Probiotic preparation for improving constipation and preparation method thereof

Abstract

The invention provides a probiotic preparation for improving constipation and a preparation method thereof, and belongs to the technical field of probiotics. Aspergillus niger is fermented to prepare zymophyte enzyme liquid, the zymophyte enzyme liquid is used for enzyme-assisted fermentation of smallanthus sonchifolius and aronia melanocarpa, filtering is conducted, a compound fruit enzyme-assisted fermentation product is prepared, and filter residues are reserved for use; hawthorn, dendrobium officinale and semen cassiae are subjected to water extraction and filtration, a water extract is prepared, and filter residues are reserved for use; the preparation method comprises the following steps: mixing the filter residues with aloe gel liquid, inoculating probiotics for fermentation, adding prebiotics to prepare synbiotics, and mixing and embedding a compound fruit enzyme co-fermentation product, a water extract and the synbiotics to prepare the probiotic preparation for improving constipation, so that the problems of gastric juice erosion and inactivation of the probiotics in adverse environments such as oxygen and high temperature can be obviously solved; the compound fertilizer contains rich active components, and can promote colonization and growth of intestinal probiotics, increase the water absorption expansion rate of food, increase the water content of excrement, promote intestinal flora balance, relax bowel and improve constipation symptoms.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and in particular to a probiotic preparation for improving constipation and a preparation method thereof. Background Art

[0002] With the change of dietary structure and the increase of mental and social pressure, constipation has become one of the problems that plague modern people, seriously affecting people's quality of life. People with constipation are prone to intestinal flora disorders, and long-term constipation can also induce various diseases. Studies have shown that various traditional drugs for treating constipation have many problems in clinical research and application. Studies have also shown that the probability of constipation patients also having colon motility disorders is very high, so it can be said that colon motility disorders are the main cause of constipation. The most common manifestation of colon motility disorders is slow colon emptying, and weakened colon motility is the main cause of this slow colon emptying. Because the intestinal contents stay in the colon for too long, water is overabsorbed, and the stool is dry and hard, which aggravates the difficulty of defecation. It is precisely because of the high incidence, wide range of causes and many hazards of constipation that this disease has received more and more widespread attention.

[0003] For the treatment of constipation, those with mild symptoms can eat more foods rich in dietary fiber, correct their bad eating or resting habits, and increase their exercise; those with severe symptoms can use osmotic or irritant laxatives, such as polyethylene glycol, lactulose or anthraquinone derivatives, but these laxatives often cause dependence and even side effects such as nausea, abdominal pain, and diarrhea. At present, a treatment method with few side effects and good effects is to take probiotic-related preparations. Therefore, the method of using probiotics to relieve constipation needs to be studied and thought about more deeply.

[0004] Patent CN1314344C discloses a kind of Coptis chinensis flower or / and leaf tea, which is composed of: 1000 parts of dried Coptis chinensis flower or / and Coptis chinensis leaf, 0.1-100 parts of sweetener (sucralose, AK sugar, stevia sugar, sweet tea, etc.), 0.1-200 parts of spices (rose, chrysanthemum, etc.), and the remainder of flavoring agent. First, the Coptis chinensis flower or / and Coptis chinensis leaf are picked and then killed; the sweetener is prepared into an aqueous solution, and the Coptis chinensis flower or / and Coptis chinensis leaf are soaked in the sweetener aqueous solution, or the sweetener aqueous solution is evenly sprayed on the Coptis chinensis flower or / and Coptis chinensis leaf, and then dried; finally, the spices are added to the Coptis chinensis flower or / and Coptis chinensis leaf, and then packaged; in this patent, since the Coptis chinensis flower itself tastes bitter, sweeteners, flavoring agents, spices, etc. need to be added during the preparation process, which increases the cost and is not good for human health.

[0005] Patent CN105394773B discloses a composite probiotic and dietary fiber preparation for improving constipation, which is composed of the following components: Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, apple or jujube dietary fiber, wherein the ratio of the number of live bacteria of Bifidobacterium breve, Lactobacillus acidophilus, and Lactobacillus plantarum is: 1-3 to 1-4 to 1-5, and the total number of live bacteria of the preparation is 120-400 billion / g, and the preparation contains 0.1-0.3g / g of apple or jujube dietary fiber. Although the preparation has a reasonable structure, including dietary fiber and probiotics, after the preparation is taken orally, the probiotics will be inactivated in large quantities when entering the gastric juice and cannot enter the intestine smoothly, which greatly affects the therapeutic effect of the probiotics. Therefore, the improvement effect of the preparation is not good. Summary of the invention

[0006] The purpose of the present invention is to provide a probiotic preparation for improving constipation and a preparation method thereof, which can significantly improve the probiotics' resistance to gastric juice erosion and inactivation in adverse environments such as oxygen and high temperature, contains rich active ingredients, can promote the colonization and growth of intestinal probiotics, increase the expansion rate of food water absorption, increase the water content of feces, promote the balance of intestinal flora, moisturize the intestine and relieve constipation, and improve constipation symptoms.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The invention provides a preparation method of a probiotic preparation for improving constipation. The method comprises the following steps: fermenting Aspergillus niger to obtain a fermentation bacterial enzyme liquid, applying the fermentation liquid to yacon and arbutus nigra, filtering to obtain a composite fruit enzyme-assisted fermentation product, and retaining the filter residue; extracting hawthorn, dendrobium officinale and cassia seed with water, filtering to obtain a water extract, and retaining the filter residue; mixing the filter residue with aloe vera gel, inoculating probiotics for fermentation, adding prebiotics to obtain synbiotics, and mixing and embedding the composite fruit enzyme-assisted fermentation product, the water extract and the synbiotics to obtain the probiotic preparation for improving constipation.

[0009] As a further improvement of the present invention, the following steps are included:

[0010] S1. Preparation of Aspergillus niger fermentation enzyme liquid: bran, malt extract, sucrose, inorganic salts, peptone and water are stirred and mixed uniformly, sterilized, inoculated with Aspergillus niger spore suspension, fermented, the product is filtered, and Aspergillus niger is washed with sterile water to obtain a fermentation enzyme liquid;

[0011] S2. Enzyme-assisted fermentation of composite fruit: Wash the yacon and black-fruited glandular ribs Aronia ovata, crush, homogenize, add the fermentation enzyme solution prepared in step S1, enzyme-assisted fermentation, filter, dry the solid residue and retain it, sterilize and kill the enzyme in the filtrate to obtain a composite fruit enzyme-assisted fermentation product;

[0012] S3. Water extraction of edible and medicinal components: Wash hawthorn, Dendrobium officinale, and Cassia seed separately, dry, crush, mix, and prepare a combined powder, add water, heat to boiling and extract, repeat the operation 2-3 times, filter, combine the filtrate, dry the solid residue and retain it, and dry the filtrate to obtain a water extract;

[0013] S4. Preparation of aloe vera gel: Peel the aloe vera and take the aloe vera gel, add water and homogenize to obtain aloe vera gel;

[0014] S5. Preparation of bacterial seed solution: Bifidobacterium lactis and Lactobacillus reuteri were inoculated into Gao's medium, activated and cultured to obtain bacterial seed solution;

[0015] S6. Preparation of synbiotics: the solid residue in step S2, the solid residue in step S3, the aloe vera gel solution obtained in step S4 and water are mixed evenly, sterilized, inoculated with the Bifidobacterium lactis and Lactobacillus reuteri seed solution obtained in step S5, fermented and cultured for a first period of time, added with prebiotics, fermented and cultured for a second period of time, filtered, washed with sterile water to remove the probiotics, and the obtained fermentation product is freeze-dried to obtain synbiotics;

[0016] S7. Preparation of a probiotic preparation for improving constipation: add the complex fruit enzyme-assisted fermentation product prepared in step S2, the water extract prepared in step S3, the synbiotics prepared in step S6, inulin, konjac glucomannan, sodium alginate, lecithin and soy protein isolate into water, stir and homogenize to obtain a homogenous liquid, add into fish oil, perform rapid membrane emulsification, add calcium chloride solution dropwise, solidify at room temperature, centrifuge, wash and dry to obtain a probiotic preparation for improving constipation.

