food and medicines. The polysaccharide can act on immune system in multiple ways and multiple layers, and a large number of experiments prove that the polysaccharide can activate macrophages
Monocytes, neutrophils, NK, DC and other immune cells are also involved in regulating the secretion of cytokines such as interleukins, interferons and the like, and activating the expression of relevant immune channels. The polysaccharide of fructus Schisandrae Sphenantherae can effectively promote proliferation of chicken T-lymphocyte, B-lymphocyte and abdominal cavity macrophage IL-2, IFN-gamma and TNF-alpha; the litchi polysaccharide can obviously increase NK cytotoxicity, effectively promote the release of IFN-gamma, inhibit the release of IL-4, promote the expression of a specific transcription factor T-beta of Th1, inhibit the expression of a specific transcription factor GATA-3 of Th2 and promote lymphocytes to enter S phase. The mulberry leaf polysaccharide can effectively promote the proliferation of mouse abdominal cavity macrophages (RAW264.7), improve the activity of RAW264.7 acid phosphatase and the capability of the RAW264.7 acid phosphatase for phagocytosing neutral red, and also can effectively improve the release amount of NO and cell factors TNF-alpha, IFN-gamma and IL-1 beta.
The polysaccharide belongs to a biological macromolecule with a relatively complex structure, and because the human genome does not encode enough carbohydrate metabolic enzymes, the polysaccharide cannot be digested and absorbed by digestive enzymes in oral cavity, stomach and small intestine, and enters the rear section of the large intestine to be fermented and utilized by intestinal microorganisms. In recent years, the role of polysaccharides in regulating intestinal microorganisms has been widely studied and reported. Polysaccharides derived from plants, microorganisms and marine algae can improve intestinal flora structure and stimulate growth of beneficial bacteria such as Bacillus bifidus and lactobacillus. Meanwhile, the intestinal bacteria have a large number of genes which are deficient in the human body and metabolize plant polysaccharides, and the polysaccharides are fermented by the genes to generate a large number of metabolites such as short-chain fatty acids (SCFAs). SCFAs produced by fermentation of dietary polysaccharides participate in different metabolism and exert different activities in human body. For example, acetic acid, which is absorbed and utilized by the host and provides about 10% of the total daily energy of the human body, is one of the important sources of energy of the host. Propionic acid, after being absorbed by the intestinal parietal cells, reaches the liver for catabolism and is involved in the process of reversal of pyruvate to glucose and possibly in the inhibition of fat synthesis. Butyric acid is primarily absorbed by epithelial cells and serves as the primary energy source for epithelial cells. Therefore, the anti-obesity, anti-inflammatory, anti-diabetes, anti-tumor and cardiovascular and nervous system protection effects of dietary polysaccharides can be achieved by regulating intestinal microorganisms and their metabolites. At present, the cell model is utilized to evaluate the activity of the polysaccharide, and the cell is directly intervened mainly by a dietary polysaccharide solution, so that the biological activity of the dietary polysaccharide cannot be accurately reflected. Therefore, the establishment of a method for evaluating the activity of metabolites after intestinal microbial fermentation based on dietary polysaccharides is urgently needed.
