阅读说明：本技术 一种铁皮石斛叶微生态制剂的制备方法 (Preparation method of dendrobium officinale leaf microecological preparation ) 是由 邵平 方静宇 林杨 陈碧莲 冯思敏 于 2021-05-26 设计创作，主要内容包括：本发明公开了一种铁皮石斛叶微生态制剂的制备方法,所述制作方法包括如下步骤：(1)由铁皮石斛叶提取得到纯化的分子量在50-100kDa之间的铁皮石斛叶多糖；(2)取步骤(1)得到的铁皮石斛叶多糖与肠粘附材料于水中混匀,喷雾干燥后即得调节糖尿病结肠菌群的铁皮石斛叶微生态制剂；所述的肠粘附材料为抗消化淀粉和卡波姆的组合,所述铁皮石斛叶多糖与抗消化淀粉、卡波姆的投料质量比为0.2-0.3：0.02-0.03：0.05-0.06。本发明在制备过程中,筛选出了高降血糖活性的石斛叶多糖,将其和结肠粘附材料混合制成制剂,使制剂在结肠处释放给药,增加了多糖在结肠中的作用时间,从而达到治疗肠道菌群失调引起的肠道功能紊乱疾病的目的。(The invention discloses a preparation method of a dendrobium officinale leaf microecological preparation, which comprises the following steps: (1) extracting purified Dendrobium officinale leaf polysaccharide with molecular weight of 50-100kDa from Dendrobium officinale leaf; (2) uniformly mixing the dendrobium officinale leaf polysaccharide obtained in the step (1) and an intestinal adhesion material in water, and performing spray drying to obtain the dendrobium officinale leaf microecological preparation for regulating the colonic flora for diabetes; the intestinal adhesion material is a combination of anti-digestion starch and carbomer, and the feeding mass ratio of the dendrobium officinale leaf polysaccharide to the anti-digestion starch to the carbomer is 0.2-0.3: 0.02-0.03: 0.05-0.06. In the preparation process, the dendrobium leaf polysaccharide with high hypoglycemic activity is screened out and mixed with the colon adhesion material to prepare the preparation, so that the preparation is released and administered at the colon, the action time of the polysaccharide in the colon is prolonged, and the aim of treating intestinal dysfunction diseases caused by intestinal dysbacteriosis is fulfilled.)

1. A preparation method of a dendrobium officinale leaf microecological preparation comprises the following steps:

(1) extracting purified Dendrobium officinale leaf polysaccharide with molecular weight of 50-100kDa from Dendrobium officinale leaf;

(2) uniformly mixing the purified dendrobium officinale leaf polysaccharide with the molecular weight of 50-100kDa obtained in the step (1) and an intestinal adhesion material in water, and performing spray drying to obtain the dendrobium officinale leaf microecological preparation; the intestinal adhesion material is a combination of anti-digestion starch and carbomer, and the feeding mass ratio of the dendrobium officinale leaf polysaccharide to the anti-digestion starch to the carbomer is 0.2-0.3: 0.02-0.03: 0.05-0.06.

2. The method of claim 1, wherein: the preparation method also comprises the following steps: sequentially adding anhydrous ethanol and the dendrobium officinale leaf polysaccharide obtained in the step (1) in sequence by adopting a wet milling method, adding agate balls, sealing and wet milling at the wet milling temperature of 30-35 ℃ for 50-60 minutes to prepare suspension, performing rotary evaporation and freeze drying to obtain nano dendrobium leaf polysaccharide, and applying the nano dendrobium leaf polysaccharide to the step (2); the feeding ratio of the absolute ethyl alcohol to the dendrobium officinale leaf polysaccharide is 160-200 ml: 8-10 g.

3. The method of claim 1 or 2, wherein: the step (1) is specifically performed as follows:

(1-1) taking a proper amount of dendrobium officinale leaves, drying, crushing, sieving with a 20-40-mesh sieve, soaking with 85-90 vol% ethanol for 8-12h, repeating for 3-4 times, and drying in a fume hood;

(1-2) adding pure water with the volume 10-20 times of the mass of the dried powder in the step (1), decocting at 70-80 ℃ for 2-3 times, each time for 2-3h, merging supernate to obtain dendrobium polysaccharide water extract, removing protein by a sevage method, precipitating with 75-80 vol% alcohol, and centrifuging to obtain precipitate;