[0017] As a further improvement of the present invention, the mass ratio of bran, malt juice, sucrose, inorganic salt, peptone and water in step S1 is 10-12:20-40:3-5:2-4:5-7:400-500, the inorganic salt includes potassium dihydrogen phosphate, magnesium sulfate, sodium nitrate, iron sulfate, manganese sulfate, and calcium chloride, and the mass ratio is 2-3:2-4:15-20:0.05-0.1:0.1-0.2:1-2, the sugar content of the malt juice is 5-7 degrees, and the concentration of the Aspergillus niger spore suspension is 10 8 -10 9 cfu / mL, the inoculation amount of the Aspergillus niger spore suspension is 2-3v / v%, the fermentation conditions are 30-32°C, 100-150r / min, the fermentation culture is 48-56h, and the sterile water used for washing is 1-2 times the volume of the filtrate.

[0018] As a further improvement of the present invention, the mass ratio of yacon, Aronia melanocarpa and fermentation enzyme solution in step S2 is 5-7:10-12:100, and the conditions of enzyme-assisted fermentation are 35-40°C, 100-150r / min, and the enzyme-assisted fermentation culture is 36-48h.

[0019] As a further improvement of the present invention, the mass ratio of hawthorn, dendrobium officinale and cassia seed in step S3 is 3-5:7-10:2-4, the solid-liquid ratio of the combined powder and water is 1:5-7 g / mL, and the heating to boiling extraction time is 1-3 h.

[0020] As a further improvement of the present invention, the mass ratio of aloe vera gel to water in step S4 is 3-5:10; the conditions for activation culture in step S5 are 37-39°C, 70-100r / min, activation culture 28-30h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL.

[0021] As a further improvement of the present invention, the mass ratio of the solid residue in step S2, the solid residue in step S3, the aloe gel solution and the water in step S6 is 7-10:10-15:30-50:100, the inoculation amounts of the Bifidobacterium lactis and the Lactobacillus reuteri species seed solutions are 2-4v / v% and 1-3v / v%, respectively, the fermentation culture conditions are 37-39°C, 70-100r / min, the first time period is 36-48h, the second time period is 24-36h, the prebiotics include konjac glucomannan and arabinose, the mass ratio is 10-15:2-4, and the amount of the prebiotics added is 7-10wt% of the total mass of the system.

[0022] As a further improvement of the present invention, the mass ratio of the complex fruit enzyme-assisted fermentation product, the water extract, the synbiotic, the inulin, the sodium alginate, the lecithin and the soy protein isolate in step S7 is 10-15:7-10:25-30:5-7:22-25:1-2:12-15, the mass ratio of the homogenized liquid and the fish oil is 3-5:7-10, the membrane pore size of the rapid membrane emulsification is 100-200 μm, the concentration of the calcium chloride solution is 5-7wt%, and the time for room temperature curing is 20-30 min.

[0023] The present invention further protects a probiotic preparation for improving constipation prepared by the above preparation method.

[0024] The present invention further protects the use of the above-mentioned probiotic preparation for improving constipation in the preparation of food and medicine for treating and improving symptoms of acute or chronic constipation.

[0025] The present invention has the following beneficial effects:

[0026] Plant cell walls are mainly composed of cellulose, xylan and pectin, which protect cells from harm together, and also hinder the digestion and utilization of nutrition by the human body. Usually, the joint action of cellulase, xylanase and pectinase can degrade plant cell walls into monosaccharides or oligosaccharides, eliminate anti-nutritional factors, promote the release of intracellular nutrition and active substances, and at the same time, convert plant cell walls into sugar compounds that are easy to digest and absorb, and promote the absorption, utilization, reproduction and growth of human intestinal probiotics. The present invention cultivates and ferments Aspergillus niger in a culture medium containing rich nutrients, and can produce a variety of biological enzymes, including a variety of complex and abundant biologically active enzymes such as cellulase, xylanase, pectinase, endoglucanase, etc., so that the obtained fermentation bacteria enzyme liquid contains abundant biologically active enzymes and Aspergillus niger fermentation bacteria.

[0027] Yacon contains a lot of water and sugar, mainly oligofructose, and contains phenolic acid, flavonoids, mushrooms, volatile oils, amino acids, and potassium, calcium, zinc, iron, magnesium and other elements. Because it is rich in water-soluble dietary fiber and oligofructose, the sugar cannot be metabolized in the human body and directly enters the large intestine. It is preferentially used by the human intestinal probiotics bifidobacteria, thereby promoting digestion, regulating the gastrointestinal tract, promoting the effective proliferation of beneficial bacteria in the intestine, producing bifidobacteria factors, clearing hyperlipidemia, controlling cholesterol and diabetes, relieving constipation, preventing and treating diarrhea, reducing fire and detoxification, preventing and treating facial acne and acne, and improving immunity. Bifidobacteria ferment in the intestine to produce a large amount of short-chain fatty acids, stimulate intestinal peristalsis, increase fecal moisture and maintain a certain osmotic pressure, and prevent constipation. Black fruit glandular rib aronia is rich in polyphenols, anthocyanins, chlorogenic acid, quercetin, etc., and has high antioxidant properties. Among them, anthocyanins can improve the composition of intestinal microorganisms, polyphenols can be decomposed into metabolites with biological activity, regulate the composition and activity of intestinal flora, improve intestinal flora imbalance, increase the content of beneficial flora, reduce the number of harmful flora, and can also improve gastrointestinal health by inhibiting inflammatory factors. Therefore, the composite fruit composed of the two components added in the present invention can improve the intestinal flora environment, promote the growth of beneficial intestinal bacteria, improve antioxidant and anti-inflammatory effects, stimulate intestinal peristalsis, and increase fecal moisture, thereby improving and preventing the formation of constipation.

[0028] The invention inoculates the prepared fermentation bacteria enzyme liquid into a homogenate of a composite fruit (including yacon and aronia nigra), which can effectively promote the rupture of plant cell walls, accelerate the dissolution of active ingredients, reduce the content of anti-nutritional factors, promote the degradation of complex polysaccharides into small molecular monosaccharides and oligosaccharides, and greatly improve the conversion of the components contained therein into effective active components, thereby obtaining a composite fruit enzyme-assisted fermentation product with high anti-constipation activity.

[0029] Hawthorn, Dendrobium officinale and Cassia seed are traditional Chinese medicines that are both edible and medicinal, and have excellent effects of clearing heat, reducing internal heat, strengthening the spleen, and warming the body. The rich dietary fiber they contain can promote the colonization and growth of intestinal probiotics, increase the water content of feces by increasing the expansion rate of food water absorption, thereby balancing the intestinal flora, moisturizing the intestines and promoting bowel movements, and improving constipation symptoms.

[0030] Aloe vera is rich in aloe gel, which includes aloe polysaccharides, calcium isocitrate, superoxide dismutase, peroxidase, vitamin C, amino acids, vitamins, minerals, etc. It can promote intestinal peristalsis and has the effects of stomachic, anti-inflammatory, bactericidal, anti-allergic, and promoting blood circulation.

