CN116970530B - Bifidobacterium breve FPHC4024 for preventing and treating infantile diarrhea and application thereof - Google Patents
Bifidobacterium breve FPHC4024 for preventing and treating infantile diarrhea and application thereof Download PDFInfo
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- CN116970530B CN116970530B CN202310984734.7A CN202310984734A CN116970530B CN 116970530 B CN116970530 B CN 116970530B CN 202310984734 A CN202310984734 A CN 202310984734A CN 116970530 B CN116970530 B CN 116970530B
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Abstract
The invention provides bifidobacterium breve FPHC4024 for preventing and treating infantile diarrhea and application thereof, and belongs to the technical field of probiotics. The bifidobacterium breve FPHC4024 is classified and named as bifidobacterium breve Bifidobacterium breve, the preservation number is GDMCC No.62945, the preservation date is 2022, 11 months and 3 days, and the preservation unit is the microorganism strain preservation center in Guangdong province, address: building 5, guangzhou city, first, china, no. 100, university, no. 59, university of Guangdong university, institute of microorganisms, post code: 510070. the invention provides the bifidobacterium breve FPHC4024 which can cause the absorption of excessive moisture in colon, has good effects of preventing, relieving and treating infantile diarrhea, and has the advantages of good curative effect, quick response, safety, no side effect, short treatment period and the like.
Description
Technical Field
The invention relates to the technical field of probiotics, in particular to bifidobacterium breve FPHC4024 for preventing and treating infantile diarrhea and application thereof.
Background
Diarrhea is the main symptom of enteritis, and clinically, the frequency of defecation is obviously higher than that of daily habit, the stool is thin, the water content is increased, and the daily defecation amount is large, or undigested food or pus blood and mucus are contained. Some patients have fever and tenesmus, if not properly treated, complications such as bleeding, perforation, toxic intestinal dilatation, polyp hyperplasia or canceration can occur, and the like, and the health hazard to the human body is great. Wherein, the infantile viral enteritis, also called autumn diarrhea, is permeability diarrhea caused by small intestine microvilli absorption dysfunction and disaccharide enzyme activity reduction caused by invasion of Rotavirus (RV) into small intestine mucous membrane epithelial cells, thereby causing cell structure change, imbalance of water and electrolyte transport in intestinal tracts. The infantile viral enteritis is well developed in infants from 6 months to 6 years old, 48.4% -77.1% of the yearly diarrhea cases are related to RV, and RV is the most common cause of diarrhea in autumn and winter of the infants. Clinical symptoms are usually mainly acute diarrhea, fever and upper respiratory tract infection, and serious patients can have complications such as dehydration, acidosis and the like, thus endangering the lives of children.
At present, the method for clinically treating diarrhea mainly uses antibiotics and prevents dehydration, acidosis and other symptomatic treatments, and has poor treatment effect; in particular to diarrhea caused by viral enteritis, no effective treatment means exists, and probiotics are a method for effectively preventing and treating infantile diarrhea, and the method has good effect, no side effect and safer.
Disclosure of Invention
The invention aims to provide the bifidobacterium breve FPHC4024 for preventing and treating the infantile diarrhea and the application thereof, and the bifidobacterium breve FPHC4024 is used in combination with superfine powder, probiotics, prebiotics and Zn-Ca enzymolysis products, so that the gastrointestinal mucosa of an infant is repaired, the harmful bacteria resistance of organisms is enhanced, the gastrointestinal flora state of the infant is improved, the organism immunity of the infant is finally enhanced, the infant can be recovered in a short time, and the application prospect is wide.
The technical scheme of the invention is realized as follows:
the invention provides a bifidobacterium breve FPHC4024 for preventing and treating diarrhea, wherein the bifidobacterium breve FPHC4024 is classified and named as bifidobacterium breve Bifidobacterium breve, the preservation number is GDMCC NO.62945, the preservation date is 2022, 11 months and 3 days, the preservation unit is the Guangdong province microorganism strain preservation center, and the address is: building 5, guangzhou city, first, china, no. 100, university, no. 59, university of Guangdong university, institute of microorganisms, post code: 510070.
As a further improvement of the invention, the 16S rRNA sequence of the bifidobacterium breve FPHC4024 is shown in SEQ ID No. 1.
As a further improvement of the invention, the bifidobacterium breve FPHC4024 is a rod-shaped bacterium of gram positive bacteria, and bacterial colonies on an MRS culture medium are round, regular in edge, milky white, convex, smooth in surface and regular in edge.
The invention further provides a microbial preparation containing the bifidobacterium breve FPHC4024, wherein the viable count of the bifidobacterium breve FPHC4024 in the viable preparation is 10 9 -10 12 cfu/g。
The invention further provides an application of the bifidobacterium breve FPHC4024 in preparing products for preventing and treating infantile diarrhea.