(1-3) redissolving the precipitate obtained in the step (2) with distilled water of which the volume is 10-20 times that of the precipitate, loading the redissolved precipitate onto a DEAE-cellulose column, eluting with distilled water at a flow rate of 4.0-5.0 ml/min, eluting with 0.5-1M sodium chloride solution at a flow rate of 4.0-5.0 ml/min to obtain a polysaccharide solution, and lyophilizing to obtain polysaccharide powder;

(1-4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain 2-5g/L polysaccharide solution, performing ultrafiltration by using an ultrafiltration cup to obtain polysaccharide solution with the molecular weight of 50-100kDa, dialyzing the obtained polysaccharide solution by using a dialysis bag with the molecular weight of 3.5-5kDa for 16-24 hours, and freeze-drying to obtain purified dendrobium officinale leaf polysaccharide with the molecular weight of 50-100 kDa.

4. The method of claim 3, wherein: in the step (1-2), the protein removal by the sevage method is performed as follows: adding mixed solution of chloroform and n-butanol into herba Dendrobii polysaccharide water extract, shaking thoroughly, retaining water phase, repeatedly treating for 3-5 times, and retaining water phase.

5. The method of claim 4, wherein: in the step (1-2), in the chloroform-n-butanol mixed solution used for removing the protein by the sevage method, the volume ratio of chloroform to n-butanol is 4-5: 1.

6. The method of claim 3, wherein: in the step (1-4), ultrafiltration is performed at 0.15-0.2 MPa.

(I) technical field

The invention belongs to the field of food processing, and particularly relates to a preparation method of a dendrobium officinale leaf microecological preparation.

(II) technical background

The incidence of diabetes of people is increased year by year due to the unhealthy modern fast-paced life and eating habits, and researches in recent years find that the imbalance of intestinal flora is closely related to the diabetes. The colon is the largest bacterial reservoir of the human body, and the intestinal microecological balance is the basis for the normal function of the intestinal mucosa. Among them, short-chain fatty acids play a very important role, mainly produced by the anaerobic bacteria glycolysis of undigested fiber, polysaccharide, etc. in the colon, while the butyrate content of diabetic patients is significantly lower than normal. At present, antibiotics and medicines for treating intestinal flora disturbance and regulating low synthesis of short-chain fatty acids in intestines exist, but the antibiotics and medicines have strong toxic and side effects on the intestines to a certain extent. And the existing microecological regulator for treating dysbacteriosis at home and abroad is mainly a gastric soluble probiotic preparation, and the medicament is released when reaching the stomach during treatment, is influenced by factors such as gastric acid and the like, and can not ensure that sufficient prebiotics enter the colon.

The polysaccharide is difficult to be digested by the stomach and small intestine of a human body, the blood sugar value of diabetes cannot be obviously improved, and the polysaccharide has almost no toxic or side effect, so that the polysaccharide with the blood sugar reducing activity in natural products has the potential of being developed into health-care food and medicines. Dendrobe is used as a traditional medicine and food dual-purpose resource, and the functional activity of the main active component dendrobe polysaccharide draws wide attention of scholars at home and abroad. However, the traditional medicinal part of dendrobium at present is stem, and the root, leaf, flower and other parts of dendrobium are usually abandoned, which causes huge waste of raw materials.

CN111919964A discloses a micro-ecological preparation based on forsythia suspense leaves, which can reduce diarrhea rate and enhance immune function. However, the fructus forsythiae leaves are simply ground and added, and the effect is general. CN111494495A discloses an application of litchi pulp dietary fiber-bonded phenol adduct in preparation of a preparation for improving intestinal microecology, which can increase the content of short-chain fatty acids in intestinal tract and is helpful for regulating the diversity, abundance and dominant flora distribution of intestinal flora, but the litchi pulp has complex components and is difficult to explain functional components and adverse components. And the influence of the dendrobe leaf polysaccharide on the generation of the intestinal flora short-chain fatty acid is not researched at present, and the influence of the dendrobe leaf polysaccharide on the intestinal flora imbalance caused by diabetes is not researched.

Disclosure of the invention

The invention aims to solve the technical problem of providing a preparation method of a dendrobium officinale leaf microecological preparation for regulating diabetic colon flora so as to solve the problem that the traditional polysaccharide has poor effects of reducing blood sugar and regulating intestinal flora.