[0031] The probiotics added by the present invention include Bifidobacterium lactis and Lactobacillus reuteri, which can compete with pathogenic bacteria in the intestine to reduce the colonization of pathogenic bacteria and the inflammatory response caused by them, change the metabolic effect of bile salts and restore the normal fermentation of the intestine. Bifidobacterium lactis has a positive effect on promoting intestinal motility and can improve the viscosity of feces. Lactobacillus reuteri can produce a large number of metabolites, such as short-chain fatty acids, which lead to a decrease in intestinal pH, and the positive feedback regulation promotes the colonization of beneficial intestinal bacteria, inhibits the colonization of conditional pathogens and pathogens, and balances the structure of intestinal flora; short-chain fatty acids stimulate small intestinal contraction and peristalsis, and by combining with intestinal surface cell receptors, promote the increase of 5-hydroxytryptamine secretion in the intestinal system of the body, leading to enhanced intestinal water retention capacity, enhanced intestinal internal osmotic pressure, effectively reducing the residence time of feces in the small intestine, and relieving constipation symptoms; under the synergistic effect of the two, it can well alleviate the imbalance of intestinal flora, thereby improving and treating constipation problems.

[0032] The present invention also adds prebiotics during the fermentation process to form synbiotics in combination with probiotics. Prebiotics can effectively promote colon motility in patients with chronic constipation, improve stool hardness, defecation frequency, abdominal distension and other symptoms. Due to the characteristic that they cannot be absorbed by the intestines, their osmotic effect can also hinder the intestinal wall's reabsorption of water and electrolytes, thereby playing a cathartic role to a certain extent, thereby improving constipation symptoms. At the same time, prebiotics can also selectively promote the growth and colonization of intestinal probiotics, thereby regulating constipation symptoms by improving intestinal flora.

[0033] Among them, konjac mannan can slow down or prevent the digestion and absorption of fat and carbohydrates, increase satiety, and is a soluble and indigestible fiber that can promote the production of bifidobacteria and lactobacillus in rat feces. Arabinose can promote the growth of bifidobacteria, improve intestinal flora, and play a role in moisturizing the intestines and promoting bowel movements. Therefore, the synergistic effect of the combined prebiotics can significantly improve the symptoms of human constipation.

[0034] However, probiotics are easily affected by unfavorable factors such as oxygen, pH value, temperature, moisture, gastric acid and bile salts, which reduces the survival rate and vitality. In order to ensure that the probiotics can reach the intestine smoothly and the number is higher than the minimum viable bacteria count standard for exerting the probiotic function, the present invention uses microencapsulation technology to encapsulate the prepared product (including probiotics) to improve its resistance to harsh environment and its survival and colonization in the intestine.

[0035] In the wall material selected for embedding, sodium alginate has the effect of being easy to cross-link to form a shell layer, but it is not acid-alkali resistant and is easy to break in an environment with high humidity or low pH. Therefore, the present invention also adds inulin, which has gelling properties, solubility, low viscosity, and high glass transition temperature. It can form a stable gel network structure during the microencapsulation process. The core material and the wall material are closely combined to form a stable glass protective layer to reduce oxidative damage. In addition, soy protein isolate is added. Under the action of composite multi-crosslinking of multiple wall materials, the prepared microcapsules can be stably present in low or high pH and harsh environments to prevent gastric juice erosion and reduce the damage of the core material in unfavorable environments such as oxygen and high temperature. At the same time, inulin is also a good prebiotic. After being degraded in the intestine, it can be absorbed and utilized by probiotics to promote the rapid growth and colonization of probiotics.

[0036] The probiotic preparation for improving constipation prepared by the present invention can significantly improve the problem of probiotics being eroded by gastric juice and being inactivated in adverse environments such as oxygen and high temperature, contains rich active components, can promote the colonization and growth of intestinal probiotics, increase the expansion rate of food water absorption, increase the water content of feces, promote the balance of intestinal flora, moisturize the intestine and promote bowel movements, and improve constipation symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0038] Figure 1 This is a comparison chart of the survival rates of each group in Test Example 1 of the present invention;

[0039] Figure 2 This is a comparison chart of the release rates of each group in Test Example 1 of the present invention. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Aspergillus niger was purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd.; Bifidobacterium lactis, 10 billion cfu / g, and Lactobacillus reuteri, 10 billion cfu / g, were purchased from Zhongke Jiayi Bioengineering Technology Co., Ltd.

[0042] Preparation of Aspergillus niger spore suspension: Inoculate Aspergillus niger into Goulburn medium and culture at 30°C, 100 r / min, for 48 h to obtain a concentration of 10 8 -10 9 cfu / mL Aspergillus niger spore suspension.

[0043] Example 1

[0044] This embodiment provides a method for preparing a probiotic preparation for improving constipation, comprising the following steps:

[0045] S1. Preparation of Aspergillus niger fermentation enzyme liquid: 10 parts by weight of bran, 20 parts by weight of malt juice with a sugar content of 5 degrees, 3 parts by weight of sucrose, 2 parts by weight of inorganic salts, 5 parts by weight of peptone and 400 parts by weight of water were stirred and mixed for 20 minutes, sterilized, and inoculated at a concentration of 10 9 cfu / mL Aspergillus niger spore suspension, inoculation amount of 2v / v%, 30℃, 100r / min, fermentation culture for 48h, filter the product, and wash the Aspergillus niger with 1 times the volume of the filtrate, to obtain a fermentation enzyme liquid;

[0046] The inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, sodium nitrate, iron sulfate, manganese sulfate, and calcium chloride in a mass ratio of 2:2:15:0.05:0.1:1;

[0047] S2. Enzyme-assisted fermentation of composite fruit: 5 parts by weight of yacon and 10 parts by weight of black fruit glandular ribs Aronia ovata were washed, crushed, homogenized at 10000r / min for 10min, 100 parts by weight of the fermentation enzyme solution prepared in step S1 were added, 35°C, 100r / min, enzyme-assisted fermentation was cultured for 36h, filtered, the solid residue was dried and retained, the filtrate was sterilized and enzyme-killed to obtain a composite fruit enzyme fermentation product;

[0048] S3. Water extraction of edible and medicinal components: 3 parts by weight of hawthorn, 7 parts by weight of Dendrobium officinale, and 2 parts by weight of Cassia seed were washed, dried, crushed, mixed, and the obtained combined powder was added to water, the solid-liquid ratio of the combined powder to water was 1:5 g / mL, heated to boiling and extracted for 1 h, the operation was repeated 2 times, filtered, the filtrate was combined, the solid residue was dried and retained, and the filtrate was dried to obtain a water extract;

[0049] S4. Preparation of aloe gel solution: Peel the aloe vera and take aloe gel, add 10 parts by weight of water to 3 parts by weight of aloe gel, homogenize at 10000r / min for 10min to obtain aloe gel solution;

[0050] S5. Preparation of bacterial seed solution: Bifidobacterium lactis and Lactobacillus reuteri were inoculated into Gao's medium respectively, and activated and cultured at 37°C, 70r / min for 28h to obtain a bacterial content of 10 8 cfu / mL of bacterial seed solution;

[0051] S6. Preparation of synbiotics: 7 parts by weight of the solid residue in step S2, 10 parts by weight of the solid residue in step S3, 30 parts by weight of the aloe vera gel obtained in step S4 and 100 parts by weight of water are mixed evenly, sterilized, and inoculated with the Bifidobacterium lactis and Lactobacillus reuteri species seed liquid obtained in step S5, wherein the inoculation amounts of the Bifidobacterium lactis and Lactobacillus reuteri species seed liquid are 2 v / v% and 1 v / v%, respectively, at 37° C., 70 r / min, and fermented for 36 h, and prebiotics are added in an amount of 7wt% of the total mass of the system, and the fermentation is continued for 24 h, filtered, and the probiotics are washed and dropped with sterile water, and the fermentation product obtained is freeze-dried to obtain synbiotics;

[0052] The prebiotics include konjac glucomannan and arabinose in a mass ratio of 10:2;

[0053] S7. Preparation of a probiotic preparation for improving constipation: add 10 parts by weight of the complex fruit enzyme-assisted fermentation product prepared in step S2, 7 parts by weight of the water extract prepared in step S3, 25 parts by weight of the synbiotic prepared in step S6, 5 parts by weight of inulin, 22 parts by weight of sodium alginate, 1 part by weight of lecithin and 12 parts by weight of soy protein isolate to 200 parts by weight of water, homogenize at 10000 r / min for 10 min to obtain a homogenous solution, add 30 parts by weight of the homogenous solution to 70 fish oil, perform rapid membrane emulsification, the membrane pore size is 100 μm, add 5wt% calcium chloride solution dropwise, solidify at room temperature for 20 min, centrifuge, wash, and dry to obtain a probiotic preparation for improving constipation.

