阅读说明：本技术 一种植物多糖健胃咖啡 (Plant polysaccharide stomach-invigorating coffee ) 是由 王伟明 霍金海 李梦雪 董文婷 庄岩 魏文峰 于 2021-09-08 设计创作，主要内容包括：本发明公开了一种植物多糖健胃咖啡,包括用于抑制胃酸分泌并且同时提高胃蛋白酶活性的玉米须多糖和用于改善咖啡乳化性的南瓜粗多糖,其中所述玉米须多糖使用纤维素酶-南瓜粗多糖联用酶解法得到。玉米须多糖冻干粉中多糖分子量为35000～50000,用于抑制胃酸分泌并且同时提高胃蛋白酶活性的玉米须多糖和用于改善咖啡乳化性的南瓜粗多糖的质量比为1：1；用于抑制胃酸分泌并且同时提高胃蛋白酶活性的玉米须多糖和用于改善咖啡乳化性的南瓜粗多糖组合物与咖啡的质量比为1：5。本发明提供的植物多糖健胃咖啡通过玉米须多糖抑制胃酸的分泌并且同时提高胃蛋白酶活性,可有效避免或减少饮用咖啡所导致的反酸、烧心、胃胀等胃部不适。(The invention discloses a plant polysaccharide stomach-invigorating coffee, which comprises corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously improving pepsin activity and pumpkin crude polysaccharide for improving coffee emulsibility, wherein the corn stigma polysaccharide is obtained by a cellulase-pumpkin crude polysaccharide combined enzymolysis method. The molecular weight of polysaccharide in the corn stigma polysaccharide freeze-dried powder is 35000-50000, and the mass ratio of the corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously improving pepsin activity to the pumpkin crude polysaccharide for improving coffee emulsibility is 1: 1; the mass ratio of the corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously improving pepsin activity and the pumpkin crude polysaccharide composition for improving coffee emulsibility to coffee is 1: 5. the plant polysaccharide stomach-invigorating coffee provided by the invention inhibits gastric acid secretion through the corn stigma polysaccharide and simultaneously improves the activity of pepsin, so that stomach discomfort such as acid regurgitation, heartburn, gastrectasia and the like caused by coffee drinking can be effectively avoided or reduced.)

1. A vegetable polysaccharide stomach-invigorating coffee, which is characterized by comprising corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously improving pepsin activity and pumpkin crude polysaccharide for improving coffee emulsibility, wherein the corn stigma polysaccharide is obtained by a cellulase-pumpkin crude polysaccharide combined enzymolysis method.

2. The plant polysaccharide stomach-invigorating coffee as claimed in claim 1, wherein the corn stigma polysaccharide has a molecular weight of 35000-50000.

3. The plant polysaccharide stomach-invigorating coffee as claimed in claim 2, wherein the corn polysaccharide comprises the following components in proportion: glucose: galactose: l-arabinose: rhamnose: pentosan: mannan: xylan: polyfructose: chitosan 25: 20: 15: 10: 8: 7: 6: 5: 4.

4. the plant polysaccharide stomach-invigorating coffee as claimed in claim 3, wherein the mass ratio of the corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously increasing pepsin activity to the pumpkin crude polysaccharide for improving coffee emulsifiability is 1: 1.

5. the plant polysaccharide stomach-invigorating coffee as claimed in claim 4, wherein the mass ratio of the corn stigma polysaccharide and the pumpkin crude polysaccharide composition for inhibiting gastric acid secretion and simultaneously increasing pepsin activity and improving coffee emulsifiability to coffee is 1: 5.