The technical scheme of the invention is specifically explained as follows:

the invention provides a preparation method of a dendrobium officinale leaf microecological preparation, which comprises the following steps:

(1) extracting purified Dendrobium officinale leaf polysaccharide with molecular weight of 50-100kDa from Dendrobium officinale leaf;

(2) uniformly mixing the purified dendrobium officinale leaf polysaccharide with the molecular weight of 50-100kDa obtained in the step (1) and an intestinal adhesion material in water, and performing spray drying to obtain the dendrobium officinale leaf microecological preparation; the intestinal adhesion material is a combination of anti-digestion starch and carbomer, and the feeding mass ratio of the dendrobium officinale leaf polysaccharide to the anti-digestion starch to the carbomer is 0.2-0.3: 0.02-0.03: 0.05-0.06.

Preferably, the preparation method further comprises the following steps: sequentially adding anhydrous ethanol and the dendrobium officinale leaf polysaccharide obtained in the step (1) in sequence by adopting a wet milling method, adding agate balls, sealing and wet milling at the wet milling temperature of 30-35 ℃ for 50-60 minutes to prepare suspension, performing rotary evaporation and freeze drying to obtain nano dendrobium leaf polysaccharide, and applying the nano dendrobium leaf polysaccharide to the step (2); the feeding ratio of the absolute ethyl alcohol to the dendrobium officinale leaf polysaccharide is 160-200 ml: 8-10 g.

Preferably, the step (1) is specifically performed as follows:

(1-1) taking a proper amount of dendrobium officinale leaves, drying, crushing, sieving with a 20-40-mesh sieve, soaking with 85-90 vol% ethanol for 8-12h, repeating for 3-4 times, and drying in a fume hood;

(1-2) adding pure water with the volume 10-20 times of the mass of the dried powder in the step (1), decocting at 70-80 ℃ for 2-3 times, each time for 2-3h, merging supernate to obtain dendrobium polysaccharide water extract, removing protein by a sevage method, precipitating with 75-80 vol% alcohol, and centrifuging to obtain precipitate;

(1-3) redissolving the precipitate obtained in the step (2) with distilled water of which the volume is 10-20 times that of the precipitate, loading the redissolved precipitate onto a DEAE-cellulose column, eluting with distilled water at a flow rate of 4.0-5.0 ml/min, eluting with 0.5-1M sodium chloride solution at a flow rate of 4.0-5.0 ml/min to obtain a polysaccharide solution, and lyophilizing to obtain polysaccharide powder;

(1-4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain 2-5g/L polysaccharide solution, performing ultrafiltration by using an ultrafiltration cup to obtain polysaccharide solution with the molecular weight of 50-100kDa, dialyzing the obtained polysaccharide solution by using a dialysis bag with the molecular weight of 3.5-5kDa for 16-24 hours, and freeze-drying to obtain purified dendrobium officinale leaf polysaccharide with the molecular weight of 50-100 kDa.

In the step (1-2), the removal of protein by the sevage method can be performed according to conventional procedures, such as: adding mixed solution of chloroform and n-butanol (preferably volume ratio of chloroform to n-butanol is 4-5:1) into herba Dendrobii polysaccharide water extract, shaking thoroughly, retaining water phase, repeating for 3-5 times, and retaining water phase.

As a further preferred, in the step (1-4), ultrafiltration is carried out at 0.15-0.2 MPa.

The dendrobium officinale leaf microecological preparation prepared by the invention can be used for regulating diabetic colon flora and treating intestinal dysfunction diseases caused by intestinal flora imbalance.

The advantages and the beneficial effects of the invention are as follows:

(1) in the preparation process, the dendrobe leaf polysaccharide with high hypoglycemic activity is screened out, and the dendrobe leaf polysaccharide is prepared into the nano dendrobe leaf polysaccharide, so that the conditions that the polysaccharide in a common preparation is incompletely released and cannot be effectively absorbed are improved, and the preparation method is high in safety and good in effect.

Specifically, not all polysaccharides in the dendrobium leaves have the effect of reducing blood sugar, the required polysaccharide mixture (free of flavone, polyphenol, protein and the like) is extracted to a greater extent under the condition of ensuring the activity by a proper extraction mode, the section where the target polysaccharide is located is separated (50-100kDa, the butyrate production in the intestinal tract of a diabetic mouse can be increased by more than 50%, the ratio of bacteroides/firmicutes is obviously reduced), and the effect of the prepared product is improved.