Disclosure of Invention
The invention aims to provide a method for evaluating the activity of dietary polysaccharide, and the specific scheme for realizing the invention is as follows:
(1) and (3) replacing the carbon source glucose in the microorganism basic culture medium with dietary polysaccharide, namely the culture medium comprises: 10g/L of dietary polysaccharide, 2.0g/L of peptone, 2.0g/L of yeast extract, 0.1g/L of sodium chloride, 0.04g/L of dipotassium hydrogen phosphate, 0.04g/L of monopotassium phosphate, 0.01g/L of magnesium sulfate, 0.01g/L of calcium chloride, 2.0g/L of sodium bicarbonate, 0.02g/L of hemin, 0.5g/L of cysteine hydrochloride, 0.5g/L of bile salt, 1.0g/L of Resazurin, 802.0 mL/L of Tween and vitamin K110μL/L；
(2) In vitro fermented stool samples were provided from 4 healthy volunteers (2 men and 2 women, age 22 to 28 years) who had not taken antibiotics or probiotic products and had no gastrointestinal disease within 3 months. Collecting fresh feces, mixing in equal amount, adding 9 times of sterilized normal saline (NaCl 8.5g/L, cysteine hydrochloride 0.5g/L), stirring, and centrifuging (250rmp) to obtain 10% feces suspension;
(3) adding the fecal suspension obtained in the step (2) into the culture medium which is obtained in the step (1) and takes the dietary polysaccharide as a carbon source in a ratio of 1: 9;
(4) culturing the culture system prepared in the step (3) in an anaerobic environment at 37 ℃ for 24 hours, and centrifuging to pass through a 0.22 mu m film to obtain a dietary polysaccharide fermentation liquid;
(5) and (4) analyzing the dietary polysaccharide fermentation liquor obtained in the step (4) in a liquid phase, a gas phase or metabonomics way and the like to identify the metabolic products of the dietary polysaccharide.
(6) Caco-2 cells were mock small intestine monolayer cell model in the upper chamber of the Transwell plate, and cells evaluated for polysaccharide activity in the lower chamber. And (3) adding the dietary polysaccharide fermentation liquor obtained in the step (4) into an upper chamber of a Transwell plate for culturing for 24h, determining which products can pass through a Caco-2 monolayer cell model by using methods such as a liquid phase method, a gas phase method or a metabonomics method, and determining lower chamber cell related indexes to evaluate the activity of the dietary polysaccharide.
Compared with the existing polysaccharide activity evaluation method, the activity evaluation method has the following advantages:
(1) the existing polysaccharide in-vitro cell activity evaluation methods are all polysaccharide and cell co-culture, but the dietary polysaccharide cannot be directly absorbed and utilized by a human body and cannot reach a target organ, so that the method utilizes in-vitro intestinal microbial fermentation and a Caco-2 cell model to simulate the digestion, absorption and activity expression of the polysaccharide in the human body, can more accurately reflect the biological activity of the dietary polysaccharide, and is more scientific;
(2) compared with in vivo experiments, the invention simulates the metabolism, absorption and activity of polysaccharide in human body by using in vitro fermentation and cell experiments, and has the advantages of simple experiment, low cost and the like.
Drawings
FIG. 1 is a flow chart of a method for evaluating the activity of dietary polysaccharides
FIG. 2 shows the effect of tea polysaccharide metabolites on NO secretion of RAW264.7 cells and the expression of iNOS in mRNA
FIG. 3 shows the effect of polysaccharide metabolites of Hibiscus manihot on fat droplets in HepG-2 cells
Detailed Description
The invention is further described below by way of examples.
Example 1:
(1) changing carbon source glucose in the microorganism basic culture medium into tea polysaccharide, namely the culture medium comprises: 10g/L of tea polysaccharide, 2.0g/L of peptone, 2.0g/L of yeast extract, 0.1g/L of sodium chloride, 0.04g/L of dipotassium hydrogen phosphate, 0.04g/L of monopotassium phosphate, 0.01g/L of magnesium sulfate, 0.01g/L of calcium chloride, 2.0g/L of sodium bicarbonate, 0.02g/L of hemin, 0.5g/L of cysteine hydrochloride, 0.5g/L of bile salt, 1.0g/L of Resazurin, 802.0 mL/L of Tween and 110 mu L/L of vitamin K;
(2) in vitro fermented stool samples were provided from 4 healthy volunteers (2 men and 2 women, age 22 to 28 years) who had not taken antibiotics or probiotic products and had no gastrointestinal disease within 3 months. 5g of fresh feces were collected from each volunteer, mixed with 45g of physiological saline (NaCl 8.5g/L, cysteine hydrochloride 0.5g/L) and stirred well, and centrifuged (250rmp) to obtain a 10% feces suspension;
(3) adding 2mL of the feces suspension obtained in the step (2) into 18mL of a culture medium taking the tea polysaccharide obtained in the step (1) as a carbon source;
(4) culturing the culture system prepared in the step (3) in an anaerobic environment at 37 ℃ for 24 hours, and centrifuging to pass through a 0.22 mu m film to obtain tea polysaccharide fermentation liquor;
(5) and (4) carrying out metabonomics analysis on the tea polysaccharide fermentation liquor obtained in the step (4) to identify that the fermentation product of the tea polysaccharide is short-chain fatty acid (acetic acid, propionic acid and butyric acid).