6. the plant polysaccharide stomach-invigorating coffee as claimed in claim 5, wherein the specific process of using corn stigma crude sugar used by cellulase-pumpkin crude polysaccharide combined enzymolysis method is as follows:

drying corn stigma at 80 deg.C to constant weight, pulverizing, sieving with 60 mesh sieve, weighing corn stigma powder 100g, soaking in 30 times of distilled water for 1 hr, treating with 500w of microwave for 5min, performing 100 deg.C constant temperature water bath for 1 hr, filtering, storing extractive solution, adding the same amount of water into the residue, and repeating the above extraction process for at least 4 times;

mixing extractive solutions, centrifuging at 6000 r.min for 20min, collecting supernatant, rotary evaporating for concentrating to 1/3 of original volume, adding anhydrous ethanol until ethanol concentration in the concentrated solution reaches 80%, standing at 4 deg.C for 24 hr for ethanol precipitation, centrifuging at 4000 r.min for 15min, separating ethanol and precipitate, sequentially adding 2 times volume of anhydrous ethanol and acetone into the precipitate, washing, evaporating solvent in boiling water bath, and oven drying at 50 deg.C to constant weight to obtain corn stigma crude sugar.

7. The plant polysaccharide stomach-invigorating coffee as claimed in claim 6, wherein the specific process of using the pumpkin crude polysaccharide by cellulase-pumpkin crude polysaccharide combined enzymolysis method is as follows:

drying pumpkin meat, crushing, sieving with a 60-mesh sieve, weighing 100g of sieved pumpkin powder, adding 5-7 times of distilled water, soaking for 2h, heating in a constant-temperature water bath kettle at 100 ℃ for 6h, filtering, preserving an extracting solution, adding the same amount of water into filter residue, and repeating the extracting process at least twice;

mixing the extracting solutions, centrifuging for 15min at 4000 r.min, taking supernatant, performing rotary evaporation and concentration to 1/3 of the original volume, adding absolute ethyl alcohol until the concentration of the ethyl alcohol in the concentrated solution reaches 60-80%, standing at 4 ℃ for 24h for alcohol precipitation, centrifuging for 15min at 4000 r.min, separating the ethyl alcohol from the precipitate, adding 2 times of 95% ethyl alcohol in volume into the precipitate obtained by alcohol precipitation, washing, taking the precipitate, evaporating the solvent on a boiling water bath, and drying at 50 ℃ until the weight is constant to obtain the pumpkin crude polysaccharide.

8. The plant polysaccharide stomach-invigorating coffee as claimed in claim 7, wherein the specific process for preparing the corn stigma polysaccharide by using cellulase-pumpkin crude polysaccharide combined enzymolysis method comprises the following steps:

preparing a corn silk crude sugar solution with the concentration of 0.1g/ml, adjusting the pH value to be in the range of 4.5-5.5, adding 40000U/g of cellulase according to the mass fraction of 1.0%, adding a pumpkin crude polysaccharide solution with the concentration of 0.1g/ml according to the mass fraction of 1.0%, carrying out enzymolysis at 45 ℃ in a water bath environment, inactivating enzymes in boiling water after complete reaction, cooling, centrifuging, taking the supernatant, and drying to obtain the corn silk polysaccharide.

9. The plant polysaccharide stomach-invigorating coffee as claimed in claim 8, wherein the pumpkin crude polysaccharide for improving coffee emulsibility is mixed with dextrin adjuvant in an amount of 1-5% by weight of the pumpkin crude polysaccharide, and then spray-dried to obtain spray-dried granules.

Technical Field

The invention belongs to the technical field of functional beverages, and particularly relates to vegetable polysaccharide stomach-invigorating coffee, which can effectively avoid or reduce stomach discomfort such as acid regurgitation, heartburn, gastrectasia and the like caused by drinking coffee by inhibiting secretion of gastric acid through corn stigma polysaccharide and improving activity of pepsin at the same time.

Background

Coffee with unique aroma and taste is one of three major beverages in the world, and along with the continuous integration of western culture, more and more people in China accept the coffee as a social beverage. In addition, caffeine in coffee can excite central nerves, improve mood, has a nerve-concentrating effect, and becomes a work partner for many employees who need to concentrate attention. However, due to differences in the digestive systems of the chinese and western countries and differences in diet, a significant portion of the countries may experience gastric reactions such as acid regurgitation, heartburn, bloating, etc. from drinking coffee or from drinking coffee in excess.