[0054] Example 2

[0055] This embodiment provides a method for preparing a probiotic preparation for improving constipation, comprising the following steps:

[0056] S1. Preparation of Aspergillus niger fermentation enzyme liquid: 12 parts by weight of bran, 40 parts by weight of malt juice with a sugar content of 7 degrees, 5 parts by weight of sucrose, 4 parts by weight of inorganic salts, 7 parts by weight of peptone and 500 parts by weight of water were stirred and mixed for 20 minutes, sterilized, and inoculated at a concentration of 10 9 cfu / mL Aspergillus niger spore suspension, inoculation amount of 3v / v%, 32°C, 150r / min, fermentation culture for 56h, the product is filtered, and the Aspergillus niger is washed with sterile water of 2 times the volume of the filtrate to obtain a fermentation enzyme liquid;

[0057] The inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, sodium nitrate, iron sulfate, manganese sulfate, and calcium chloride in a mass ratio of 3:4:20:0.1:0.2:2;

[0058] S2. Enzyme-assisted fermentation of composite fruit: 7 parts by weight of yacon and 12 parts by weight of black fruit glandular ribs Aronia ovata were washed, crushed, homogenized at 10000r / min for 10min, 100 parts by weight of the fermentation enzyme solution prepared in step S1 were added, 40 ° C, 150r / min, enzyme-assisted fermentation was cultured for 48h, filtered, the solid residue was dried and retained, the filtrate was sterilized and enzyme-inactivated to obtain a composite fruit enzyme fermentation product;

[0059] S3. Water extraction of edible and medicinal components: 5 parts by weight of hawthorn, 10 parts by weight of Dendrobium officinale, and 4 parts by weight of Cassia seed were washed, dried, crushed, mixed, and the obtained combined powder was added to water, the solid-liquid ratio of the combined powder to water was 1:7 g / mL, heated to boiling and extracted for 3h, the operation was repeated 3 times, filtered, the filtrate was combined, the solid residue was dried and retained, and the filtrate was dried to obtain a water extract;

[0060] S4. Preparation of aloe gel solution: Peel the aloe vera and take aloe gel, add 10 parts by weight of water to 5 parts by weight of aloe gel, homogenize at 10000r / min for 10min to obtain aloe gel solution;

[0061] S5. Preparation of bacterial seed solution: Bifidobacterium lactis and Lactobacillus reuteri were inoculated into Gao's medium, respectively, at 39°C, 100 r / min, activated and cultured for 30 h to obtain a bacterial content of 10 9 cfu / mL of bacterial seed solution;

[0062] S6. Preparation of synbiotics: 10 parts by weight of the solid residue in step S2, 15 parts by weight of the solid residue in step S3, 50 parts by weight of the aloe vera gel obtained in step S4 and 100 parts by weight of water are mixed evenly, sterilized, and inoculated with the Bifidobacterium lactis and Lactobacillus reuteri species seed solution obtained in step S5, wherein the inoculation amounts of the Bifidobacterium lactis and Lactobacillus reuteri species seed solution are 4 v / v% and 3 v / v%, respectively, at 39° C., 100 r / min, and fermented for 48 h, and prebiotics are added in an amount of 10 wt% of the total mass of the system, and the fermentation is continued for 36 h, filtered, and the probiotics are washed with sterile water and dropped, and the fermentation product obtained is freeze-dried to obtain synbiotics;

[0063] The prebiotics include konjac glucomannan and arabinose in a mass ratio of 15:4;

[0064] S7. Preparation of a probiotic preparation for improving constipation: add 15 parts by weight of the complex fruit enzyme-assisted fermentation product prepared in step S2, 10 parts by weight of the water extract prepared in step S3, 30 parts by weight of the synbiotic prepared in step S6, 7 parts by weight of inulin, 25 parts by weight of sodium alginate, 2 parts by weight of lecithin and 15 parts by weight of soy protein isolate to 200 parts by weight of water, homogenize at 10000 r / min for 10 min to obtain a homogenous solution, add 50 parts by weight of the homogenous solution to 100 ml of fish oil, perform rapid membrane emulsification, the membrane pore size is 200 μm, add 7wt% calcium chloride solution dropwise, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain a probiotic preparation for improving constipation.

[0065] Example 3

[0066] This embodiment provides a method for preparing a probiotic preparation for improving constipation, comprising the following steps:

[0067] S1. Preparation of Aspergillus niger fermentation enzyme liquid: 11 parts by weight of bran, 30 parts by weight of malt juice with a sugar content of 6 degrees, 4 parts by weight of sucrose, 3 parts by weight of inorganic salts, 6 parts by weight of peptone and 450 parts by weight of water were stirred and mixed for 20 minutes, sterilized, and inoculated at a concentration of 10 9 cfu / mL Aspergillus niger spore suspension, inoculation amount of 2.5v / v%, 31°C, 120r / min, fermentation culture for 52h, product filtration, and Aspergillus niger washed with 1.5 times the volume of filtrate sterile water to obtain fermentation enzyme liquid;

[0068] The inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, sodium nitrate, iron sulfate, manganese sulfate, and calcium chloride in a mass ratio of 2.5:3:17:0.07:0.15:1.5;

[0069] S2. Enzyme-assisted fermentation of composite fruit: 6 parts by weight of yacon and 11 parts by weight of black fruit glandular ribs Aronia ovata were washed, crushed, homogenized at 10000r / min for 10min, 100 parts by weight of the fermentation enzyme solution prepared in step S1 were added, 37°C, 120r / min, enzyme-assisted fermentation was cultured for 42h, filtered, the solid residue was dried and retained, the filtrate was sterilized and enzyme-killed to obtain a composite fruit enzyme fermentation product;

[0070] S3. Water extraction of edible and medicinal components: 4 parts by weight of hawthorn, 8.5 parts by weight of Dendrobium officinale, and 3 parts by weight of Cassia seed were washed, dried, crushed, mixed, and the obtained combined powder was added to water, the solid-liquid ratio of the combined powder to water was 1:6 g / mL, heated to boiling and extracted for 2h, the operation was repeated 3 times, filtered, the filtrate was combined, the solid residue was dried and retained, and the filtrate was dried to obtain a water extract;

[0071] S4. Preparation of aloe gel solution: Peel the aloe vera and take aloe gel, add 10 parts by weight of water to 4 parts by weight of aloe gel, homogenize at 10000r / min for 10min to obtain aloe gel solution;

[0072] S5. Preparation of bacterial seed solution: Bifidobacterium lactis and Lactobacillus reuteri were inoculated into Gao's medium, respectively, at 38°C, 85r / min, activated and cultured for 29h to obtain a bacterial content of 10 9 cfu / mL of bacterial seed solution;

[0073] S6. Preparation of synbiotics: 8.5 parts by weight of the solid residue in step S2, 12 parts by weight of the solid residue in step S3, 40 parts by weight of the aloe vera gel obtained in step S4 and 100 parts by weight of water were mixed evenly, sterilized, and inoculated with the Bifidobacterium lactis and Lactobacillus reuteri species seed liquid obtained in step S5, wherein the inoculation amounts of the Bifidobacterium lactis and Lactobacillus reuteri species seed liquid were 3 v / v% and 2 v / v%, respectively, at 38°C, 85 r / min, and fermented for 42 h, and prebiotics were added in an amount of 8.5 wt% of the total mass of the system, and the fermentation was continued for 30 h, filtered, and the probiotics were washed with sterile water and dropped, and the fermentation product obtained was freeze-dried to obtain synbiotics;