(6) Caco-2 cells in the upper chamber of the Transwell plate mimic the monolayer cell model, and RAW264.7 in the lower chamber. And (3) adding the tea polysaccharide fermentation liquor obtained in the step (4) into a Transwell plate upper chamber for culturing for 24h to ensure that the content of polysaccharide metabolites in the culture medium is 50, 100 and 200 mug/mL, determining which products can pass through a Caco-2 monolayer cell model by utilizing metabonomics, and determining the influence of the tea polysaccharide fermentation liquor on the proliferation and phagocytosis of RAW264.7 cells and the secretion of NO, TNF-alpha and IL-6. The results show that the tea polysaccharide metabolites are mainly acetic acid, propionic acid and butyric acid, and the metabolites can remarkably increase the phagocytic index of RAW264.7 cells and promote the secretion of NO, TNF-alpha and IL-6. FIG. 2 shows that tea polysaccharide metabolites increase NO secretion in RAW264.7 cells and expression of iNOS at mRNA level.
Example 2:
(1) changing carbon source glucose in a microorganism basic culture medium into golden flower fungus polysaccharide, namely the culture medium comprises: 10g/L of golden flower fungus polysaccharide, 2.0g/L of peptone, 2.0g/L of yeast extract, 0.1g/L of sodium chloride, 0.04g/L of dipotassium phosphate, 0.04g/L of potassium dihydrogen phosphate, 0.01g/L of magnesium sulfate, 0.01g/L of calcium chloride, 2.0g/L of sodium bicarbonate, 0.02g/L of hemin, 0.5g/L of cysteine hydrochloride, 0.5g/L of bile salt, 1.0g/L of Resazurin, 802.0 mL/L of Tween and 110 mu L/L of vitamin K;
(2) in vitro fermented stool samples were provided from 4 healthy volunteers (2 men and 2 women, age 22 to 28 years) who had not taken antibiotics or probiotic products and had no gastrointestinal disease within 3 months. 5g of fresh feces were collected from each volunteer, mixed with 45g of physiological saline (NaCl 8.5g/L, cysteine hydrochloride 0.5g/L) and stirred well, and centrifuged (250rmp) to obtain a 10% feces suspension;
(3) adding 2mL of the feces suspension obtained in the step (2) into 18mL of a culture medium taking the golden flower fungus polysaccharide obtained in the step (1) as a carbon source;
(4) culturing the culture system prepared in the step (3) in an anaerobic environment at 37 ℃ for 24 hours, and centrifuging to pass through a 0.22-micrometer film to obtain a golden flower fungus polysaccharide fermentation liquid;
(5) and (4) carrying out metabonomics analysis on the golden flower fungus polysaccharide fermentation liquor obtained in the step (4) to identify fermentation products (acetic acid and propionic acid) of the golden flower fungus polysaccharide.
(6) Caco-2 cells were mock monolayer cells in the upper chamber and HepG-2 cells in the lower chamber of the Transwell plate. Adding the golden flower fungus polysaccharide fermentation liquor obtained in the step (4) into a Transwell plate upper chamber for culturing for 24 hours, determining which products can pass through a Caco-2 single-layer cell model by utilizing metabonomics, and determining the influence of the golden flower fungus polysaccharide fermentation liquor on the lipid metabolism of a HepG-2 cell, wherein the results show that the acetic acid and the propionic acid of the golden flower fungus polysaccharide metabolite can obviously regulate the lipid metabolism of the HepG-2 cell, and fig. 3 shows that the influence of different doses of the golden flower fungus polysaccharide metabolite on fat drops in the HepG-2 cell can discover that the golden flower fungus polysaccharide metabolite can obviously reduce the content of the fat drops in the HepG-2 cell.