Through research, several main substances causing stomach discomfort of Chinese people in coffee are found to include: (1) caffeine: as a powerful psychostimulant, the composition can increase the frequency of contraction of the digestive tract, so that gastric acid is increased, and people who are sensitive to gastric acid generate acid regurgitation; (2) chlorogenic acid, also known as caffeoylquinic acid, is depside composed of caffeic acid and quinic acid, is a phenolic compound accounting for 6-12% of coffee components, and can promote secretion of cholecystokinin and gastrin, thereby increasing secretion of gastric acid. Abnormal increase of gastric acid can irritate the gastric mucosa, causing discomfort symptoms such as heartburn, hiccups, nausea, and vomiting. If there is a gastritis-gastric ulcer, the stomach discomfort is further aggravated by excessive stomach acid, resulting in stomach pain.

The corn stigma is called Yumai stigma, is a dry style and stigma of the gramineous crop corn, is in a shape of thread with needles and has wavy folds at the edge. The stigma Maydis extract contains various bioactive substances including flavonoids, polysaccharides, fatty oil, sitosterol and stigmasterol, and also contains common alkaloids, volatile oil, saponin, tartaric acid, and oxalic acid. The corn silk polysaccharide content accounts for 4.87% of the dry weight of the corn silk, and is the substance with the highest content in the corn silk functional factors. The stigma Maydis polysaccharide has effects of reducing blood sugar, reducing blood lipid, lowering blood pressure, regulating immunity, resisting tumor, clearing heat, promoting bile flow, protecting liver, and resisting oxidation.

Pumpkin is also called pumpkin, squash, pumpkin, etc., is an annual vine herb plant in the pumpkin genus of the Cucurbitaceae family, has sweet taste and warm nature, and has the functions of strengthening the middle-jiao and replenishing qi, moistening lung and benefiting heart. The pumpkin is rich and comprehensive in nutrition and is rich in pectin, pentosan, various amino acids, vitamins, alkaloid, soluble cellulose and other physiological active substances; the pumpkin polysaccharide has the functions of enhancing the immunity of organisms, reducing blood sugar, reducing blood fat and the like, and is an important active component in pumpkin.

At present, plant polysaccharides represented by corn stigma polysaccharide and pumpkin polysaccharide are mostly used in the fields of cancer treatment, virus infection resistance and the like, particularly, the corn stigma polysaccharide is not used for systematic research on improvement of functions of digestive tracts, and particularly, specific applicability research on stomach discomfort caused by application of the corn stigma polysaccharide to coffee is lacked.

Disclosure of Invention

Aiming at the stomach side effect caused by coffee, the invention provides the plant polysaccharide stomach-invigorating coffee, which inhibits the secretion of gastric acid and simultaneously improves the activity of pepsin by combining the corn polysaccharide and the pumpkin polysaccharide on the basis of not influencing the coffee concentrating effect and the taste, thereby effectively avoiding or reducing the stomach discomfort caused by drinking coffee.

A vegetable polysaccharide stomach-invigorating coffee comprises corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously increasing pepsin activity and pumpkin crude polysaccharide for improving coffee emulsibility, wherein the corn stigma polysaccharide is obtained by cellulase-pumpkin crude polysaccharide combined enzymolysis.

Further, the corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously improving the activity of pepsin has the molecular weight of 35000-50000.

Further, the corn polysaccharide comprises the following components in proportion: glucose: galactose: l-arabinose: rhamnose: pentosan: mannan: xylan: polyfructose: chitosan 25: 20: 15: 10: 8: 7: 6: 5: 4.

further, the mass ratio of the corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously improving pepsin activity to the pumpkin crude polysaccharide for improving coffee emulsibility is 1: 1.

further, the mass ratio of the corn stigma polysaccharide for inhibiting gastric acid secretion and simultaneously improving pepsin activity and the pumpkin crude polysaccharide composition for improving coffee emulsibility to coffee is 1: 5.