In comparison, the content of pigment in dendrobium officinale leaves is high, the polysaccharide in the leaves has a structure lower than that in dendrobium stems but more varieties, and the polysaccharide extraction mode adopted by the invention is changed on the basis of the existing commonly used extraction mode of stem polysaccharide so as to obtain a polysaccharide mixture with the required effect and remove the carbohydrate with small molecular weight.

In addition, the invention prepares the dendrobium leaf polysaccharide into the nano dendrobium leaf polysaccharide by a wet grinding method under the conditions of not damaging the structure of an active fragment of the polysaccharide and not generating reducing sugar and other substances for increasing blood sugar, so that the dendrobium leaf polysaccharide can be uniformly distributed on other adhesive materials and can be more easily utilized by flora in colon, and the problems that the dendrobium leaf polysaccharide powder is easy to agglomerate and absorb moisture, is difficult to redissolve after drying and is difficult to be uniformly mixed with other materials in the preparation process are solved. In addition, the polysaccharide in the common polysaccharide preparation is incompletely released (usually, the polysaccharide is easy to agglomerate and influence the effect), and the nano polysaccharide prepared by the method can improve the release rate and greatly improve the utilization rate of a human body on the nano polysaccharide.

(2) The invention mixes the dendrobium leaf polysaccharide with high hypoglycemic activity and the colon adhesion material, so that the medicine is released and administered at the colon, and the action time of the polysaccharide in the colon is prolonged, thereby achieving the purposes of regulating the colon flora of diabetes and treating intestinal dysfunction diseases caused by imbalance of intestinal flora.

Specifically, the present invention incorporates a indigestible polysaccharide powder with a digestion resistant starch and a carbomer. The carbomer is a high molecular polymer of acrylic acid bonded allyl sucrose or pentaerythritol allyl ether, has excellent bioadhesion, but has high viscosity after being dissolved in water. When the three are dissolved in water, the volume of the nano-leaf polysaccharide is small (about 1: 10-100) relative to the volume of the material, and the polysaccharide can be fully adhered to the surface of the nano-leaf polysaccharide. The obtained composite system can prevent partial structure of dendrobium leaf polysaccharide from being partially decomposed by alpha-amylase and alpha-glucosidase in small intestine, thereby preventing small molecular saccharide substances from generating adverse effect on blood sugar of diabetic patients. The adhesive material is adhered to the colon mucosa wall after being fully swelled to release the medicine, so that the colon is positioned and adhered, and the dendrobium leaf polysaccharide is continuously released to act on the intestinal tract.

(IV) description of the drawings

FIG. 1 is a flow chart of the production process of the microecologics of the present invention.

(V) detailed description of the preferred embodiment

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1:

(1) taking a proper amount of dendrobium officinale leaves (purchased from cooperative society for planting Dendrobii caulis in Tengling of Leqing), drying, crushing, sieving with a 20-mesh sieve, soaking in 85 vol% ethanol for 8h, repeating for three times, and drying in a fume hood;

(2) adding pure water with the volume 15 times of the mass of the powder after drying in the step (1), decocting for 3 times at 70 ℃ for 3 hours each time, combining supernate to obtain dendrobe polysaccharide extract, adding Sevage reagent (chloroform: n-butyl alcohol is 4:1(V/V)) with the volume of 1/4 solution into the polysaccharide solution, fully shaking, retaining a water phase, repeating for 5 times, precipitating with 80% alcohol, and centrifuging to obtain a precipitate;

(3) redissolving the precipitate obtained in step (2) with 10 times volume of distilled water, loading the redissolved precipitate onto a DEAE-cellulose column, eluting with 500 ml of distilled water at a flow rate of 4.0 ml/min, eluting with 500 ml of 0.5M sodium chloride solution at a flow rate of 4.0 ml/min to obtain a polysaccharide solution, and lyophilizing the polysaccharide solution into powder;

(4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain 3g/L polysaccharide solution, then performing ultrafiltration by using an ultrafiltration cup under 0.15-0.2MPa by using a membrane with MWCO of 50kDa and 100kDa, collecting to obtain polysaccharide solution with the molecular weight of 50-100kDa, dialyzing (3.5kDa cut-off membrane) after rotary evaporation for 24 hours, and freeze-drying to obtain purified dendrobium leaf polysaccharide;