The invention further provides a preparation method of the composition for preventing and treating infantile diarrhea, which comprises the following steps:
s1, activating strains: inoculating probiotics into a Gao's culture medium, and performing activation culture to obtain strain seed liquid;
s2, fermenting: cleaning radix Scutellariae, rhizoma Coptidis and rhizoma Dioscoreae, drying, pulverizing, adding into water, sterilizing, inoculating the probiotic strain seed liquid obtained in step S1, fermenting, culturing, filtering, retaining solid, and lyophilizing the filtrate to obtain fermented product;
s3, protease enzymolysis: adding protease into the solid obtained in the step S2 for enzymolysis, inactivating enzyme, filtering, reserving the solid, and freeze-drying the filtrate to obtain an enzymolysis product;
Complexing of Zn-Ca: dissolving soluble zinc salt and soluble calcium salt in water, adding the enzymolysis product obtained in the step S3, stirring, mixing, reacting, and freeze drying to obtain Zn-Ca complex enzymolysis product;
s5, mixing superfine powder: drying, crushing and sieving the solid in the step S3 to obtain solid fine powder, and uniformly mixing the solid fine powder with montmorillonite powder to obtain superfine powder;
s6, preparation of prebiotics: uniformly mixing soybean oligosaccharide and isomaltooligosaccharide to prepare prebiotics;
s7, preparing a composition for preventing and treating infantile diarrhea: adding the fermentation product obtained in the step S2 into water, adding sodium alginate, sodium carboxymethylcellulose and an emulsifying agent, stirring and mixing uniformly, adding into fish oil, emulsifying by a rapid membrane, dripping a calcium chloride solution, solidifying at normal temperature, freeze-drying to obtain fermentation product microspheres, and mixing uniformly with the Zn-Ca complex enzymolysis product obtained in the step S4, the superfine powder obtained in the step S5 and the prebiotics obtained in the step S6 to obtain the composition for preventing and treating infantile diarrhea.
As a further improvement of the invention, the temperature of the activation culture in the step S1 is 36-38 ℃ and the time is 18-24 hours, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu/mL; in the step S2, the mass ratio of the baical skullcap root to the golden thread to the Chinese yam to the water is 7-10:5-7:5-7:120-150, the fermentation culture condition is 36-38 ℃,50-70r/min, and the fermentation culture is carried out for 48-72h; the protease in the step S3 is at least one selected from trypsin, pepsin, neutral protease, alkaline protease, papain, ficin and bromelain, preferably is a mixture of trypsin and alkaline protease, the mass ratio is 5-7:3, the enzymolysis temperature is 35-40 ℃, and the enzymolysis time is 2-3h; in the step S4, at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc acetate, zinc citrate and zinc gluconate is selected as the soluble zinc salt, at least one of calcium chloride, calcium gluconate, calcium nitrate and calcium bicarbonate is selected as the soluble calcium salt, the soluble calcium salt and enzymolysis products are in a mass ratio of 3-5:1-2:15-20, and the stirring and mixing reaction time is 20-30min; the mesh number of the screened screen mesh in the step S5 is 500-1000 meshes, and the mass ratio of the solid fine powder to the montmorillonite powder is 10-12:15-20; the mass ratio of the soybean oligosaccharide to the isomaltooligosaccharide in the step S6 is 3-5:7, preparing a base material; in the step S7, the mass ratio of the fermentation product, sodium alginate to sodium carboxymethyl cellulose and the emulsifier is 12-15:7-10:3-5:0.5-1, the emulsifier is at least one selected from lecithin, sodium stearoyl lactate, calcium stearoyl lactate, diacetyl tartaric acid monoglyceride, sucrose fatty ester and distilled monoglyceride, the pore diameter of the membrane of the rapid membrane is 1-5 microns, the normal temperature curing time is 20-30min, and the mass ratio of the fermentation product microsphere, zn-Ca complex enzymolysis product, superfine powder and prebiotic is 15-20:7-12:12-15:2-3.
As a further improvement of the invention, the probiotics is bifidobacterium breve FPHC4024, and the inoculation amount of the seed liquid of the probiotics strain is 2.5-3.5%.
As a further improvement of the invention, the probiotics are a composition of bifidobacterium breve FPHC4024, lactobacillus bulgaricus and streptococcus thermophilus, and the seed liquid of the probiotics strain has the following inoculation amount: the inoculum sizes of the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus strain seed liquid are respectively 2-3%, 1-2% and 0.5-1.2%.
The invention further provides a composition for preventing and treating infantile diarrhea prepared by the preparation method.
The invention has the following beneficial effects:
the bifidobacterium breve FPHC4024 has high gastric acid resistant survival rate, can cause the absorption of excessive moisture in colon, has good effects of preventing, relieving and treating infantile diarrhea, promoting the proliferation and colonization of probiotics in vivo, inhibiting the growth of harmful bacteria, constructing a healthy biological barrier on the surface layer of gastrointestinal mucosa, preventing the invasion of the harmful bacteria, improving the intestinal environment of the infant, avoiding the continuous reproduction and growth of the harmful bacteria, finally improving the state of the gastrointestinal flora of the infant, relieving diarrhea symptoms, reducing the incidence and severity of diarrhea, and has the advantages of good curative effect, quick response, safety, no side effect, short treatment period and the like.