further, the specific process of the corn stigma crude sugar used by the cellulase-pumpkin crude polysaccharide combined enzymolysis method comprises the following steps:

drying corn stigma at 80 deg.C to constant weight, pulverizing, sieving with 60 mesh sieve, weighing corn stigma powder 100g, soaking in 30 times of distilled water for 1 hr, treating with 500w of microwave for 5min, performing 100 deg.C constant temperature water bath for 1 hr, filtering, storing extractive solution, adding the same amount of water into the residue, and repeating the above extraction process for at least 4 times;

mixing extractive solutions, centrifuging at 6000 r.min for 20min, collecting supernatant, rotary evaporating for concentrating to 1/3 of original volume, adding anhydrous ethanol until ethanol concentration in the concentrated solution reaches 80%, standing at 4 deg.C for 24 hr for ethanol precipitation, centrifuging at 4000 r.min for 15min, separating ethanol and precipitate, sequentially adding 2 times volume of anhydrous ethanol and acetone into the precipitate, washing, evaporating solvent in boiling water bath, and oven drying at 50 deg.C to constant weight to obtain corn stigma crude sugar.

Further, the specific process of the pumpkin crude polysaccharide by the cellulase-pumpkin crude polysaccharide combined enzymolysis method comprises the following steps:

drying pumpkin meat, crushing, sieving with a 60-mesh sieve, weighing 100g of sieved pumpkin powder, adding 5-7 times of distilled water, soaking for 2h, heating in a constant-temperature water bath kettle at 100 ℃ for 6h, filtering, preserving an extracting solution, adding the same amount of water into filter residue, and repeating the extracting process at least twice;

mixing the extracting solutions, centrifuging for 15min at 4000 r.min, taking supernatant, performing rotary evaporation and concentration to 1/3 of the original volume, adding absolute ethyl alcohol until the concentration of the ethyl alcohol in the concentrated solution reaches 60-80%, standing at 4 ℃ for 24h for alcohol precipitation, centrifuging for 15min at 4000 r.min, separating the ethyl alcohol from the precipitate, adding 2 times of 95% ethyl alcohol in volume into the precipitate obtained by alcohol precipitation, washing, taking the precipitate, evaporating the solvent on a boiling water bath, and drying at 50 ℃ until the weight is constant to obtain the pumpkin crude polysaccharide.

Further, the specific process for preparing the corn stigma polysaccharide by using the cellulase-pumpkin crude polysaccharide combined enzymolysis method comprises the following steps:

preparing a corn silk crude sugar solution with the concentration of 0.1g/ml, adjusting the pH value to be in the range of 4.5-5.5, adding 40000U/g of cellulase according to the mass fraction of 1.0%, adding a pumpkin crude polysaccharide solution with the concentration of 0.1g/ml according to the mass fraction of 1.0%, carrying out enzymolysis at 45 ℃ in a water bath environment, inactivating enzymes in boiling water after complete reaction, cooling, centrifuging, taking the supernatant, and drying to obtain the corn silk polysaccharide.

Furthermore, the pumpkin crude polysaccharide for improving the coffee emulsibility is mixed with dextrin auxiliary materials accounting for 1-5% of the weight of the pumpkin crude polysaccharide, and then spray drying is carried out to obtain spray-dried particles.

The technical effects obtained by the invention are as follows:

1) pharmacological tests prove that the corn stigma polysaccharide has an obvious gastric mucosa protection effect, the purified corn stigma polysaccharide component is extracted and purified for multiple times, the corn stigma polysaccharide can be instantly dissolved in water, loose powder is obtained after freeze drying treatment, the dispersibility is good, the corn stigma polysaccharide and coffee are integrated, the taste is fine and smooth, the particle size is small, and no granular sensation exists.

2) In the process of deproteinizing the corn stigma polysaccharide, a cellulose protease-pumpkin polysaccharide combination method is adopted, so that the protein removal rate is high, and the polysaccharide retention rate is highest; the extracted pumpkin polysaccharide is spray-dried to obtain orange pumpkin polysaccharide powder which can be quickly dissolved in water, has good emulsification effect and improves the bitter and sour taste of the pure coffee.

3) The pumpkin polysaccharide spray-dried powder and the corn stigma polysaccharide freeze-dried powder are brewed together with pure coffee according to the proportion of 1:1, can be dissolved with coffee at the same time, are fragrant and sweet, can better alleviate the sour taste and the bitter taste of the coffee, cannot float on the surface of the coffee, can be completely mixed with coffee solution, cannot generate the phenomena of layering and turbidity, and has good stability and solubility.