(5) sequentially adding 200ml of absolute ethyl alcohol and 10g of dendrobe leaf polysaccharide obtained in the subsequent step (4) by adopting a wet grinding method, adding agate balls, sealing and wet grinding to prepare suspension, performing rotary evaporation and freeze drying at the temperature of 35 ℃ for 50 minutes to prepare nano dendrobe leaf polysaccharide;

(6) and (3) uniformly mixing 0.3g of the nano dendrobe leaf polysaccharide obtained in the step (5) with 0.02g of digestion resistant starch (Koch Biotech, Shanghai) Co., Ltd.) and 0.05g of carbomer (purchased from Bailijia science, Ltd., Guangzhou) in water, and performing spray drying to obtain the dendrobe leaf polysaccharide microecological regulator.

Example 2:

(1) taking appropriate amount of Dendrobium officinale leaf (purchased from cooperative society of Dendrobii planting in Leqing Yeteng), oven drying, pulverizing, sieving with 20 mesh sieve, soaking in 85% ethanol for 12 hr, repeating for 4 times, and drying in a fume hood;

(2) adding pure water with the volume 15 times of the mass of the powder after drying in the step (1), decocting for 2 times at 80 ℃ for 3 hours each time, merging supernate to obtain dendrobe polysaccharide water extract, adding Sevage reagent (chloroform: n-butyl alcohol is 5:1(V/V)) with the volume of 1/5 of polysaccharide solution, fully shaking, retaining a water phase, repeating for 3 times, precipitating with 75% alcohol, and centrifuging to obtain a precipitate;

(3) redissolving the precipitate obtained in the step (2) with distilled water of which the volume is 10 times that of the precipitate, loading the redissolved precipitate onto a DEAE-cellulose column, eluting with 500 ml of distilled water at the flow rate of 5.0 ml/min, eluting with 500 ml of 0.5M sodium chloride solution at the flow rate of 4.0 ml/min to obtain a polysaccharide solution, and freeze-drying the polysaccharide solution into powder;

(4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain a polysaccharide solution of 3g/L, performing ultrafiltration by using an ultrafiltration cup under 0.15-0.2MPa by using a membrane with MWCO of 50kDa and 100kDa, collecting a trapped fluid of a 100kDa permeate passing through the 50kDa membrane to obtain a polysaccharide solution with the molecular weight of 50-100kDa, dialyzing the obtained polysaccharide solution after rotary evaporation (the trapped membrane of 3.5 kDa) for 24 hours, and freeze-drying to obtain purified dendrobium leaf polysaccharide;

(5) sequentially adding 160ml of absolute ethyl alcohol and 8g of the dendrobium leaf polysaccharide obtained in the subsequent step (4) by adopting a wet grinding method, adding agate balls, sealing and wet grinding to prepare suspension, performing rotary evaporation and freeze drying at the temperature of 35 ℃ for 50 minutes to prepare nano dendrobium leaf polysaccharide;

(6) and (3) uniformly mixing 0.4g of the nano dendrobe leaf polysaccharide obtained in the step (5) with 0.03g of the intestinal adhesion material, namely, digestive starch (Koch Biotech, Ltd.) and 0.06g of carbomer (purchased from Bailijia science, Ltd., Guangzhou) in water, and performing spray drying to obtain the dendrobe leaf polysaccharide microecological regulator.

Comparative example 1: polysaccharide molecular weight <50kDa

(1) Taking appropriate amount of Dendrobium officinale leaf (purchased from cooperative society of Dendrobii planting in Leqing Yeteng), oven drying, pulverizing, sieving with 20 mesh sieve, soaking in 85% ethanol for 12 hr, repeating for 4 times, and drying in a fume hood;

(2) adding pure water with the volume 15 times of the mass of the dried powder in the step (1), decocting for 2 times at 80 ℃ for 3 hours each time, mixing supernate to obtain dendrobe polysaccharide extract, adding 1/4 volumes of chloroform-n-butanol mixed solution (4:1) into the polysaccharide solution, sufficiently shaking, retaining a water phase, repeating for 3 times, precipitating with 75% alcohol, and centrifuging to obtain a precipitate;