Claims (6)
1. A method for evaluating the activity of dietary polysaccharides, characterized in that it comprises the following operating steps:
(1) and (3) replacing the carbon source glucose in the microorganism basic culture medium with dietary polysaccharide, namely the culture medium comprises: 10g/L of dietary polysaccharide, 2.0g/L of peptone,2.0g/L of yeast extract, 0.1g/L of sodium chloride, 0.04g/L of dipotassium hydrogen phosphate, 0.04g/L of monopotassium phosphate, 0.01g/L of magnesium sulfate, 0.01g/L of calcium chloride, 2.0g/L of sodium bicarbonate, 0.02g/L of hemin, 0.5g/L of cysteine hydrochloride, 0.5g/L of bile salt, 1.0g/L of resazurin, 802.0 mL/L of Tween and vitamin K110μL/L；
(2) In vitro fermented stool samples were provided from 4 healthy volunteers (2 men and 2 women, age 22 to 28 years) who had not taken antibiotics or probiotic products and had no gastrointestinal disease within 3 months. Collecting fresh feces, mixing in equal amount, adding 9 times of sterilized normal saline (NaCl 8.5g/L, cysteine hydrochloride 0.5g/L), stirring, and centrifuging (250rmp) to obtain 10% feces suspension;
(3) adding the fecal suspension obtained in the step (2) into the culture medium which is obtained in the step (1) and takes the dietary polysaccharide as a carbon source in a ratio of 1: 9;
(4) culturing the culture system prepared in the step (3) in an anaerobic environment at 37 ℃ for 24 hours, and centrifuging to pass through a 0.22 mu m film to obtain a dietary polysaccharide fermentation liquid;
(5) and (4) carrying out metabonomics analysis on the dietary polysaccharide fermentation liquor obtained in the step (4) to identify the fermentation product of the dietary polysaccharide.
(6) Caco-2 cells were mock monolayer cell models in the upper chamber of the Transwell plate and cells evaluated for polysaccharide activity in the lower chamber. And (3) adding the dietary polysaccharide fermentation liquor obtained in the step (4) into an upper chamber of a Transwell plate for culturing for 24h, determining which products can pass through a Caco-2 monolayer cell model by utilizing metabonomics, and determining related indexes of lower chamber cells to evaluate the activity of the dietary polysaccharide.
2. A method of assessing the activity of dietary polysaccharides according to claim 1. The method is characterized in that carbon sources in a microorganism basic culture medium are changed into dietary polysaccharides for anaerobic fermentation. The culture medium comprises: 10g/L of dietary polysaccharide, 2.0g/L of peptone, 2.0g/L of yeast extract, 0.1g/L of sodium chloride, 0.04g/L of dipotassium hydrogen phosphate, 0.04g/L of monopotassium phosphate, 0.01g/L of magnesium sulfate, 0.01g/L of calcium chloride, 2.0g/L of sodium bicarbonate, 0.02g/L of hemin, 0.5g/L of cysteine hydrochloride, 0.5g/L of bile salt, 1.0g/L of Resazurin, 802.0 mL/L of Tween and vitaminBiotin K110μL/L。
3. A method of assessing the activity of dietary polysaccharides according to claim 1. The method is characterized in that the activity evaluation is carried out by utilizing dietary polysaccharide intestinal microorganism fermentation liquor.
4. A method of assessing the activity of dietary polysaccharides according to claim 1. The method is characterized in that the dietary polysaccharide microbial metabolites are identified by utilizing liquid phase, gas phase or metabonomics and the like.
5. The method for evaluating the activity of the dietary polysaccharide according to claim 1, wherein a Caco-2 monolayer cell model is used to explore which metabolites in the dietary polysaccharide fermentation broth can pass through the Caco-2 monolayer cell model.
6. A method of assessing the activity of dietary polysaccharides according to claim 1, characterized in that: the biological activity of metabolites across the Caco-2 monolayer cell model was explored using different cell models cultured in the Transwell lower chamber.
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Application publication date: 20201023 |