The invention provides a composition for preventing and treating infantile diarrhea, wherein:
the Scutellariae radix contains baicalein, wogonin, benzoic acid, beta-sitosterol, protein, polysaccharide, etc., and has effects of inhibiting microorganism such as Bacillus dysenteriae, diphtheria bacillus, staphylococci, pseudomonas aeruginosa, streptococcus, etc., and has antiinflammatory and antiallergic effects, wogonin has spasmolytic effects, and baicalin has antiviral and tranquilizing effects. The rhizoma Coptidis contains berberine, coptisine, palmitoleine, phellodendrine, protein, polysaccharide, etc., and has antibacterial and acetylcholine antagonism effects, and also has antipyretic, antiviral, cholagogic, antidiuretic, local anesthetic, tranquilizing, and analgesic effects. The Chinese yam contains rich nutrients such as protein, fat, carbohydrate, cellulose, vitamin C, vitamin B6, magnesium, calcium and the like, also contains special mucin, has the effect of protecting gastric mucosa, can relieve gastrointestinal discomfort, contains Chinese yam saponin with good anti-inflammatory, antioxidant and antitumor effects, and has the effect of regulating immunity, enhancing immunity and improving organism resistance. Under the synergistic effect of the components, the health-care food has the effects of protecting gastric mucosa, resisting inflammation and oxidation, relieving spasm and fever, relieving gastrointestinal discomfort, providing rich protein sources and having an immunoregulation effect.
The probiotic composition of the invention breaks the cell wall of the traditional Chinese medicine preferentially through fermenting the traditional Chinese medicine composition, promotes the dissolution of active components in the baikal skullcap root, the coptis chinensis and the Chinese yam, improves the drug effect, simultaneously, in the fermentation process, greatly proliferates the probiotic, greatly generates beneficial products, improves the anti-inflammatory effect and the immunocompetence of patients, can maintain the balance of gastrointestinal tract strains of the patients after entering the bodies of the patients, improves the internal environment of the intestinal tract, repairs the intestinal mucosa, obviously improves diarrhea symptoms, controls the regeneration of pathogenic bacteria in the bodies of the patients, eliminates residual bacteria in the bodies, and shortens the recovery time and the disease course time of the patients.
The bifidobacterium breve FPHC4024 has high gastric acid-resistant survival rate, can cause the absorption of excessive moisture in colon, has good effects of preventing, relieving and treating infantile diarrhea, promoting the proliferation and colonization of probiotics in vivo, inhibiting the growth of harmful bacteria, constructing a healthy biological barrier on the surface layer of gastrointestinal mucosa, preventing the invasion of the harmful bacteria, improving the intestinal environment of the infant, avoiding the continuous reproduction and growth of the harmful bacteria, finally improving the state of the intestinal flora of the infant, relieving diarrhea symptoms, reducing the incidence and severity of diarrhea, and has the advantages of good curative effect, quick response, safety, no side effect, short treatment period and the like;
The Lactobacillus bulgaricus can ferment sugar in food into lactic acid, reduce pH value in intestinal canal, inhibit growth of bad bacteria, decompose complex carbohydrate in food, provide nutrients for other beneficial bacteria in intestinal canal, increase number of beneficial bacteria in intestinal canal, reduce proliferation of harmful bacteria, improve intestinal flora balance, promote normal function of immune system, and enhance body resistance
The streptococcus thermophilus can ferment saccharides to produce organic acids such as lactic acid, reduce the pH value of the environment, can produce polysaccharide, bacteriocin and lactic acid, has the effects of resisting inflammation and oxidization, can help lactose intolerant people to decompose lactose, can help children to decompose lactose, can reduce diarrhea caused by lactose intolerance, has symbiotic effect with lactobacillus bulgaricus, can cooperatively produce more beneficial substances, and can promote intestinal health.
Under the synergistic effect of the single bifidobacterium breve FPHC4024 or the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus, the activity of the decomposition products is beneficial to gastrointestinal peristalsis, and the harmful substances immobilized by the montmorillonite powder can be discharged from the intestinal tract, meanwhile, the defect that constipation is easily caused by excessive montmorillonite powder is avoided, the immune dysfunction of children patients is improved, and the safety of the composition is improved. Meanwhile, the number of beneficial bacteria in the body can be increased, the probiotic composition can be combined with other anaerobic bacteria to construct a biological barrier on the gastrointestinal mucosa surface layer of the infant to prevent invasion of harmful bacteria, acetic acid and lactic acid can be secreted from the probiotic composition, the intestinal tract environment of the infant can be improved, continuous reproduction and growth of harmful bacteria are avoided, the gastrointestinal flora state of the infant is improved, and diarrhea symptoms are relieved.