(3) redissolving the precipitate obtained in the step (2) with distilled water of 10 times volume, loading the redissolved precipitate onto a DEAE-cellulose column, eluting with 500 ml of distilled water at a flow rate of 5.0 ml/min, eluting with 500 ml of 0.5M sodium chloride solution to obtain a polysaccharide solution, and freeze-drying the polysaccharide solution into powder;

(4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain a polysaccharide solution of 3g/L, performing ultrafiltration by using an ultrafiltration cup with an MWCO 50kDa membrane under 0.15-0.2MPa, collecting a permeate to obtain a polysaccharide solution with the molecular weight of less than 50kDa, dialyzing the obtained polysaccharide solution after rotary evaporation (3.5kDa cut-off membrane) for 24 hours, and freeze-drying to obtain purified dendrobium leaf polysaccharide;

(5) sequentially adding 160ml of absolute ethyl alcohol and 8g of the dendrobium leaf polysaccharide obtained in the subsequent step (4) by adopting a wet grinding method, adding agate balls, sealing and wet grinding to prepare suspension, performing rotary evaporation and freeze drying at the temperature of 35 ℃ for 50 minutes to prepare nano dendrobium leaf polysaccharide;

(6) and (3) uniformly mixing 0.4g of the nano dendrobe leaf polysaccharide obtained in the step (5) with 0.03g of digestive starch (Koch Biotech, Shanghai) limited) and 0.06g of carbomer (purchased from Bailijia science, Inc., Guangzhou) in water, and performing spray drying to obtain the dendrobe leaf polysaccharide microecological regulator.

Comparative example 2: polysaccharide molecular weight >100kDa

(1) Taking appropriate amount of Dendrobium officinale leaf (purchased from cooperative society of Dendrobii planting in Leqing Yeteng), oven drying, pulverizing, sieving with 20 mesh sieve, soaking in 85% ethanol for 12 hr, repeating for 4 times, and drying in a fume hood;

(2) adding pure water with the volume 15 times of the mass of the dried powder in the step (1), decocting for 2 times at 80 ℃ for 3 hours each time, mixing supernate to obtain dendrobe polysaccharide extract, adding 1/4 volumes of chloroform-n-butanol mixed solution (4:1) into the polysaccharide solution, sufficiently shaking, retaining a water phase, repeating for 3 times, precipitating with 75% alcohol, and centrifuging to obtain a precipitate;

(3) redissolving the precipitate obtained in step (2) with 10 times volume of distilled water, loading the redissolved precipitate onto a DEAE-cellulose column, eluting the redissolved precipitate with 500 ml of distilled water at a flow rate of 5.0 ml/min, eluting the redissolved precipitate with 500 ml of 0.5M sodium chloride solution to obtain a polysaccharide solution, and freeze-drying the polysaccharide solution into powder;

(4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain a polysaccharide solution of 3g/L, performing ultrafiltration by using an ultrafiltration cup under 0.15-0.2MPa by using a membrane with MWCO of 100kDa, collecting trapped fluid to obtain a polysaccharide solution with the molecular weight of more than 100kDa, dialyzing the obtained polysaccharide solution (the trapped membrane with the molecular weight of 3.5 kDa) after rotary evaporation for 24 hours, and freeze-drying to obtain purified dendrobium leaf polysaccharide;

(5) sequentially adding 160ml of absolute ethyl alcohol and 8g of the dendrobium leaf polysaccharide obtained in the subsequent step (4) by adopting a wet grinding method, adding agate balls, sealing and wet grinding to prepare suspension, performing rotary evaporation and freeze drying at the temperature of 35 ℃ for 50 minutes to prepare nano dendrobium leaf polysaccharide;

(6) and (3) uniformly mixing 0.4g of the nano dendrobe leaf polysaccharide in the step (5) with 0.03g of the intestinal adhesion material, namely digestive starch (Koch Biotech, Shanghai) Co., Ltd.) and 0.06g of carbomer (purchased from Bailijia science, Ltd., Guangzhou) in water, and performing spray drying to obtain the dendrobe leaf polysaccharide microecological regulator.

