CN115105577A

CN115105577A - Multifunctional probiotic composition and application thereof

Info

Publication number: CN115105577A
Application number: CN202210785020.9A
Authority: CN
Inventors: 杨惠玲; 林子洪
Original assignee: Evergreen Biological Guangzhou Co ltd
Current assignee: Evergreen Biological Guangzhou Co ltd
Priority date: 2022-07-05
Filing date: 2022-07-05
Publication date: 2022-09-27

Abstract

The invention provides a multifunctional probiotic composition and application thereof. The probiotic composition comprises probiotic powder, prebiotic powder and herbal material; the probiotic powder consists of lactobacillus acidophilus, bifidobacterium lactis, lactobacillus paracasei and lactobacillus rhamnosus; the prebiotics powder comprises fructo-oligosaccharide powder and oligomeric semi-lactose powder; the Chinese herbal materials include Cordyceps militaris, Lentinus Edodes or their extracts, and Curcuma rhizome. Compared with the prior art, in the technical scheme of the invention, the types of the prebiotics are adjusted, the types and the number of the probiotics are reduced, the effect of the probiotics is assisted and enhanced by various Chinese herbal medicine materials, the cost is reduced, and the curative effect is ensured; meanwhile, the combination of probiotics and Chinese herbal medicine materials is increased, so that the effect of preventing and treating alcoholic gastric ulcer is improved; in addition, the invention can play a role in inhibiting cancers and the like besides aiming at antibiotic diarrhea and preventing alcoholic gastric ulcer.

Description

Multifunctional probiotic composition and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a multifunctional probiotic composition and application thereof.

Background

Antibiotic-associated diarrheal (AAD) refers to diarrhea that occurs with the use of antibiotics and cannot be explained for other reasons. The alcoholic gastric ulcer is a common acute gastric injury disease, and the oral intake of a large amount of alcohol can directly damage epithelial cells of gastric mucosa, destroy the barrier of the gastric mucosa, and cause acute congestion, erosion and even superficial ulcer of the gastric mucosa. The pathological process of alcoholic gastric ulcer generally involves several aspects of inflammatory reaction, oxidative stress and apoptosis.

In the prior art, there are many solutions for the treatment or prevention of the above-mentioned diseases, such as treatment or prevention by probiotics.

As published in the prior art for antibiotic diarrhea, CN 112057479 a discloses a probiotic composition capable of alleviating antibiotic-induced diarrhea, comprising lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus paracasei, bifidobacterium bifidum, resistant dextrin, maltodextrin, inulin, and stachyose, wherein the probiotic is more diverse and more costly.

In the published prior art aiming at alcoholic gastric ulcer, CN 110731432A discloses a seaweed prebiotics stomach-protecting beverage and a preparation method thereof, wherein the beverage comprises the following raw materials in percentage by mass: 20-30 parts of sodium alginate, 13-25 parts of guar gum extract, 5-15 parts of seaweed extract, 1-6 parts of guar gum, 80-100 parts of white granulated sugar, 1-8 parts of citric acid, 5-20 parts of concentrated hawthorn juice and 800-1000 parts of soft water. But it is too single in function.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a multifunctional probiotic composition, which can help to enhance the effect of probiotics by adjusting the types or amounts of probiotics or prebiotics and using various Chinese herbal medicines (or extracts thereof), reduce the cost and ensure the curative effect, and can exert the effects of inhibiting cancer/relieving inflammatory bowel disease, etc. in addition to the effects of treating antibiotic diarrhea and preventing alcoholic gastric ulcer.

In order to solve the above technical problems, the present invention provides a multifunctional probiotic composition, comprising probiotic powder, prebiotic powder and herbal material; the probiotic powder comprises lactobacillus acidophilus, bifidobacterium lactis, lactobacillus paracasei and lactobacillus rhamnosus, the prebiotics powder comprises fructo-oligosaccharide powder and oligomeric semilactose powder, and the Chinese herbal medicine materials comprise cordyceps militaris (extract), shiitake mushroom (extract) and turmeric.

For the content of the components, preferably, per 15g of the probiotic composition, comprises:

22-24 hundred million CFU of lactobacillus acidophilus, 42-44 hundred million CFU of bifidobacterium lactis, 18-20 hundred million CFU of lactobacillus paracasei, 14-16 hundred million CFU of lactobacillus rhamnosus and 100 hundred million CFU of total bacterial load;

10-12g of fructo-oligosaccharide powder (95S), 1-1.5g of oligomeric half lactose powder (57S); 0.2-0.4g of cordyceps militaris extract, 0.2-0.4g of shiitake extract and 0.1-0.3g of turmeric.