Zinc is a trace element, and can improve the growth and development effects of children, strengthen the immunity of human bodies and promote the absorption of vitamin A. The zinc element is lacking in the body, the damage to the growth of metabolic cells of the human body can be caused, the diarrhea condition of children is very easy to occur, meanwhile, zinc is also an important electrolyte, the zinc demand is relatively large in the growth process of infants, and a large amount of zinc element can be lost after diarrhea diseases are generated, so that diarrhea symptoms are more serious. The invention uses proteinase enzymolysis to make the protein enzymolysis in the traditional Chinese medicine component produce a large amount of active substances such as small molecular protein peptide, short peptide, amino acid and the like, has good chelating and fixing effects on metal zinc ions and calcium ions, so that the composition contains abundant zinc elements and calcium elements, the increase of the zinc elements can prevent and treat infantile diarrhea, the calcium elements contain not less than 100 enzymes and activating factors, and can be directly used for synthesizing nucleic acid and protein, can also play roles of oxidation reduction, energy metabolism, humoral and cellular immunity and the like, and is one of main components for forming small intestines. By supplementing zinc and calcium elements, the method can promote the improvement of human T helper cells and T suppressor cells, improve the immunity of human bodies, improve diarrhea sensitivity, strengthen cellular immunity, protect intestinal mucosa, inhibit harmful bacteria in intestinal tracts, ensure that local intestinal tracts have an immune function, and effectively remove bacteria.
The montmorillonite powder has the effect of strengthening the covering capacity of the digestive tract mucosa, can effectively inhibit viruses, germs, toxins and the like in the digestive tract, so that the barrier defense function of the digestive tract mucosa of the infant is improved, and the gastrointestinal function recovery of the infant is promoted, but the problems of constipation and the like are easily caused, therefore, the invention dries and superfine pulverizes the solid residue after the fermentation and extraction of the traditional Chinese medicine to obtain the traditional Chinese medicine fine powder, uniformly mixes the traditional Chinese medicine fine powder with the montmorillonite powder, and adds the traditional Chinese medicine fine powder into the composition, can cooperate with the montmorillonite powder to strengthen the covering capacity of the digestive tract mucosa, and simultaneously, the traditional Chinese medicine fiber can promote intestinal peristalsis, relieve the constipation problem caused by the montmorillonite powder, and discharge the harmful substances fixed by the montmorillonite powder from the intestinal tract, thereby ensuring that the composition is safer and more effective.
The superfine powder, probiotics, prebiotics and Zn-Ca enzymolysis products are jointly applied, so that the gastrointestinal mucosa of the infant is repaired, the harmful bacteria resistance of the organism is enhanced, the gastrointestinal flora state of the infant is improved, the organism immunity of the infant is finally enhanced, the infant can be promoted to recover in a short time, and the infant has a wide application prospect.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a colony morphology of Bifidobacterium breve FPHC 4024;
fig. 2 is a bacterial cell morphology diagram of bifidobacterium breve FPHC 4024.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a bifidobacterium breve FPHC4024, which is classified and named as bifidobacterium breve Bifidobacterium breve, and has the preservation number of GDMCC No.62945, the preservation date of 2022, 11 and 3 days, and the preservation unit of the microorganism strain preservation center in Guangdong province, and addresses: building 5, guangzhou city, first, china, no. 100, university, no. 59, university of Guangdong university, institute of microorganisms, post code: 510070.
MRS liquid culture medium comprises the following components: 10g/L of peptone, 5g/L of beef extract, 4g/L of yeast extract, 20g/L of glucose, 2g/L of dipotassium hydrogen phosphate, 2g/L of triammonium citrate, 5g/L of sodium acetate, 80 1mL of Tween, 0.2g/L of magnesium sulfate and 0.05g/L of manganese sulfate, which are purchased from Beijing Soy Bao technology Co.
MRS solid culture medium comprises the following components: 10g/L of peptone, 8g/L of beef extract, 4g/L of yeast extract, 20g/L of glucose, 2g/L of dipotassium hydrogen phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14g/L of agar and 80 1mL of Tween, which are purchased from Qingdao sea Bo biotechnology Co.
Example 1 screening and identification of Bifidobacterium breve FPHC4024
1. Screening of bifidobacterium breve FPHC4024
1.1 sample Source
The strain used by the invention is all collected from the fermented mare milk product of inner Mongolia, namely self-made mare milk, namely the self-made mare milk from inner Mongolia is taken as a sample.