[0074] The prebiotics include konjac glucomannan and arabinose in a mass ratio of 12:3;

[0075] S7. Preparation of a probiotic preparation for improving constipation: 12 parts by weight of the complex fruit enzyme-assisted fermentation product prepared in step S2, 8.5 parts by weight of the water extract prepared in step S3, 27 parts by weight of the synbiotic prepared in step S6, 6 parts by weight of inulin, 23.5 parts by weight of sodium alginate, 1.5 parts by weight of lecithin and 13.5 parts by weight of soy protein isolate are added to 200 parts by weight of water, homogenized at 10,000 r / min for 10 minutes to obtain a homogenous solution, 40 parts by weight of the homogenous solution is added to 85 fish oil, rapid membrane emulsification is performed, the membrane pore size is 150 μm, 6wt% calcium chloride solution is added dropwise, solidified at room temperature for 25 minutes, centrifuged, washed, and dried to obtain a probiotic preparation for improving constipation.

[0076] Example 4

[0077] Compared with Example 3, the difference is that the Bifidobacterium lactis seed solution is not inoculated in step S6. The inoculation amount of the Lactobacillus reuteri seed solution is 5 v / v%.

[0078] Example 5

[0079] Compared with Example 3, the difference is that the Lactobacillus reuteri seed solution is not inoculated in step S6. The inoculation amount of the Bifidobacterium lactis seed solution is 5 v / v%.

[0080] Example 6

[0081] Compared with Example 3, the difference lies in that the prebiotic is a single konjac glucomannan.

[0082] Example 7

[0083] Compared with Example 3, the difference is that the prebiotic is single arabinose.

[0084] Comparative Example 1

[0085] Compared with Example 3, the difference is that in step S1, the Aspergillus niger is not washed with sterile water of 1.5 times the volume of the filtrate to obtain the crude enzyme solution.

[0086] The details are as follows:

[0087] S1. Preparation of crude enzyme solution of Aspergillus niger: 11 parts by weight of bran, 30 parts by weight of malt extract with a sugar content of 6 degrees, 4 parts by weight of sucrose, 3 parts by weight of inorganic salts, 6 parts by weight of peptone and 450 parts by weight of water were stirred and mixed for 20 minutes, sterilized, and inoculated at a concentration of 10 9 cfu / mL Aspergillus niger spore suspension, inoculation amount of 2.5v / v%, 31°C, 120r / min, fermentation culture for 52h, product filtration to obtain crude enzyme solution;

[0088] The inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, sodium nitrate, iron sulfate, manganese sulfate, and calcium chloride, with a mass ratio of 2.5:3:17:0.07:0.15:1.5.

[0089] Comparative Example 2

[0090] Compared with Example 3, the difference is that the fermentation bacterial enzyme solution in step S2 is replaced by a mixed solution containing 100 U / mL of cellulase and 50 U / mL of pectinase of equal mass.

[0091] The details are as follows:

[0092] S2. Enzymatic hydrolysis of composite fruit: 6 parts by weight of yacon and 11 parts by weight of aronia solani were washed, crushed, homogenized at 10000 r / min for 10 min, added with 100 parts by weight of a mixture containing 100 U / mL of cellulase and 50 U / mL of pectinase, and hydrolyzed for 42 h at 37°C and 120 r / min. The mixture was filtered, the solid residue was dried and retained, and the enzyme was inactivated in the filtrate to obtain the composite fruit enzymatic hydrolysis product.

[0093] Comparative Example 3

[0094] Compared with Example 3, the difference is that water extraction is not performed in step S3.

[0095] The details are as follows:

[0096] S3. Mixing of medicinal and edible components: 4 parts by weight of hawthorn, 8.5 parts by weight of dendrobium officinale, and 3 parts by weight of cassia seed were washed, dried, crushed, and mixed to prepare a combination powder.

[0097] Comparative Example 4

[0098] Compared with Example 3, the difference is that no aloe vera gel is added in step S6.

[0099] The details are as follows:

[0100] S6. Preparation of synbiotics: 8.5 parts by weight of the solid residue in step S2, 12 parts by weight of the solid residue in step S3 and 140 parts by weight of water were mixed evenly, sterilized, and inoculated with the Bifidobacterium lactis and Lactobacillus reuteri species seed solution prepared in step S5, wherein the inoculation amounts of the Bifidobacterium lactis and Lactobacillus reuteri species seed solution were 3 v / v% and 2 v / v%, respectively, at 38°C, 85 r / min, and fermented for 42 h, and prebiotics were added in an amount of 8.5 wt% of the total mass of the system, and the fermentation was continued for 30 h, filtered, and the probiotics were washed with sterile water and dropped, and the fermentation product obtained was freeze-dried to obtain a synbiotic;

[0101] The prebiotics include konjac glucomannan and arabinose in a mass ratio of 12:3.

[0102] Comparative Example 5

[0103] Compared with Example 3, the difference is that no prebiotics are added in step S6.

[0104] The details are as follows:

[0105] S6. Preparation of fermentation product: 8.5 parts by weight of the solid residue in step S2, 12 parts by weight of the solid residue in step S3, 40 parts by weight of the aloe vera gel solution prepared in step S4 and 100 parts by weight of water are mixed evenly, sterilized, and inoculated with the Bifidobacterium lactis and Lactobacillus reuteri seed solutions prepared in step S5, wherein the inoculation amounts of the Bifidobacterium lactis and Lactobacillus reuteri seed solutions are 3 v / v% and 2 v / v%, respectively. The fermentation culture is carried out at 38°C and 85 r / min for 72 hours, filtered, the probiotics are washed and dropped with sterile water, and the obtained fermentation product is freeze-dried to obtain a fermentation product.

[0106] Comparative Example 6

[0107] Compared with Example 3, the difference is that in step S6, Bifidobacterium lactis and Lactobacillus reuteri seed liquid are not inoculated.

[0108] The details are as follows:

[0109] S6. Preparation of a mixture: 8.5 parts by weight of the solid residue in step S2, 12 parts by weight of the solid residue in step S3, 40 parts by weight of the aloe vera gel obtained in step S4 and 100 parts by weight of water were mixed uniformly, and a prebiotic was added in an amount of 8.5wt% of the total mass of the system to obtain a mixture;

[0110] The prebiotics include konjac glucomannan and arabinose in a mass ratio of 12:3.

[0111] Comparative Example 7

[0112] Compared with Example 3, the difference is that inulin is not added in step S7.

[0113] The details are as follows:

[0114] S7. Preparation of a probiotic preparation for improving constipation: 12 parts by weight of the complex fruit enzyme-assisted fermentation product prepared in step S2, 8.5 parts by weight of the water extract prepared in step S3, 27 parts by weight of the synbiotic prepared in step S6, 29.5 parts by weight of sodium alginate, 1.5 parts by weight of lecithin and 13.5 parts by weight of soy protein isolate are added to 200 parts by weight of water, homogenized at 10,000 r / min for 10 minutes to obtain a homogenous solution, 40 parts by weight of the homogenous solution is added to 85 fish oil, rapid membrane emulsification is performed, the membrane pore size is 150 μm, 6 wt% calcium chloride solution is added dropwise, solidified at room temperature for 25 minutes, centrifuged, washed, and dried to obtain a probiotic preparation for improving constipation.

[0115] Comparative Example 8

[0116] Compared with Example 3, the difference is that in step S7, the components are simply mixed without being embedded.