More preferably, per 15g of probiotic composition, comprises:

lactobacillus acidophilus 23 hundred million CFU, Bifidobacterium lactis 43 hundred million CFU, Lactobacillus paracasei 19 hundred million CFU, Lactobacillus rhamnosus 15 hundred million CFU;

12g of fructo-oligosaccharide powder and 1.33g of oligomeric half lactose powder;

0.3g of cordyceps militaris extract, 0.3g of shiitake mushroom extract and 0.2g of turmeric.

Cordyceps militaris extract, production batch number: ZLSC 2020111303;

cordyceps militaris extract, production batch number: ZLYT 2021110402;

mushroom extract, production lot No.: ZLYT 2021121120101;

the extracts are obtained from Biotech, Inc. of Nanjing Zeylang, and Curcuma rhizome from food industry, Inc. of Zhuhai Yafuxing, respectively.

In the invention, the probiotics are specifically lactobacillus acidophilus NCFM, bifidobacterium lactis HN019, lactobacillus paracasei LPC37 and lactobacillus rhamnosus HN 001.

The efficacy of the components in this application:

(1) probiotics

1) Lactobacillus acidophilus: lactobacillus acidophilus is one of the most commonly used probiotics, has been used in a variety of fermented foods for a long time, and has fewer adverse reactions. Studies have suggested that lactobacillus acidophilus acts in anti-inflammatory and antioxidant ways to reduce the formation of atherosclerotic lesions by inhibiting oxidative stress and reducing inflammatory responses; administration of lactobacillus acidophilus helps to reduce cholesterol levels; in addition, it can prevent and reduce diarrhea associated with various diseases.

2) Bifidobacterium lactis: bifidobacteria are important bacteria in the intestinal tract, and 72 bifidobacteria subspecies have been discovered and sequenced, wherein various types, including bifidobacterium lactis, are proved to be the major bifidobacteria subspecies existing in the digestive tract of mammals, play an important role in the intestinal function and even the human body homeostasis, and have a plurality of functions of improving the gastrointestinal function and preventing diseases.

3) Lactobacillus paracasei: the paracasei lactic acid bacteria is gram-positive heterotype fermentation lactic acid bacteria, can be separated from intestinal tracts, oral cavities, silage and various dairy products of human bodies, and is facultative anaerobic, motionless and bacillus-free bacillus or long bacillus. Research suggests that lactobacillus paracasei can balance intestinal flora and treat irritable bowel syndrome patients; and has strong inhibition effect on various common food-borne pathogenic bacteria. In addition, it also has blood pressure lowering, cholesterol lowering and antitumor effects.

4) Lactobacillus rhamnosus: the lactobacillus rhamnosus is a common probiotic and has the biological characteristics of gastric acid resistance, bile salt resistance, multiple antibiotic resistance and the like. Research has shown that lactobacillus rhamnosus has good adhesion and colonization ability in human gastrointestinal tract. Can be used for preventing and treating various gastrointestinal microecological disorder related diseases, such as antibiotic-associated diarrhea, helicobacter pylori infection, infectious diarrhea, inflammatory bowel disease, etc., by enhancing intestinal barrier function, improving adhesion to intestinal mucosa, inhibiting pathogen adhesion, competitively repelling pathogenic microorganism, producing antibiotic-like substance, and regulating immune system. In addition, it has tumor inhibiting effect.

(2) Prebiotics

1) Fructo-oligosaccharide powder: fructo-oligosaccharide is a novel glycogen which accords with the modern health concept and is widely existed in various fruits and vegetables as a typical prebiotic and excellent water-soluble dietary fiber. The fructo-oligosaccharide has various biological activities, and has obvious effects in regulating intestinal flora of human body, promoting growth of beneficial bacteria, promoting mineral absorption, enhancing immunity, reducing cholesterol and blood sugar, regulating intestinal function, promoting gastrointestinal motility, and inhibiting colon cancer.

2) Oligomeric half lactose powder: galactooligosaccharides are increasingly used as a prebiotic in functional foods. Galactooligosaccharides can selectively proliferate probiotics in intestinal tracts to ferment to generate short-chain fatty acids, and cooperate with intestinal microorganisms to participate in physiological function mechanism regulation, thereby playing an important role in the aspects of mechanisms of protecting intestinal tract health, regulating immune systems, inhibiting tumor cell generation, improving mineral absorption of human bodies, improving cardiovascular diseases and the like.

(3) Chinese herbal medicine material

1) Cordyceps militaris: cordyceps militaris is also called cordyceps militaris, the species is a model species of cordyceps, is an edible and medicinal fungus combined with insect fungi, has rich nutritional value and medicinal value, and can be used as one of substitutes of cordyceps militaris. The main active ingredients of the compound comprise cordycepin, cordycepic acid, polysaccharide and the like.