1.2 isolation of strains
1g of the sample was placedAdding into 10mL MRS liquid culture medium, mixing, culturing at 36deg.C, collecting enrichment solution 1mL in ultra clean bench, performing ten-fold gradient dilution, and selecting 10 -5 、10 -6 、10 -7 Three bacterial solutions with dilution gradient of 100 mu L are coated on a culture dish containing a sterile MRS solid culture medium, and are subjected to static culture for 24-48 hours at 38 ℃ under the aerobic condition until obvious single colonies are formed, a plate with 50-90 single colonies growing on the culture medium is selected, typical colonies are selected, and the culture medium is subjected to repeated streak purification on the MRS solid plate culture medium until the colony morphology of the whole plate is consistent, so that the culture is obtained, and the strain identification of the culture is performed.
1.3 preservation of strains
Single colonies were selected and cultured in MRS liquid medium at 37℃for 36 hours, 500. Mu.L of the bacterial liquid was aspirated and added to 0.8mL of 60% (v/v) glycerol tubes, and the resulting culture was frozen at-80 ℃.
2. Identification of bifidobacterium breve FPHC4024
2.1 colony characterization
After culturing bifidobacterium breve FPHC4024 in MRS solid medium for 24 hours, the bifidobacterium breve FPHC4024 is round, and has neat edges, milky white, convex, smooth surfaces and neat edges, as shown in FIG. 1.
2.2 morphology under microscope
Bifidobacterium breve FPHC4024 colony smear: gram staining was positive and the cells were rod-shaped, single, paired, as shown in figure 2.
2.3 16S rRNA identification
Identification unit: the microbiological analysis and detection center in Guangdong province.
Identification sequence:
GGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCATCACACCGCATGGTGTGTTGGGAAAGCCTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGCACTTTGTGTTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGATCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGTGCGAGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGT。
identification result: sample bifidobacterium breve FPHC4024 (GDMCC 62945) the 16S rRNA gene sequence obtained by sequencing was compared with the registered sequence for nucleotide homology of 100% to Bifidobacterium breve (bifidobacterium breve) and the selected strain was identified as Bifidobacterium breve (bifidobacterium breve).
Example 2 preparation of Bifidobacterium breve FPHC4024 powder
Taking out glycerol tube containing Bacillus brevis FPHC4024 from-80deg.C, and performing activation culture to obtain strain with a bacterial content of 10 8 After diluting cfu/g strain seed liquid by 100 times, inoculating into MRS liquid culture medium with an inoculum size of 2wt%, anaerobically culturing for 24h (environment with carbon dioxide concentration of 5v/v% and nitrogen concentration of 95 v/v%) at 37 ℃, centrifuging the strain liquid, freeze-drying to obtain bifidobacterium breve FPHC4024 powder, and measuring the viable count of bifidobacterium breve FPHC4024 strain to be 10 11 cfu/g。
The conditions for the activation culture and the fermentation culture in the following examples were anaerobic conditions, and the carbon dioxide concentration was 5v/v% and the nitrogen concentration was 95v/v%.
Lactobacillus bulgaricus, 100 hundred million cfu/g, purchased from Wilkihai Sisi (Shandong) bioengineering Co., ltd; streptococcus thermophilus, STN-26, 100 hundred million cfu/g, purchased from Jia Yi bioengineering Co., ltd; trypsin, 4000U/g, alkaline protease, 100000U/g, available from Nanning Donghenghua biological technology Co., ltd; montmorillonite powder with content >99% was purchased from inner Mongolian Chuangjia pasture biotechnology Co.
Test example 1 antibiotic resistance test
The sensitivity of the bifidobacterium breve FPHC4024 to the antibacterial drugs is detected, and the Minimum Inhibitory Concentration (MIC) value is obtained according to the detection of the direct feeding microorganism and fermentation product production strain identification and safety evaluation guidelines of agricultural production and grazing [ 2021 ] 43.
The results are shown in Table 1.
TABLE 1
As is clear from the above table, the bifidobacterium breve FPHC4024 has good drug resistance to the antibacterial drugs.