[0117] The details are as follows:

[0118] S7. Preparation of a probiotic preparation for improving constipation: 12 parts by weight of the complex fruit enzyme-assisted fermentation product prepared in step S2, 8.5 parts by weight of the water extract prepared in step S3, 27 parts by weight of the synbiotic prepared in step S6, and 6 parts by weight of inulin are mixed evenly to prepare a probiotic preparation for improving constipation.

[0119] Comparative Example 9

[0120] Compared with Example 3, the difference is that no compound fruit enzyme-assisted fermentation product is added in step S7.

[0121] The details are as follows:

[0122] S7. Preparation of a probiotic preparation for improving constipation: 8.5 parts by weight of the water extract prepared in step S3, 27 parts by weight of the synbiotic prepared in step S6, 6 parts by weight of inulin, 23.5 parts by weight of sodium alginate, 1.5 parts by weight of lecithin and 13.5 parts by weight of soy protein isolate are added to 200 parts by weight of water, homogenized at 10,000 r / min for 10 minutes to obtain a homogenous solution, 40 parts by weight of the homogenous solution are added to 85 fish oil, rapid membrane emulsification is performed, the membrane pore size is 150 μm, 6 wt% calcium chloride solution is added dropwise, and the mixture is cured at room temperature for 25 minutes. The mixture is centrifuged, washed and dried to obtain a probiotic preparation for improving constipation.

[0123] Comparative Example 10

[0124] Compared with Example 3, the difference is that no synbiotics are added in step S7.

[0125] The details are as follows:

[0126] S7. Preparation of a probiotic preparation for improving constipation: add 12 parts by weight of the complex fruit enzyme-assisted fermentation product prepared in step S2, 8.5 parts by weight of the water extract prepared in step S3, 6 parts by weight of inulin, 23.5 parts by weight of sodium alginate, 1.5 parts by weight of lecithin and 13.5 parts by weight of soy protein isolate to 200 parts by weight of water, homogenize at 10000 r / min for 10 min to obtain a homogenous solution, add 40 parts by weight of the homogenous solution to 85 fish oil, perform rapid membrane emulsification, the membrane pore size is 150 μm, add 6wt% calcium chloride solution dropwise, solidify at room temperature for 25 min, centrifuge, wash, and dry to obtain a probiotic preparation for improving constipation.

[0127] Test Example 1

[0128] Preparation of artificial simulated saliva: Mix 0.75 g potassium chloride, 0.15 g dipotassium hydrogen phosphate, 0.99 g sodium bicarbonate, 0.03 g magnesium chloride hexahydrate and 0.01 g ammonium carbonate in 500 mL sterile water and adjust the pH to 7.00.

[0129] Preparation of artificial simulated gastric fluid: Mix 0.26 g potassium chloride, 0.02 g potassium dihydrogen phosphate, 1.05 g sodium bicarbonate, 1.40 g sodium chloride, 0.01 g magnesium chloride dihydrate, 0.04 g ammonium carbonate and 0.01 g calcium chloride dihydrate in 500 mL sterile water, adjust the pH to 2.00, and keep the pepsin at 3.20 g / L.

[0130] Preparation of artificial simulated intestinal fluid: Mix 0.25g potassium chloride, 0.01g potassium dihydrogen phosphate, 3.27g sodium bicarbonate, 1.12g sodium chloride, 6.13g magnesium chloride hexahydrate and 0.03g calcium chloride dihydrate in 500mL sterile water, adjust the pH to 7.40, keep the pancreatic enzyme at 10.00g / L, and the bile salt at 3.00g / L.

[0131] 1 g of the probiotic preparations for improving constipation prepared in Examples 1-3 of the present invention and Comparative Examples 7 and 8 were respectively added into 20 mL of artificial simulated saliva, and reacted at 37° C. and 150 r / min for 10 min. After the reaction, the probiotic cell population was counted and weighed.

[0132] 1 g of the probiotic preparation for improving constipation prepared in Examples 1-3 of the present invention and Comparative Examples 7 and 8 was added into 20 mL of artificial simulated gastric juice and 20 mL of artificial simulated intestinal juice, respectively, and reacted at 37° C. and 150 r / min for 2 h and 3 h, respectively. After the reaction, the probiotic cell population was counted and weighed.

[0133] In addition, an equal amount of probiotic preparation for improving constipation was added to 20 mL of artificial saliva, reacted at 37°C and 150 r / min for 10 min, centrifuged, then added to 20 mL of artificial simulated gastric fluid, reacted at 37°C and 150 r / min for 2 h, centrifuged, and then added to 20 mL of artificial simulated intestinal fluid for another 3 h. After the reaction, the probiotic cell count and weight were performed.

[0134] The survival rate was calculated according to the following formula:

[0135] Survival rate (%) = N t / N0×100%

[0136] Where N t N0 is the concentration of probiotics that survive after a certain period of incubation (cfu / g), and N0 is the original concentration of probiotics added (cfu / g).

[0137] The release rate was calculated according to the following formula:

[0138] Release rate (%) = (W t -W0) / W0×100%

[0139] Where W t is the initial weight of the sample; W0 is the weight of the sample after a certain period of incubation.

[0140] Results Figure 1 and Figure 2 .

[0141] As can be seen from the figure, the probiotic preparations for improving constipation prepared in Examples 1-3 of the present invention are stable in low or high pH and harsh environments to block gastric juice erosion and reduce damage to the core material in adverse environments such as oxygen and high temperature.

[0142] Test Example 2

[0143] SPF male Kunming mice, 5-7 weeks old, 20-22 g, were selected and subcutaneously injected with 5 mg / kg loperamide saline solution at 10 am and 6 pm every day for 6 days. If the mice had less feces, dry and hard feces, constipation was successfully induced.

[0144] The mice were divided into a normal group (without induction of constipation), a model group, Example 1-7 groups, and Comparative Example 1-10 groups, with 12 mice in each group.

[0145] The Example 1-7 group and the Comparative Example 1-10 group were intragastrically administered with 2 g / kg of the probiotic preparation for improving constipation prepared in the corresponding group every day, and the normal group and the model group were intragastrically administered with an equal amount of physiological saline. During the experiment, the group had free access to water and a normal diet.

[0146] Gastrointestinal transit rate and bowel movement time tests:

[0147] After the experiment, the mice were fasted for 24 hours and orally administered 0.2 mL of 10% activated carbon dissolved in 10% gum arabic. The normal group was orally administered 10% gum arabic. 30 minutes after the first oral gavage treatment, 6 mice in each group were killed by cervical dislocation, and their small intestines were measured for length, and the intestinal propulsion rate was calculated:

[0148] Intestinal propulsion rate (%) = activated carbon propagation distance / total length of small intestine × 100%.

[0149] The remaining 6 mice in each group were used to measure the defecation time (min) to the first black stool.

[0150] The results are shown in Table 1.

[0151] Table 1

[0152] Group Intestinal propulsion rate (%) Time to first bowel movement (min) Normal group 99.02±2.21# 62.34±10.24# Model Group 40.17±1.89* 188.49±32.15* Example 1 90.26±3.14* 82.10±18.19* Example 2 91.04±3.47* 81.89±18.42* Example 3 91.57±3.32* 81.45±19.02* Example 4 85.21±2.89 89.49±19.44 Example 5 84.34±2.94 90.12±18.75 Example 6 88.95±3.14 86.57±19.20 Example 7 89.03±3.08 85.92±19.04 Comparative Example 1 85.62±3.27 89.21±18.77 Comparative Example 2 82.39±3.11 92.45±18.51 Comparative Example 3 83.41±3.34 91.39±18.17 Comparative Example 4 88.19±2.84 84.76±19.47 Comparative Example 5 86.79±3.01 87.45±20.12 Comparative Example 6 79.58±2.96 99.42±19.57 Comparative Example 7 85.75±3.07 90.24±21.10 Comparative Example 8 73.62±3.12 117.84±20.57 Comparative Example 9 80.54±3.22 96.67±20.31 Comparative Example 10 72.44±3.45 119.59±21.07

[0153] Note: # means P<0.05 compared with the normal group; * means P<0.05 compared with the model group.