Although the content of cordycepin is very low, the biological activity is very wide, and researches show that the cordycepin has the effects of resisting virus and bacteria, obviously inhibiting tumor growth, regulating the immune function of a human body, reducing blood sugar and blood fat and the like.

The cordyceps militaris polysaccharide is one of active substances with high content in cordyceps militaris, is used as a polysaccharide compound and mainly comprises monosaccharides such as galactose, mannose, rhamnose, xylose and glucose. The cordyceps militaris polysaccharide is a good immune promoting substance and has the effects of resisting tumor, resisting oxidation, resisting aging, enhancing immunity, regulating blood sugar and the like.

2) Mushroom: lentinus Edodes is Basidiomycetes of Lentinus of Pleurotaceae, contains multiple effective medicinal components, is rich in medicinal source, and can be used for treating various diseases. In recent years, multiple researches show that the compound shiitake extract has multiple biological functions of resisting virus, bacteria and tumors, regulating immune function and the like, so that the compound shiitake extract can be used for adjuvant therapy of tumors, immune regulation and the like. Researches suggest that the traditional Chinese medicine composition also has an effect on the adjuvant therapy of diabetes.

3) Turmeric: turmeric is the rhizome of turmeric of Zingiberaceae, and is produced in Sichuan, Fujian, Guangdong, Jiangxi provinces. Turmeric is bitter and pungent in taste and warm in nature, enters liver and spleen channels, has the effects of promoting qi circulation, removing blood stasis, and dredging channels to relieve pain, so that turmeric is commonly used for clinical treatment of various diseases such as chest and abdomen distending pain, shoulder and arm pain, heart pain, postpartum hematodynia, amenorrhea and the like. Curcumin is a lipophilic polyphenol extracted from traditional Chinese medicine turmeric, and also has various pharmacological effects of resisting inflammation, resisting infection, resisting oxidation, resisting tumor and the like. Turmeric shows better treatment potential in neuroinflammatory diseases, inflammatory bowel diseases, malignant tumors, rheumatic diseases, infectious diseases, atherosclerosis and myocardial infarction. In recent years, a plurality of researches suggest that the turmeric and probiotics or prebiotics are combined to assist in regulating intestinal flora and enhancing the immunity of the organism.

Compared with the prior art, the invention has the advantages that the types of the prebiotics are adjusted, the number of the types of the probiotics is reduced, compared with the quintuplet probiotic product in the prior art, the invention reduces the number of the probiotics to the tetratuplet probiotic product, and the cost is reduced and the curative effect is ensured by adopting less probiotics and simultaneously assisting the enhancement of the effect of the probiotics by means of multiple Chinese herbal medicine materials; meanwhile, the combination of probiotics and Chinese herbal medicine materials is increased, so that the effect of preventing and treating alcoholic gastric ulcer is improved; in addition, the invention can play a role in inhibiting cancers and the like besides aiming at antibiotic diarrhea and preventing alcoholic gastric ulcer.

Drawings

FIG. 1 is a graph showing the change in weight gain of mice in the antibiotic-treated group and the saline group in the model construction;

FIG. 2 is a graph showing the change of feces in the antibiotic-treated group and the saline group from the first day to the fifth day in the mouse antibiotic diarrhea model construction;

FIG. 3 is a graph showing diarrhea scores in the antibiotic-treated group and the saline group in the model construction;

FIG. 4 is a graph showing the change of feces in the treatment process of mice in the normal saline application group and the composite powder application group after successful model construction;

FIG. 5 is a graph showing the weight change of 4 mice in group A1 (saline continuous application group), group A2 (composite powder applied after saline treatment), group B1 (saline applied after antibiotic molding) and group B2 (composite powder applied after antibiotic molding) during antibiotic molding;

FIG. 6 is a graph showing the change of feces in the first to fifth days of 4 groups of mice during antibiotic molding, including group A1 (saline solution continuous application group), group A2 (saline solution treatment and composite powder application group), group B1 (saline solution application group after antibiotic molding) and group B2 (composite powder application group after antibiotic molding);

FIG. 7 is a graph showing diarrhea scores of 4 mice in groups A1 (saline continuous application group), A2 (saline treated and compound powder applied group), B1 (saline treated and compound powder applied group) and B2 (antibiotic molded and compound powder applied group) during antibiotic molding;

FIG. 8 is a graph showing the feces of 4 mice of groups A1 (continuous application of physiological saline), A2 (application of composite powder after physiological saline treatment), B1 (application of physiological saline after antibiotic molding), and B2 (application of composite powder after antibiotic molding) during composite powder treatment according to the present invention on the sixth to ninth days;