Example 3
The embodiment provides a preparation method of a composition for preventing and treating infantile diarrhea, which comprises the following steps:
s1, activating strains: taking out glycerol tube containing Bacillus brevis FPHC4024 from-80deg.C, inoculating into Gao's medium, and performing activation culture at 36deg.C for 18 hr to obtain strain with a bacterial content of 10 8 cfu/mL bifidobacterium breve FPHC4024 strain seed liquid;
s2, fermenting: cleaning 7 parts by weight of radix scutellariae, 5 parts by weight of coptis chinensis and 5 parts by weight of yam, drying, crushing, adding 120 parts by weight of water, sterilizing, inoculating the bifidobacterium breve FPHC4024 strain seed liquid prepared in the step S1, fermenting and culturing for 48 hours at 36 ℃ and 50r/min, filtering, reserving solids, and freeze-drying filtrate to obtain a fermentation product;
s3, protease enzymolysis: adding protease into the solid obtained in the step S2, carrying out enzymolysis for 2 hours at 35 ℃, inactivating the enzyme, filtering, reserving the solid, and freeze-drying the filtrate to obtain an enzymolysis product;
the protease is a mixture of trypsin and alkaline protease, and the mass ratio is 5:3;
complexing of Zn-Ca: 3 parts by weight of zinc chloride and 1 part by weight of calcium chloride are dissolved in 100 parts by weight of water, 15 parts by weight of the enzymolysis product prepared in the step S3 are added, stirred and mixed for reaction for 20min, and freeze-dried to prepare a Zn-Ca complex enzymolysis product;
S5, mixing superfine powder: drying the solid in the step S3, crushing and sieving with a 500-mesh sieve to obtain solid fine powder, and uniformly mixing 10 parts by weight of the solid fine powder and 15 parts by weight of montmorillonite powder to obtain superfine powder;
s6, preparation of prebiotics: mixing 3 parts by weight of soybean oligosaccharide and 7 parts by weight of isomaltooligosaccharide for 20min to prepare prebiotics;
s7, preparing a composition for preventing and treating infantile diarrhea: adding 12 parts by weight of the fermentation product prepared in the step S2 into 100 parts by weight of water, adding 7 parts by weight of sodium alginate, 3 parts by weight of sodium carboxymethylcellulose and 0.5 part by weight of calcium stearoyl lactylate, stirring and mixing for 20min, adding 200 parts by weight of fish oil, emulsifying by a rapid membrane, wherein the pore diameter of the rapid membrane is 1 micrometer, dripping 20 parts by weight of 5wt% calcium chloride solution, solidifying for 20min at normal temperature, freeze-drying to obtain fermentation product microspheres, and mixing 15 parts by weight of the fermentation product microspheres with 7 parts by weight of the Zn-Ca complex enzymolysis product prepared in the step S4, 12 parts by weight of the superfine powder prepared in the step S5 and 2 parts by weight of the prebiotics prepared in the step S6 for 20min to obtain the composition for preventing and treating infantile diarrhea.
Example 4
The embodiment provides a preparation method of a composition for preventing and treating infantile diarrhea, which comprises the following steps:
S1, activating strains: taking out glycerol tube containing Bacillus brevis FPHC4024 from-80deg.C, inoculating with Lactobacillus bulgaricus and Streptococcus thermophilus respectively into Gao's medium, and performing activation culture at 38deg.C for 24 hr to obtain strain with a bacterial content of 10 9 cfu/mL strain seed liquid;
s2, fermenting: cleaning, drying, crushing, adding 150 parts by weight of water, sterilizing, inoculating the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus seed liquid prepared in the step S1, wherein the inoculum sizes of the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus seed liquid are respectively 2%, 1% and 0.5%,38 ℃,70r/min, fermenting and culturing for 72 hours, filtering, reserving solids, and freeze-drying filtrate to prepare a fermentation product;
s3, protease enzymolysis: adding protease into the solid obtained in the step S2, carrying out enzymolysis for 3 hours at 40 ℃, inactivating the enzyme, filtering, reserving the solid, and freeze-drying the filtrate to obtain an enzymolysis product;
the protease is a mixture of trypsin and alkaline protease, and the mass ratio of the protease to the alkaline protease is 7:3;
complexing of Zn-Ca: dissolving 5 parts by weight of zinc nitrate and 2 parts by weight of calcium nitrate in 100 parts by weight of water, adding 20 parts by weight of the enzymolysis product prepared in the step S3, stirring and mixing for reaction for 30min, and freeze-drying to prepare a Zn-Ca complex enzymolysis product;
S5, mixing superfine powder: drying the solid in the step S3, crushing and sieving with a 1000-mesh sieve to obtain solid fine powder, and uniformly mixing 12 parts by weight of the solid fine powder with 20 parts by weight of montmorillonite powder to obtain superfine powder;
s6, preparation of prebiotics: mixing 5 parts by weight of soybean oligosaccharide and 7 parts by weight of isomaltooligosaccharide for 20min to prepare prebiotics;
s7, preparing a composition for preventing and treating infantile diarrhea: adding 15 parts by weight of the fermentation product prepared in the step S2 into 100 parts by weight of water, adding 10 parts by weight of sodium alginate, 5 parts by weight of sodium carboxymethylcellulose and 1 part by weight of sodium stearoyl lactylate, stirring and mixing for 20min, adding 200 parts by weight of fish oil, emulsifying by a rapid membrane, wherein the pore diameter of the rapid membrane is 5 microns, dripping 20 parts by weight of 5wt% calcium chloride solution, solidifying for 30min at normal temperature, freeze-drying to prepare fermentation product microspheres, and mixing 20 parts by weight of the fermentation product microspheres with 12 parts by weight of the Zn-Ca complex enzymolysis product prepared in the step S4, 15 parts by weight of the superfine powder prepared in the step S5 and 3 parts by weight of the prebiotics prepared in the step S6 for 20min to prepare the composition for preventing and treating infantile diarrhea.