Comparative example 3: direct feeding of 50-100kDa polysaccharide

(1) Taking appropriate amount of Dendrobium officinale leaf (purchased from cooperative society of Dendrobii planting in Leqing Yeteng), oven drying, pulverizing, sieving with 20 mesh sieve, soaking in 85% ethanol for 12 hr, repeating for 4 times, and drying in a fume hood;

(2) adding pure water with the volume 15 times of the mass of the dried powder in the step (1), decocting for 2 times at 80 ℃ for 3 hours each time, mixing supernate to obtain dendrobe polysaccharide extract, adding 1/4 volumes of chloroform-n-butanol mixed solution (4:1) into the polysaccharide solution, sufficiently shaking, retaining a water phase, repeating for 3 times, precipitating with 75% alcohol, and centrifuging to obtain a precipitate;

(3) redissolving the precipitate obtained in step (2) with 10 times volume of distilled water, loading the redissolved precipitate onto a DEAE-cellulose column, eluting the redissolved precipitate with 500 ml of distilled water at a flow rate of 5.0 ml/min, eluting the redissolved precipitate with 500 ml of 0.5M sodium chloride solution to obtain a polysaccharide solution, and freeze-drying the polysaccharide solution into powder;

(4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain a polysaccharide solution of 3g/L, performing ultrafiltration by using an ultrafiltration cup under 0.15-0.2MPa by using a membrane with MWCO of 50kDa and 100kDa, collecting a retentate of a 100kDa permeate passing through the 50kDa membrane to obtain a polysaccharide solution with the molecular weight of 50-100kDa, dialyzing the obtained polysaccharide solution after rotary evaporation (the 3.5kDa retentate membrane) for 24 hours, and freeze-drying to obtain the purified dendrobium leaf polysaccharide.

Comparative example 4: 50-100kDa polysaccharide non-wet milled

(1) Taking appropriate amount of Dendrobium officinale leaf (purchased from cooperative society of Dendrobii planting in Leqing Yeteng), oven drying, pulverizing, sieving with 20 mesh sieve, soaking in 85% ethanol for 12 hr, repeating for 4 times, and drying in a fume hood;

(2) adding pure water with the volume 15 times of the mass of the dried powder in the step (1), decocting for 2 times at 80 ℃ for 3 hours each time, mixing supernate to obtain dendrobe polysaccharide extract, adding 1/4 volumes of chloroform-n-butanol mixed solution (4:1) into the polysaccharide solution, sufficiently shaking, retaining a water phase, repeating for 3 times, precipitating with 75% alcohol, and centrifuging to obtain a precipitate;

(3) redissolving the precipitate obtained in the step (2) with distilled water of 10 times volume, loading the redissolved precipitate onto a DEAE-cellulose column, eluting with 500 ml of distilled water at a flow rate of 5.0 ml/min, eluting with 500 ml of 0.5M sodium chloride solution to obtain a polysaccharide solution, and freeze-drying the polysaccharide solution into powder;

(4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain 3g/L polysaccharide solution, performing ultrafiltration by using an ultrafiltration cup under 0.15-0.2MPa by using a membrane with MWCO of 50kDa and 100kDa, collecting retentate of 100kDa permeate passing through the 50kDa membrane to obtain polysaccharide solution with molecular weight of 50-100kDa, dialyzing the obtained polysaccharide solution (3.5kDa retention membrane) after rotary evaporation for 24 hours, and freeze-drying to obtain purified dendrobium leaf polysaccharide;

(5) and (3) uniformly mixing 0.4g of the dendrobe leaf polysaccharide obtained in the step (4) with 0.03g of digestion resistant starch (Koch Biotech, Ltd.) and 0.06g of carbomer (purchased from Bailijia science, Ltd., Guangzhou) in water, and performing spray drying to obtain the dendrobe leaf polysaccharide microecological regulator.

Comparative example 5: different colon adhesion materials

(1) Taking appropriate amount of Dendrobium officinale leaf (purchased from cooperative society of Dendrobii planting in Leqing Yeteng), oven drying, pulverizing, sieving with 20 mesh sieve, soaking in 85% ethanol for 12 hr, repeating for 4 times, and drying in a fume hood;

(2) adding pure water with the volume 15 times of the mass of the dried powder in the step (1), decocting for 2 times at 80 ℃ for 3 hours each time, mixing supernate to obtain dendrobe polysaccharide extract, adding 1/4 volumes of chloroform-n-butanol mixed solution (4:1) into the polysaccharide solution, sufficiently shaking, retaining a water phase, repeating for 3 times, precipitating with 75% alcohol, and centrifuging to obtain a precipitate;