[0154] As can be seen from the above table, the probiotic preparations for improving constipation prepared in Examples 1-3 of the present invention significantly increased the intestinal propulsion rate and shortened the first defecation time.

[0155] Stool water content and bowel movement frequency tests:

[0156] After the remaining mice were treated with the drug for 7 consecutive days, the feces were collected, weighed, dried, weighed, and the fecal water content (%) in the feces was calculated.

[0157] Feces moisture content (%) = (feces wet weight - feces dry weight) / feces wet weight × 100%.

[0158] Calculate the amount of stool per hour.

[0159] The results are shown in Table 2.

[0160] Table 2

[0161] Group Fecal weight (g) Fecal water content (%) Normal group 0.92±0.05# 50.63±2.94# Model Group 0.28±0.02* 20.17±1.82* Example 1 0.79±0.08* 41.12±3.16* Example 2 0.80±0.06* 41.59±2.95* Example 3 0.82±0.05* 42.04±3.06* Example 4 0.71±0.03 37.49±3.11 Example 5 0.70±0.06 37.12±2.89 Example 6 0.74±0.05 38.29±3.05 Example 7 0.75±0.08 38.75±3.24 Comparative Example 1 0.73±0.04 37.21±2.78 Comparative Example 2 0.69±0.02 35.69±3.11 Comparative Example 3 0.68±0.07 38.59±2.89 Comparative Example 4 0.72±0.06 33.19±2.84 Comparative Example 5 0.72±0.03 37.86±2.96 Comparative Example 6 0.63±0.05 34.23±3.01 Comparative Example 7 0.67±0.09 38.04±3.21 Comparative Example 8 0.61±0.07 30.75±3.15 Comparative Example 9 0.64±0.04 33.10±3.06 Comparative Example 10 0.57±0.06 30.19±2.89

[0162] Note: # means P<0.05 compared with the normal group; * means P<0.05 compared with the model group.

[0163] As can be seen from the above table, the probiotic preparations for improving constipation prepared in Examples 1-3 of the present invention significantly improved the feces quality and feces water content of mice.

[0164] Nitric oxide, glutathione and malondialdehyde levels tested:

[0165] The remaining mice were treated with the drug continuously for 14 days and then killed. The small intestinal tissues of the mice were collected and prepared into tissue homogenates. The contents of nitric oxide (NO), glutathione (GSH) and malondialdehyde (MDA) at the small intestinal level were detected using kits.

[0166] The results are shown in Table 3.

[0167] Table 3

[0168]

[0169]

[0170] Note: # means P<0.05 compared with the normal group; * means P<0.05 compared with the model group.

[0171] It can be seen from the above table that the probiotic preparations for improving constipation prepared in Examples 1-3 of the present invention significantly reduced the contents of NO and MDA, and increased the content of GSH.

[0172] Embodiment 4,5 Compared with Example 3, Bifidobacterium lactis strain seed liquid or Lactobacillus reuteri strain seed liquid was not inoculated in step S6.Comparative Example 6 Compared with Example 3, Bifidobacterium lactis and Lactobacillus reuteri strain seed liquid were not inoculated in step S6.Comparative Example 10 Compared with Example 3, synbiotics were not added in step S7.Intestinal propulsion rate decreased, first defecation time was extended, feces weight was reduced, feces moisture content decreased, NO, MDA content increased, GSH content decreased.The probiotics added by the present invention include Bifidobacterium lactis and Lactobacillus reuteri, which can compete with pathogenic bacteria in the intestinal tract to reduce the colonization of pathogenic bacteria and the inflammatory response caused by them, change the metabolic effect of bile salts and restore the normal fermentation of the intestinal tract. Bifidobacterium lactis has a positive effect on promoting intestinal motility and can improve the viscosity of feces. Lactobacillus reuteri can produce a large number of metabolites, such as short-chain fatty acids, which lead to a decrease in intestinal pH. The positive feedback regulation effect promotes the colonization of beneficial intestinal bacteria, inhibits the colonization of conditional pathogens and pathogens, and balances the intestinal flora structure. Short-chain fatty acids stimulate the contraction and peristalsis of the small intestine, and by binding to the intestinal surface cell receptors, promote the increase in the secretion of 5-hydroxytryptamine in the body's intestinal system, resulting in enhanced intestinal water retention capacity and internal intestinal osmotic pressure, effectively reducing the residence time of feces in the small intestine and relieving constipation symptoms. The synergistic effect of the two can effectively alleviate the imbalance of intestinal flora, thereby improving and treating constipation problems.

[0173] Compared with Example 3, the prebiotics in Example 6 and 7 are single konjac glucomannan or arabinose.Comparative Example 5 is compared with Example 3, and no prebiotics are added in step S6.Intestinal propulsion rate decreases, first defecation time is extended, feces weight decreases, and feces moisture content decreases.The present invention also adds prebiotics during fermentation, and forms synbiotics with probiotics. Prebiotics can effectively promote colonic motility in patients with chronic constipation, improve symptoms such as stool hardness, defecation frequency and abdominal distension, and due to the characteristics that it cannot be absorbed by the intestine, its osmotic effect can also hinder the intestinal wall from reabsorbing water and electrolytes, thereby playing a cathartic effect to a certain extent, thereby also being able to improve constipation symptoms, while prebiotics can also well selectively promote the growth and colonization of intestinal probiotics, thereby regulating constipation symptoms by improving intestinal flora.Wherein, konjac mannan can slow down or prevent fat, carbohydrate digestion and absorption, increase satiety, is soluble and indigestible fiber, can promote the production of bifidobacteria and lactobacillus in rat stool. Arabinose can promote the growth of bifidobacteria, improve intestinal flora, and play a role in moistening the intestines and promoting bowel movements. Therefore, the synergistic effect of combined prebiotics can significantly improve the symptoms of constipation in the human body.

[0174] Comparative Example 1 Compared with Example 3, the aspergillus niger was not washed and dropped with sterile water of 1.5 times the volume of filtrate in step S1 to obtain a crude enzyme solution. Comparative Example 2 Compared with Example 3, the fermentation bacteria enzyme solution in step S2 was replaced by a mixed solution of 100U / mL of cellulase and 50U / mL of pectinase of equal mass. Comparative Example 9 Compared with Example 3, the composite fruit enzyme fermentation product was not added in step S7. The intestinal propulsion rate decreased, the first defecation time was extended, the feces weight was reduced, the feces moisture content decreased, the NO and MDA contents were increased, and the GSH content decreased. The present invention cultured and fermented aspergillus niger in a culture medium containing rich nutrients, and a variety of biological enzymes can be produced, including a variety of complex and abundant biologically active enzymes such as cellulase, xylanase, pectinase, endoglucanase, etc., so that the obtained fermentation bacteria enzyme solution contains abundant biologically active enzymes and aspergillus niger fermentation bacteria.

[0175] Comparative Example 3 Compared with Example 3, water extraction was not performed in step S3. The intestinal propulsion rate decreased, the first defecation time was prolonged, the feces weight decreased, the NO and MDA contents increased, and the GSH content decreased. Hawthorn, Dendrobium Officinale, and Cassia Seed are traditional Chinese medicines that are both medicinal and edible, and have good effects of clearing heat, reducing fire, strengthening the spleen, and warming and nourishing. The rich dietary fiber they contain can promote the colonization and growth of intestinal probiotics, and increase the water content of feces by increasing the expansion rate of food absorption, thereby balancing the intestinal flora, moistening the intestines and relieving constipation, and improving constipation symptoms.