4) Animal experiments prove that the pure coffee combined corn stigma freeze-dried powder and pumpkin crude polysaccharide composition can inhibit gastric acid secretion, can increase the activity of pepsin, and can protect the gastrointestinal health of people with excessive gastric acid on the basis of promoting digestion function, and particularly, the pure coffee combined corn stigma polysaccharide cut-off liquid and pumpkin combined effect is optimal, wherein the molecular weight of the pure coffee combined corn stigma freeze-dried powder and the molecular weight of the corn stigma polysaccharide cut-off liquid are 35000-50000.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the plant polysaccharide stomach-invigorating coffee provided by the present invention is described in detail below with reference to the examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

Example 1 preparation of lyophilized powder of stigma Maydis polysaccharide

Firstly, preparing corn stigma crude polysaccharide:

removing impurities from stigma Maydis, oven drying at 80 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve. Accurately weighing 100g of stigma Maydis powder, soaking in 30 times of distilled water for 1 hr, irradiating with 500w microwave for 5min, heating in 100 deg.C constant temperature water bath for 1 hr, filtering, and storing the extractive solution. The same amount of water was added to the residue and the process was repeated 4 times. Mixing extractive solutions, centrifuging at high speed of 6000 r.min for 20min, collecting supernatant, rotary evaporating to concentrate to 1/3, adding anhydrous ethanol to the concentrate to make the ethanol concentration in the concentrate reach 80%, standing at 4 deg.C for 24 hr for alcohol precipitation, centrifuging at 4000 r.min for 15min, separating ethanol and precipitate, and sequentially adding 2 times of anhydrous ethanol and acetone into the precipitate to wash. And (3) evaporating the solvent of the precipitate on a boiling water bath to almost complete degree, putting the precipitate into a drying oven, and drying at 50 ℃ to constant weight to obtain the crude corn stigma polysaccharide fluid extract.

Secondly, preparing pumpkin crude polysaccharide and pumpkin crude polysaccharide spray-dried particles:

taking a proper amount of pumpkin, cleaning, peeling, removing pulp, slicing, drying, crushing, and sieving with a 60-mesh sieve. Accurately weighing 100g of sieved substances in a beaker, adding 5-7 times of distilled water, soaking for 2h, heating in a 100 ℃ constant-temperature water bath kettle for 6h, filtering, preserving the extracting solution, adding the same amount of water into filter residues, and repeating the above process for extraction for 2 times. Mixing the extracting solutions, filtering impurities, centrifuging at a high speed of 4000 r.min for 15min, taking supernatant, concentrating by rotary evaporation to 1/3 of the original volume, adding absolute ethyl alcohol into the concentrated solution to enable the concentration of the ethyl alcohol in the concentrated solution to reach 60% -80%, standing at a low temperature of 4 ℃ for 24h for alcohol precipitation, centrifuging at 4000 r.min for 15min, separating the ethyl alcohol and precipitate, adding 2 times of 95% ethyl alcohol in volume into the precipitate obtained by alcohol precipitation for washing respectively, taking the precipitate, evaporating the solvent almost completely on a boiling water bath, putting the precipitate into a drying box, and drying at a temperature of 50 ℃ to constant weight to obtain the pumpkin crude polysaccharide fluid extract.

One part of the pumpkin crude polysaccharide fluid extract is prepared into a pumpkin crude polysaccharide solution with the concentration of 0.1g/ml, and the pumpkin crude polysaccharide solution is used for purifying the corn stigma polysaccharide protein.

And (3) putting the other part of the pumpkin crude polysaccharide fluid extract into a spray dryer, starting the spray dryer, and taking dextrin as an auxiliary material, wherein the use amount of the dextrin is 1-5% of the weight of the fluid extract. The temperature of the spray chamber was adjusted at 20000-. Obtaining pumpkin coarse polysaccharide spray-dried particles. Spray-dried particles, uniformly dispersed, smooth surface.