FIG. 9 is a graph showing the degree of gastric ulcer in 4 groups of mice treated differently, namely group A1 (saline-continuous application group), group A2 (composite powder-treated saline-continuous application group), group B1 (saline-treated antibiotic-molded application group), and group B2 (composite powder-treated antibiotic-molded application group);

FIG. 10 is a graph showing the results of MDA detection of serum in 4 groups of mice, which were treated differently, in group A1 (saline solution treatment group), group A2 (saline solution treatment group and composite powder treatment group), group B1 (saline solution treatment group and antibiotic molding group), and group B2 (composite powder treatment group and antibiotic molding group);

FIG. 11 is a graph showing the growth inhibition curves of HCT116 cells for 24 hours of treatment with treatment solutions of different concentrations of the composite powder of the present invention;

FIG. 12 shows the growth inhibition curves of MC38 cells in 48 hours after treatment with different concentrations of the composite powder of the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

A multifunctional probiotic composition, comprising per 15g of probiotic composition:

The preferred embodiments of the present invention are as follows:

per 15g of probiotic composition, comprising:

And (3) experimental verification:

the overall effect is as follows:

treatment against antibiotic diarrhea and prevention of alcoholic gastric ulcers:

(1) against antibiotic diarrhea: dividing mice into A, B groups, and continuously performing intragastric administration for 5 days by adopting physiological saline (A) or cefuroxime (0.26g/kg) and levofloxacin (0.20g/kg) (B) to construct and verify a stable mouse model of antibiotic-associated diarrhea; after a clear modeling success, A, B groups of mice were randomly subdivided into two groups (a1, a2, B1, B2) and gavage was continued for 4 days with saline (a1, B1) or a composite powder of the probiotic composition of the invention (a2, B2). During this period mice were observed for fecal status, diarrhea scores and weight changes as follows:

1) mice in group B (antibiotic group) showed listlessness during antibiotic gavage, increased water intake, decreased food intake, increased frequency of defecation, and significantly lower weight gain compared to group A (normal saline group) (FIG. 1). After 5 days of gastric lavage, mice in group B (antibiotic group) all have diarrhea, the feces gradually become soft, most of the feces have obviously light color and are thin and soft, loose and amorphous occasionally, and feces adhere to the anus in severe cases (figure 2); diarrhea scores were seen to be significantly higher in group B (antibiotic group) diarrhea scores compared to group a (saline group) (fig. 3).

2) After the continuous gavage treatment is carried out for four days by using physiological saline (A1, B1) or the composite powder (A2, B2) respectively, compared with a group B1 (a group of physiological saline is applied after antibiotic molding), the feces of mice in a group B2 (a group of composite powder is applied after antibiotic molding) are gradually molded and hardened, while the feces of mice in a group B1 (a group of physiological saline is applied after antibiotic molding) are still soft and light in color and occasionally loose and amorphous (figure 4), which indicates that the application of the composite powder can improve antibiotic diarrhea; secondly, compared with the group A1 (group continuously applying normal saline), the group A2 (group applying the composite powder after normal saline treatment) has no obvious change of the stool characters (figure 4), and the defecation frequency is not obviously increased, which indicates that the composite powder of the invention has no obvious influence on the stool characters and the defecation habits of mice, and is not easy to cause adverse reactions such as diarrhea and the like.

(2) For alcoholic gastric ulcer: dividing mice into A, B groups, and performing intragastric gavage for 5 consecutive days by adopting physiological saline (A) or cefuroxime (0.26g/kg) and levofloxacin (0.20g/kg) (B) to construct and verify a stable mouse model of antibiotic-associated diarrhea; after the clear modeling was successful, A, B groups of mice were randomly divided into two groups (a1, a2, B1, B2) respectively, and gavage was continued for 4 days with physiological saline (a1, B1) or a composite powder of the probiotic composition of the present invention (a2, B2); during which time mice were observed for fecal status, diarrhea scores, and weight changes. Gavage with 0.2ml of absolute ethanol on day 9, blood was taken 1h later for MDA detection, then mice were sacrificed and gastric tissue was taken, changes in gastric mucosa were observed and histopathological analysis was performed.

1) During the course of antibiotic gavage, mice in group B1/B2 (antibiotic group) showed listlessness, increased water intake, decreased food intake, increased frequency of defecation, and significantly lower weight gain compared to group A1/A2 (normal saline group) (FIG. 5). After 5 days of gastric lavage, mice in group B1/group B2 (antibiotic group) all have diarrhea, the feces gradually become soft, most of the feces become light in color and thin and soft, occasionally loose and amorphous, and feces adhere to the anus (figure 6) in severe cases; diarrhea scores were seen in the B1/B2 group (antibiotic group) with significantly higher diarrhea scores than the A1/A2 group (saline group) (FIG. 7).