(3) redissolving the precipitate obtained in the step (2) by using distilled water with the volume being 10 times that of the precipitate, loading the redissolved precipitate onto a DEAE-cellulose column, eluting the DEAE-cellulose column by using 500 ml of distilled water at the flow rate of 4.0 ml/min, eluting the DEAE-cellulose column by using 500 ml of 0.5M sodium chloride solution to obtain a polysaccharide solution, and freeze-drying the polysaccharide solution into powder;

(4) dissolving the polysaccharide obtained in the step (3) in pure water to obtain 3g/L polysaccharide solution, performing ultrafiltration by using an ultrafiltration cup under 0.15-0.2MPa by using a membrane with MWCO of 50kDa and 100kDa, collecting retentate of 100kDa permeate passing through the 50kDa membrane to obtain polysaccharide solution with molecular weight of 50-100kDa, dialyzing the obtained polysaccharide solution (3.5kDa retention membrane) after rotary evaporation for 24 hours, and freeze-drying to obtain purified dendrobium leaf polysaccharide;

(5) sequentially adding 160ml of absolute ethyl alcohol and 8g of the dendrobium leaf polysaccharide obtained in the subsequent step (4) by adopting a wet grinding method, adding agate balls, sealing and wet grinding to prepare suspension, performing rotary evaporation and freeze drying at the temperature of 35 ℃ for 50 minutes to prepare nano dendrobium leaf polysaccharide;

(6) and (3) uniformly mixing 0.4g of the nano dendrobe leaf polysaccharide obtained in the step (5) and acrylic resin (purchased from Bailijia science and technology limited company in Guangzhou), and performing spray drying to obtain the dendrobe leaf polysaccharide microecological regulator.

The finished products obtained in the above examples and comparative examples were subjected to a performance test:

test 1: the blood sugar lowering effect of the finished products prepared in the above examples and comparative examples was measured by the following method:

model group: injecting streptozotocin into the abdominal cavity of SPF grade C57 mouse fed with high fat for molding to make its blood sugar higher than 11mmol/l after fasting for 12h, and measuring fasting blood sugar on 0, 10, 20 and 30 days after molding is successful.

Normal control group: SPF grade C57 mice were taken and fasting blood glucose was measured at 0, 10, 20, 30.

Targeted formulation group: once daily administration of 200mg/kg body weight was performed, fasting blood glucose was measured at 0, 10, 20, 30 th day.

The results are shown in Table 1.

TABLE 1 Effect on fasting plasma glucose in animals

And (3) testing 2: the regulation of the metabolites of intestinal flora of animals with dysbacteriosis by the products prepared in the above examples and comparative examples was determined by the following method:

(1) the model and administration were performed in the same manner as in test 1, and 0.2g of feces per mouse were aseptically collected 28 days after administration to the mice and stored at-80 ℃.

(2) A 0.1g fecal sample was suspended in 1mL PBS solution (pH 7.6, 0.01M).

(3) After complete homogenization centrifugation for 10min, the supernatant was collected and passed through a 0.22 μm aqueous membrane.

(4) The liquid after passing through the membrane was detected by a gas chromatograph, and the amounts of acetic acid, propionic acid and n-butyric acid in feces were determined by calculating the peak area as mmol/mL. The results are shown in Table 2.

TABLE 2 Effect on short-chain fatty acid production in animals with dysbacteriosis

And (3) testing: the regulation effect of the products prepared in the above examples and comparative examples on the intestinal flora of dysbacteriosis animals is determined by the following method:

(1) the model and administration method were the same as in test 1, and after the mice were sacrificed 30 days after administration, 0.05-0.10g of colon contents of the mice were aseptically collected and stored at-80 ℃.

(2) Each colon content sample was extracted using a rapid DNA stool mini kit and the extracted genomic DNA was detected using 1% agarose gel electrophoresis.

(3) The V4 hypervariable region of the bacterial 16S rRNA gene was amplified by PCR using primers 341F (5 '-cctayggrbgcacag-3') and 806R (5 '-GGACTACNNGGGTATCTAAT-3').

(4) After purification with a DNA purification kit, PCR products were quantified by fluorescence.

(5) Statistical analysis of community structure was performed at each classification level using Illumina PE250 sequencing.

(6) The ratio of bacteroides and firmicutes to the total flora was calculated on the basis of the colony composition at the phylum level, expressed as a percentage. The results are shown in Table 3.

TABLE 3 Regulation of intestinal flora in dysbacteriosis animals