Step three, preparing corn stigma polysaccharide:

preparing a corn silk polysaccharide solution with the concentration of 0.1g/ml, adjusting the pH value of the solution to 4.5-5.5, adding 1.0% of cellulase (40000U/g) according to the mass fraction, adding 0.1g/ml of pumpkin crude polysaccharide according to the mass fraction of 1.0% for enzymolysis at 45 ℃ in a water bath environment, inactivating the enzyme through a boiling water bath for 5min after the reaction is finished, and cooling to room temperature to obtain the corn silk polysaccharide.

Fourthly, preparing corn stigma polysaccharide freeze-dried powder:

1) selecting a dialysis bag with a molecular weight of 35000, cutting the dialysis bag into small sections of 10-20 cm, boiling with 30% ethanol for 30min, and sequentially adding 0.02mol/LNaHCO₃The solution, 1mmol/LEDTA solution, was boiled for 10min and then rinsed with distilled water. At normal temperature, the obtained polysaccharide solution is filled into a dialysis bag, and is dialyzed for 12 hours by flowing distilled water. Retention of dialysateThe corn stigma polysaccharide cut-off solution with the molecular weight less than 35000 is used.

2) Selecting a dialysis bag with the molecular weight of 50000, cutting the dialysis bag into small sections of 10-20 cm, boiling with 30% ethanol for 30min, and sequentially adding 0.02mol/LNaHCO₃The solution, 1mmol/LEDTA solution, was boiled for 10min and then rinsed with distilled water. At normal temperature, the obtained polysaccharide solution is filled into a dialysis bag, and is dialyzed for 12 hours by flowing distilled water. The retentate in the dialysis bag was retained as a corn stigma polysaccharide retentate with a molecular weight greater than 50000.

3) The cut solution in the dialysis bag after dialysis for 12h with the molecular weight of 35000 in example 1 and the dialysis solution after dialysis for 12h with the molecular weight of 50000 in example 2 were mixed to obtain the cut solution of corn stigma polysaccharide 35000-50000.

The corn stigma polysaccharide freeze-dried powder is prepared from the samples obtained in 1) -3) by adopting a freeze-drying process, and the process comprises the following steps: placing the corn silk polysaccharide in the tray of a freeze dryer, placing the corn silk polysaccharide in the vacuum dryer for freeze drying, setting the parameters as shown in the following table, wherein the freeze drying is smooth, and the eutectic point test result of the vacuum freeze dryer is-26 ℃, so that the pre-freezing temperature of the corn silk extract is-40 ℃, the primary drying temperature is-30-0 ℃, and the analytic drying temperature is 10-40 ℃, and the corresponding freeze-dried powder is formed.

TABLE 1 corn silk polysaccharide freeze drying parameter settings

Example 2 preparation of vegetable polysaccharide stomach-invigorating coffee

Sample 1: mixing and matching the corn stigma polysaccharide freeze-dried powder with the molecular weight of the corn stigma polysaccharide intercepted less than 3500 and the pumpkin crude polysaccharide spray-dried particles obtained in the example 1 according to the weight ratio of 1:1 (4 g in total), and mixing and matching according to the composition: the pure coffee is mixed in a ratio of 1:5 by weight.

Sample 2: mixing lyophilized powder of stigma Maydis polysaccharide with molecular weight of more than 50000 with fructus Cucurbitae Moschatae crude polysaccharide spray-dried granule obtained in example 1 at a ratio of 1:1 (4 g in total), and mixing according to the composition: the pure coffee is mixed in a ratio of 1: 5.

Sample 3: mixing and matching the freeze-dried corn stigma polysaccharide powder with the molecular weight of the corn stigma polysaccharide intercepted between 3500 and 5000 with the pumpkin crude polysaccharide spray-dried particles obtained in example 1 according to a ratio of 1:1 (4 g in total), and mixing the components according to the following composition: the pure coffee is mixed in a ratio of 1: 5.

Sample 4: the pumpkin crude polysaccharide spray-dried powder obtained in example 1 was mixed with pure coffee in a ratio of 1: 5.

Sample 5: the lyophilized powder of corn silk polysaccharide obtained in example 1 (without dialysis) was mixed with pure coffee in a ratio of 1: 5.

The brewing method comprises the following steps: adding 180ml of water with the temperature of 90 ℃ into the samples 1-5, and brewing to examine the instant property, stability and taste.