2) Gavage was continued with physiological saline (a1, B1) or a composite powder of the probiotic composition of the invention (a2, B2) for 4 days. After four days of gastric perfusion treatment, compared with the B1 group (a normal saline group is applied after antibiotic molding), the feces of the B2 group (a composite powder group is applied after antibiotic molding) mice are gradually molded and hardened, while the feces of the B1 group (a normal saline group is applied after antibiotic molding) mice after four days are still soft and light in color, and loose and amorphous occasionally (figure 8); ② compared with the group A1 (group continuously applying normal saline), the A2 group (group applying composite powder after normal saline treatment) has no obvious change of stool character and no obvious increase of defecation frequency (figure 8).

3) After the treatment of the gastric perfusion with absolute ethanol, blood is taken for MDA detection, then the mice are sacrificed to take gastric tissue, the change of the gastric mucosa is observed and the pathological histology is analyzed. It can be seen that the mice treated prophylactically with the composite powder of probiotic composition of the invention (group a2, B2) showed substantially lower degree of ulceration than the saline-treated group (group a1, B1) (fig. 9) and MDA values lower than the saline-treated group (group a1, B1) (fig. 10).

Growth inhibitory Effect against intestinal cancer cells

In vitro experiments prove that the composite powder of the probiotic composition has the growth inhibition effect on human-derived and murine-derived colorectal cancer cells HCT116 and MC38

1. Preparation of composite powder treatment liquid

(1) Adding 0.1g of composite powder of the probiotic composition into 20ml of brain heart leaching culture medium, culturing for 48 hours in an anaerobic incubator, centrifuging for 10 minutes at 1000g of rotation speed of a centrifuge, and filtering supernatant by a 0.22um filter membrane to obtain composite powder culture solution (mainly bacterial culture supernatant);

(2) 5g of composite powder of the probiotic composition is dissolved in 20ml of PBS, and after the composite powder is completely dissolved, the composite powder is filtered by a 0.22um filter membrane to obtain composite powder dissolved solution;

(3) and mixing the composite powder culture solution and the composite powder dissolving solution according to the volume ratio of 1:1 to prepare a composite powder treatment solution.

2. Growth inhibitory effect of composite powder treatment fluid on colorectal cancer cells.

Human and murine colorectal cancer cells HCT116 and MC 3824 or 48 hours are respectively treated by composite powder treatment solutions with different volume ratios (0, 5%, 10%, 15%, 20%, 30%), and the growth inhibition effect on the colorectal cancer cells is gradually enhanced along with the increase of the concentration of the composite powder treatment solution (figures 11-12), so that the targeted delivery of the prepared capsules to the rectum for treating the colorectal cancer can be prepared.

And (4) conclusion: the composite powder treatment of the invention can improve the antibiotic diarrhea condition, has a preventive effect on alcoholic gastric ulcer, and can inhibit the growth of colorectal cancer cells.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A multifunctional probiotic composition, comprising a probiotic powder, a prebiotic powder, and a herbal material, wherein:

the probiotic powder consists of lactobacillus acidophilus, bifidobacterium lactis, lactobacillus paracasei and lactobacillus rhamnosus;

the prebiotics powder comprises fructo-oligosaccharide powder and oligomeric semi-lactose powder;

the Chinese herbal medicine materials comprise cordyceps militaris, shiitake mushroom and turmeric.

2. The multifunctional probiotic composition according to claim 1, characterized in that it comprises, per 15g of probiotic composition:

3. The multifunctional probiotic composition according to claim 2, characterized in that it comprises, per 15g of probiotic composition:

4. The multifunctional probiotic composition according to claim 1, characterized in that said probiotic powder consists of lactobacillus acidophilus NCFM, bifidobacterium lactis HN019, lactobacillus paracasei LPC37, lactobacillus rhamnosus HN 001.

5. Use of a multifunctional probiotic composition according to any one of claims 1 to 4, for the preparation of a medicament for the treatment of rectal cancer.

6. Use of a multifunctional probiotic composition according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of antibiotic diarrhea.

7. Use of a multifunctional probiotic composition according to any one of claims 1 to 4, in the preparation of a medicament for the treatment of alcoholic gastric ulcers.

8. Use of a multifunctional probiotic composition according to any one of claims 1 to 4 in the preparation of a medicament for the treatment of inflammatory bowel disease.