Example 5
The embodiment provides a preparation method of a composition for preventing and treating infantile diarrhea, which comprises the following steps:
S1, activating strains: taking out glycerol tube containing Bacillus brevis FPHC4024 from-80deg.C, inoculating with Lactobacillus bulgaricus and Streptococcus thermophilus respectively into Gao's medium, and performing activation culture at 37deg.C for 21 hr to obtain strain with a bacterial content of 10 9 cfu/mL strain seed liquid;
s2, fermenting: cleaning 8.5 parts by weight of radix scutellariae, 6 parts by weight of coptis chinensis and 6 parts by weight of yam, drying, crushing, adding into 135 parts by weight of water, sterilizing, inoculating the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus seed liquid prepared in the step S1, wherein the inoculum sizes of the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus seed liquid are respectively 2.5%, 1.5% and 0.7%,37 ℃,60r/min, fermenting and culturing for 56 hours, filtering, reserving solids, and freeze-drying filtrate to obtain a fermentation product;
s3, protease enzymolysis: adding protease into the solid obtained in the step S2, carrying out enzymolysis for 2.5 hours at 37 ℃, inactivating the enzyme, filtering, reserving the solid, and freeze-drying the filtrate to obtain an enzymolysis product;
the protease is a mixture of trypsin and alkaline protease, and the mass ratio is 6:3;
complexing of Zn-Ca: dissolving 4 parts by weight of zinc gluconate and 1.5 parts by weight of calcium gluconate in 100 parts by weight of water, adding 17 parts by weight of the enzymolysis product prepared in the step S3, stirring and mixing for reaction for 25min, and freeze-drying to obtain a Zn-Ca complex enzymolysis product;
S5, mixing superfine powder: drying the solid in the step S3, crushing and sieving with a 700-mesh sieve to obtain solid fine powder, and uniformly mixing 11 parts by weight of the solid fine powder with 17 parts by weight of montmorillonite powder to obtain superfine powder;
s6, preparation of prebiotics: mixing 4 parts by weight of soybean oligosaccharide and 7 parts by weight of isomaltooligosaccharide for 20min to prepare prebiotics;
s7, preparing a composition for preventing and treating infantile diarrhea: adding 13.5 parts by weight of the fermentation product prepared in the step S2 into 100 parts by weight of water, adding 8.5 parts by weight of sodium alginate, 4 parts by weight of sodium carboxymethylcellulose and 0.7 part by weight of lecithin, stirring and mixing for 20min, adding into 200 parts by weight of fish oil, emulsifying by a rapid membrane, dropwise adding 20 parts by weight of 5wt% calcium chloride solution with the pore diameter of 3 microns, solidifying for 25min at normal temperature, freeze-drying to obtain fermentation product microspheres, and mixing 17 parts by weight of the fermentation product microspheres with 10 parts by weight of Zn-Ca complex enzymolysis product prepared in the step S4, 13.5 parts by weight of superfine powder prepared in the step S5 and 2.5 parts by weight of prebiotics prepared in the step S6 for 20min to obtain the composition for preventing and treating infantile diarrhea.
Example 6
The difference compared to example 5 is that the protease is a single trypsin.
Example 7
The difference compared to example 5 is that the protease is a single alkaline protease.
Comparative example 1
In comparison with example 5, the difference is that no baikal skullcap root is added in step S2.
The method comprises the following steps:
s2, fermenting: 6 parts by weight of coptis chinensis and 6 parts by weight of yam are cleaned, dried, crushed, added into 135 parts by weight of water, sterilized, inoculated with bifidobacterium breve FPHC4024, lactobacillus bulgaricus and streptococcus thermophilus seed liquid prepared in the step S1, and the inoculum sizes of the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus seed liquid are respectively 2.5 percent, 1.5 percent and 0.7 percent, the temperature is 37 ℃, the speed is 60r/min, the fermentation culture is carried out for 56 hours, the filtration is carried out, the solid is reserved, and the filtrate is frozen and dried, thus obtaining the fermentation product.
Comparative example 2
In comparison with example 5, the difference is that no coptis root is added in step S2.
The method comprises the following steps:
s2, fermenting: cleaning 8.5 parts by weight of baical skullcap root and 6 parts by weight of Chinese yam, drying, crushing, adding 135 parts by weight of water, sterilizing, inoculating the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus seed liquid prepared in the step S1, wherein the inoculum sizes of the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus seed liquid are respectively 2.5%, 1.5% and 0.7%,37 ℃ and 60r/min, fermenting and culturing for 56 hours, filtering, reserving solids, and freeze-drying filtrate to obtain a fermentation product.
Comparative example 3
Compared with example 5, the difference is that no yam is added in step S2.