TABLE 2 sample Performance analysis Table

Comparative experiment 1 cellulase-pumpkin crude polysaccharide combined enzymolysis method and effect comparison of other protein removal methods

The crude corn silk polysaccharide obtained in the first step of example was used to prepare a solution with a concentration of 0.1 g/ml.

Protein removal by the Sevage method: weighing 40mL of a sample corn stigma crude polysaccharide solution, adding 10mL of a chloroform-n-butanol (4: 1) mixed solvent, mixing, fully oscillating for 20min, centrifuging, removing denatured protein at the interface of an aqueous phase and an organic phase, and repeating for 7 times until no precipitate appears. Adding water into the upper layer solution to fix the volume to 50mL, and calculating the protein removal rate and the total polysaccharide retention rate.

Deproteinization by a cellulose protease method: measuring 40mL of a sample corn stigma crude polysaccharide solution, adjusting the pH value of the solution, adding 1.0% cellulase (40000U/g) according to mass fraction, carrying out enzymolysis in a water bath environment, inactivating the enzyme in a boiling water bath for 5min after the reaction is finished, cooling to room temperature, centrifuging, and taking supernate to measure the removal rate of protein and the retention rate of total polysaccharide.

ProteinRemoval rate (%) ═ C_Front-C_{Rear end})/C_{Front side}X 100%, wherein C_{Front side}And C_{Rear end}Protein concentrations before and after deproteinization were determined, respectively.

Polysaccharide retention (%) ═ C_{Rear end}/C_{Front side}X 100%, wherein C_{Front side}And C_{Rear end}The polysaccharide contents before and after deproteinization, respectively.

TABLE 3 results after different deproteinization process treatments

Through test determination, the Sevage method is independently adopted to remove protein, the protein removal rate is highest and can reach 78.43%, but the polysaccharide retention rate is lower. After enzymolysis is carried out by adopting cellulase and pumpkin crude polysaccharide, the polysaccharide retention rate is 83.34% at most.

Comparative experiment 2 Effect of the oral coffee composition of the present invention on gastric acid and pepsin secretion in normal rats

Laboratory instruments and animals:

type S210-K PH meter (METTLER, USA)

Pepsin activity detecting kit (BC2325, Solaibao)

The clean-grade healthy SD rats are half male and female, have the body weight of 200 +/-20 g, are bred from Beijing Wintonlihua laboratory animal technology Limited company, [ SCXK (Jing) 2017-.

Experimental methods

1. Experiment grouping

50 SD rats were divided into a blank control group, a coffee group, and 1 to 5 groups of samples obtained in example 2, each group containing 10 rats, and 5 rats were housed in cages for each group.

2. Gastric acid and pepsin assay

Except for the blank group, the groups were sequentially gavaged with the stomach-filling coffee and the samples 1-5 obtained in example 2, and the gavage period was 28 days according to the gavage volume of 2ml/100g of the body weight. At 28 days, blood was taken from the abdominal aorta, and after death at the cervical spine, the rat was dissected, the stomach was separated, and the cardia and pylorus were ligated. After the stomach was irrigated with 5mL of physiological saline, the physiological saline and the contents of the stomach were collected, centrifuged for 20min, and the pH value was measured with a pH meter after food residue was precipitated.

And centrifuging the mixture of the rat stomach normal saline and the stomach contents for 20min, and taking the supernatant, and operating according to the instruction of the pepsin ELISA kit. After the reaction is finished, the ELISA plate is placed at the 450nm wavelength of an ELISA reader to measure the value A, and the value A is substituted into a regression equation (the value A of the pepsin standard substance is used for carrying out regression analysis on the mass concentration), so that the pepsin content is calculated.

3. RT-PCR method for detecting H⁺-K⁺Expression level of ATPase mRNA

RT-PCR assay for H⁺-K⁺The expression level of ATPase mRNA was determined by extracting total mRNA from the gastric mucosa of 5 groups of rats, reverse transcription to give cDNA, and PCR amplification. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 45s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, and 32 cycles; final extension 72 ℃ for 10 min. H + -K + -ATP enzyme upstream and downstream primers: an upstream primer 5'-CTCTGCTTTGCGGGACTT-3'; the downstream primer 5'-CCTTGGCTGTGATGGGAT-3', with Actin as an internal reference. The PCR product was electrophoresed through 3% agarose gel, observed, and photographed.