[0176] Comparative Example 4 Compared with Example 3, aloe gel was not added in step S6. The intestinal propulsion rate decreased, the first defecation time was prolonged, the feces moisture content decreased, the NO and MDA contents increased, and the GSH content decreased. Aloe vera contains rich aloe gel, including aloe polysaccharides, calcium isocitrate, superoxide dismutase, peroxidase, vitamin C, amino acids, vitamins, minerals, etc., which can promote intestinal peristalsis and have the effects of stomachic, anti-inflammatory, bactericidal, anti-allergic, and promoting blood circulation.

[0177] Compared with Example 3, in Comparative Example 7, inulin was not added in step S7. Compared with Example 3, in Comparative Example 8, the components were simply mixed in step S7 without embedding. The intestinal propulsion rate decreased, the first defecation time was prolonged, the feces weight was reduced, the feces moisture content was reduced, the NO and MDA contents were increased, the GSH content was decreased, the survival rate of probiotics in saliva, gastric acid and intestinal fluid was decreased, and the release rate was increased. In the wall material selected for embedding, sodium alginate has the effect of being easy to cross-link to form a shell layer, but it is not acid-alkali resistant and is easy to break in an environment with high humidity or low pH. Therefore, the present invention also adds inulin, which has gelling properties, solubility, low viscosity, and high glass transition temperature. It can form a stable gel network structure during the microencapsulation process. The core material and the wall material are closely combined to form a stable glass protective layer to reduce oxidative damage. In addition, soy protein isolate is added. Under the action of composite multi-crosslinking of multiple wall materials, the prepared microcapsules can be stably present in low or high pH and harsh environments to prevent gastric juice erosion and reduce the damage of the core material in unfavorable environments such as oxygen and high temperature. At the same time, inulin is also a good prebiotic. After being degraded in the intestine, it can be absorbed and utilized by probiotics to promote the rapid growth and colonization of probiotics.

[0178] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a probiotic preparation for improving constipation, characterized in that: The fermentation enzyme liquid prepared by Aspergillus niger fermentation is used for enzyme-assisted fermentation of yacon and Sorbus nigra, filtered to obtain a compound fruit enzyme-assisted fermentation product, and the filter residue is retained; hawthorn, Dendrobium officinale and Cassia seed are extracted with water, filtered to obtain a water extract, and the filter residue is retained; the filter residue and aloe vera gel are mixed, inoculated with probiotics for fermentation, and prebiotics are added to obtain synbiotics; the compound fruit enzyme-assisted fermentation product, the water extract and the synbiotics are mixed and embedded to obtain a probiotic preparation for improving constipation.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Preparation of Aspergillus niger fermentation enzyme liquid: bran, malt extract, sucrose, inorganic salts, peptone and water are stirred and mixed uniformly, sterilized, inoculated with Aspergillus niger spore suspension, fermented, the product is filtered, and Aspergillus niger is washed with sterile water to obtain a fermentation enzyme liquid; S2. Enzyme-assisted fermentation of composite fruit: Wash the yacon and black-fruited glandular ribs Aronia ovata, crush, homogenize, add the fermentation enzyme solution prepared in step S1, enzyme-assisted fermentation, filter, dry the solid residue and retain, sterilize and kill the enzyme in the filtrate to obtain a composite fruit enzyme-assisted fermentation product; S3. Water extraction of edible and medicinal components: Wash hawthorn, Dendrobium officinale, and Cassia seed separately, dry, crush, mix, and prepare a combined powder, add water, heat to boiling and extract, repeat the operation 2-3 times, filter, combine the filtrate, dry the solid residue and retain it, and dry the filtrate to obtain a water extract; S4. Preparation of aloe vera gel: Peel the aloe vera and take the aloe vera gel, add water and homogenize to obtain aloe vera gel; S5. Preparation of bacterial seed solution: Bifidobacterium lactis and Lactobacillus reuteri were inoculated into Gao's medium, activated and cultured to obtain bacterial seed solution; S6. Preparation of synbiotics: the solid residue in step S2, the solid residue in step S3, the aloe vera gel solution obtained in step S4 and water are mixed evenly, sterilized, inoculated with the Bifidobacterium lactis and Lactobacillus reuteri seed solution obtained in step S5, fermented and cultured for a first period of time, added with prebiotics, fermented and cultured for a second period of time, filtered, washed with sterile water to remove the probiotics, and the obtained fermentation product is freeze-dried to obtain synbiotics; S7. Preparation of a probiotic preparation for improving constipation: add the complex fruit enzyme-assisted fermentation product prepared in step S2, the water extract prepared in step S3, the synbiotics prepared in step S6, inulin, konjac glucomannan, sodium alginate, lecithin and soy protein isolate into water, stir and homogenize to obtain a homogenous liquid, add into fish oil, perform rapid membrane emulsification, add calcium chloride solution dropwise, solidify at room temperature, centrifuge, wash and dry to obtain a probiotic preparation for improving constipation.

3. The preparation method according to claim 2, characterized in that: The mass ratio of bran, malt juice, sucrose, inorganic salt, peptone and water in step S1 is 10-12:20-40:3-5:2-4:5-7:400-500, the inorganic salt includes potassium dihydrogen phosphate, magnesium sulfate, sodium nitrate, iron sulfate, manganese sulfate and calcium chloride, the mass ratio is 2-3:2-4:15-20:0.05-0.1:0.1-0.2:1-2, the sugar content of the malt juice is 5-7 degrees, the concentration of the Aspergillus niger spore suspension is 10 8 -10 9 cfu / mL, the inoculation amount of the Aspergillus niger spore suspension is 2-3v / v%, the fermentation conditions are 30-32°C, 100-150r / min, the fermentation culture is 48-56h, and the sterile water used for washing is 1-2 times the volume of the filtrate.

4. The preparation method according to claim 2, characterized in that: In step S2, the mass ratio of yacon, Aronia melanocarpa and fermentation bacteria enzyme solution is 5-7:10-12:100, and the enzyme-assisted fermentation conditions are 35-40° C., 100-150 r / min, and the enzyme-assisted fermentation culture is 36-48 hours.

5. The preparation method according to claim 2, characterized in that: In step S3, the mass ratio of hawthorn, dendrobium officinale and cassia seed is 3-5:7-10:2-4, the solid-liquid ratio of the combined powder and water is 1:5-7 g / mL, and the time for heating to boiling extraction is 1-3 h.

6. The preparation method according to claim 2, characterized in that: The mass ratio of aloe vera gel to water in step S4 is 3-5:10; the conditions of activation culture in step S5 are 37-39°C, 70-100r / min, activation culture for 28-30h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL.

7. The preparation method according to claim 2, characterized in that: The mass ratio of the solid residue in step S2, the solid residue in step S3, the aloe gel solution and the water in step S6 is 7-10:10-15:30-50:100, the inoculation amounts of the Bifidobacterium lactis and the Lactobacillus reuteri species seed solutions are 2-4v / v% and 1-3v / v%, respectively, the fermentation culture conditions are 37-39°C, 70-100r / min, the first time period is 36-48h, the second time period is 24-36h, the prebiotics include konjac glucomannan and arabinose in a mass ratio of 10-15:2-4, and the amount of the prebiotics added is 7-10wt% of the total mass of the system.

8. The preparation method according to claim 2, characterized in that: In step S7, the mass ratio of the complex fruit enzyme-assisted fermentation product, the water extract, the synbiotic, inulin, sodium alginate, lecithin and soy protein isolate is 10-15:7-10:25-30:5-7:22-25:1-2:12-15, the mass ratio of the homogenate and the fish oil is 3-5:7-10, the membrane pore size of the rapid membrane emulsification is 100-200 μm, the concentration of the calcium chloride solution is 5-7wt%, and the time for room temperature curing is 20-30 min.

9. A probiotic preparation for improving constipation obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the probiotic preparation for improving constipation as claimed in claim 9 in the preparation of food or medicine for treating or improving symptoms of acute or chronic constipation.

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