4. Data statistics and result analysis

SPSS20.0 software is adopted for data statistical treatment, the mean value plus or minus standard deviation is used for representing, the difference between groups is analyzed by single-factor variance, the difference P is less than 0.05 and has significant difference, and the difference P is less than 0.01 and has extremely significant difference.

1) Gastric acid and pepsin activity assay

The experimental results show that the pH of the rats in the coffee group is obviously reduced and has statistical difference (P is less than 0.01) compared with the blank control group as shown in the table 4; the samples 1-3 are prepared by matching the corn stigma polysaccharide, the pumpkin polysaccharide and the coffee in the same proportion, compared with a pure coffee group, the sample 4 is prepared by matching the pumpkin polysaccharide and the coffee in the same proportion, the obvious improvement effect (P is more than 0.05) is not seen, the sample 5 is prepared by matching the corn stigma total polysaccharide and the coffee in the same proportion, the effect is more obvious (P is less than 0.05), but the action strength is weaker than that of the sample 3, and the 35000-50000-molecular-weight corn stigma polysaccharide can play a greater medicinal effect.

Meanwhile, compared with a blank control group, the caffeine group has obvious increase of pepsin activity (P is less than 0.01), which indicates that the increase of the pepsin activity is stimulated along with the increase of gastric acid, so that the nausea and digestion are enhanced, and the risk of gastric ulcer is caused. The other examples, except sample 4, all had a reduction compared to the caffeine group, but all remained at a higher level of pepsin compared to the blank group.

In conclusion, the experiment results show that the sample 3 can inhibit the secretion of gastric acid and simultaneously can keep the activity of pepsin at a high level, thereby promoting the digestion function and protecting the gastrointestinal health of people with excessive gastric acid.

Table 428 d gastric acid content and pepsin activity in rats

In comparison to the blank set, the data is,^##p < 0.01, in comparison with the caffeine group<0.05。

2) RT-PCR assay for H⁺-K⁺Relative expression amount of ATPase mRNA

Compared with the blank group, the result shows that H is contained in the gastric mucosa tissue of the rat after the coffee is infused into the stomach⁺An increase in the expression of the mRNA of the K + -ATPase; the other groups showed different degree of reduction compared to the pure coffee group except that sample 4 showed no significant reduction, with sample 3 showing significant reduction (P)<0.01), sample 5 also had a decrease (P)<0.05), the results are shown in table 2.

TABLE 5H in rat gastric mucosal tissue⁺-K⁺Relative expression amount of ATPase mRNA

In comparison to the blank set, the data is,^##p < 0.01, in comparison with the caffeine group<0.05。

One of the key enzymes of gastric acid secretion is H⁺-K⁺ATP enzyme, which actively transports K + from extracellular fluids into the cell by dephosphorylation and phosphorylation thereof, and simultaneously pumps H + from the cell out of the cell, thereby achieving H⁺/K⁺Transmembrane transport and secretion of gastric acid. The study showed that H⁺-K⁺The expression change of the ATP enzyme is consistent with the gastric acid secretion capacity, the expression condition of the ATP enzyme is selected and measured to reflect the secretion condition of pepsin and gastric acid, after the rats are infused with gastric coffee, the activities of the gastric acid and pepsin are increased, and the mRNA content of H + -K + -ATP enzyme is increased. Sample 3 and sample 5 groups, H, compared to the coffee group⁺-K⁺The ATP enzyme content is reduced, the gastric acid and pepsin content are reduced, the effect of the sample 3 groups is more obvious, the combination of the corn stigma polysaccharide and the coffee with the molecular weight of 35000-50000 can reduce the gastric acid caused by the coffee, and the action mechanism and the H function are shown⁺-K⁺-an ATPase.

The present invention is not limited to the above-described examples, and various changes can be made without departing from the spirit and scope of the present invention within the knowledge of those skilled in the art.