The method comprises the following steps:
s2, fermenting: 8.5 parts by weight of baical skullcap root and 6 parts by weight of coptis root are cleaned, dried, crushed, added into 135 parts by weight of water, sterilized, inoculated with seed liquid of bifidobacterium breve FPHC4024, lactobacillus bulgaricus and streptococcus thermophilus prepared in the step S1, the inoculum sizes of the seed liquid of bifidobacterium breve FPHC4024, lactobacillus bulgaricus and streptococcus thermophilus bacterial are respectively 2.5 percent, 1.5 percent and 0.7 percent, the temperature is 37 ℃ and 60r/min, fermented and cultivated for 56 hours, filtered, reserved for solids, and the filtrate is frozen and dried to prepare a fermented product.
Comparative example 4
The difference from example 5 is that the seed solution of bifidobacterium breve FPHC4024 strain was not inoculated in step S2.
The method comprises the following steps:
s2, fermenting: cleaning 8.5 parts by weight of baical skullcap root, 6 parts by weight of golden thread and 6 parts by weight of Chinese yam, drying, crushing, adding 135 parts by weight of water, sterilizing, inoculating lactobacillus bulgaricus and streptococcus thermophilus seed liquid prepared in the step S1, wherein the inoculum sizes of lactobacillus bulgaricus and streptococcus thermophilus seed liquid are respectively 4% and 0.7%, the temperature is 37 ℃, the speed is 60r/min, fermenting and culturing for 56 hours, filtering, reserving solids, and freeze-drying filtrate to obtain a fermentation product.
Comparative example 5
In comparison with example 5, the difference is that lactobacillus bulgaricus is not inoculated in step S2.
The method comprises the following steps:
s2, fermenting: cleaning 8.5 parts by weight of radix scutellariae, 6 parts by weight of coptis chinensis and 6 parts by weight of yam, drying, crushing, adding into 135 parts by weight of water, sterilizing, inoculating the bifidobacterium breve FPHC4024 and the streptococcus thermophilus seed liquid prepared in the step S1, wherein the inoculum sizes of the bifidobacterium breve FPHC4024 and the streptococcus thermophilus seed liquid are respectively 4% and 0.7%, the temperature is 37 ℃, the speed is 60r/min, fermenting and culturing for 56 hours, filtering, reserving solids, and freeze-drying filtrate to obtain a fermentation product.
Comparative example 6
In comparison with example 5, the difference is that in step S2, fermentation was not performed, and water was added and boiling was used for extraction for 4 hours.
The method comprises the following steps:
s2, fermenting: cleaning 8.5 parts by weight of scutellaria baicalensis, 6 parts by weight of coptis chinensis and 6 parts by weight of yam, drying, crushing, adding 135 parts by weight of water, and boiling and extracting for 4 hours to obtain an extract.
Comparative example 7
In comparison with example 5, the process differs in that the enzymolysis in step S3 is not carried out, and the enzymolysis product in step S4 is replaced by the solid in step S2.
The method comprises the following steps:
complexing of Zn-Ca: and (2) dissolving 4 parts by weight of zinc gluconate and 1.5 parts by weight of calcium gluconate in 100 parts by weight of water, adding 17 parts by weight of the solid in the step (S2), stirring and mixing for reaction for 25min, and freeze-drying to obtain the Zn-Ca complex enzymatic hydrolysate.
Comparative example 8
In comparison with example 5, the difference is that zinc gluconate is not added in step S4.
The method comprises the following steps:
complexing of ca: and 5.5 parts by weight of calcium gluconate is dissolved in 100 parts by weight of water, 17 parts by weight of the enzymolysis product prepared in the step S3 is added, and the mixture is stirred and mixed for 25 minutes to prepare the Ca complex enzymolysis product by freeze drying.
Comparative example 9
In comparison with example 5, the difference is that no calcium gluconate is added in step S4.
The method comprises the following steps:
complexing of Zn: and 5.5 parts by weight of zinc gluconate is dissolved in 100 parts by weight of water, 17 parts by weight of the enzymolysis product prepared in the step S3 is added, and the mixture is stirred and mixed for 25 minutes to prepare the Zn complex enzymolysis product by freeze drying.
Comparative example 10
In comparison with example 5, the difference is that step S4 is not performed, and the Zn-Ca complex enzyme hydrolysis product is replaced by an enzyme hydrolysis product in step S7.
The method comprises the following steps:
s7, preparing a composition for preventing and treating infantile diarrhea: adding 13.5 parts by weight of the fermentation product prepared in the step S2 into 100 parts by weight of water, adding 8.5 parts by weight of sodium alginate, 4 parts by weight of sodium carboxymethylcellulose and 0.7 part by weight of lecithin, stirring and mixing for 20min, adding into fish oil, emulsifying by a rapid membrane, dropwise adding 20 parts by weight of 5wt% calcium chloride solution with the pore diameter of the rapid membrane of 3 microns, solidifying at normal temperature for 25min, freeze-drying to obtain fermentation product microspheres, and mixing 17 parts by weight of the fermentation product microspheres with 10 parts by weight of the enzymolysis product prepared in the step S3, 13.5 parts by weight of the superfine powder prepared in the step S5 and 2.5 parts by weight of the prebiotics prepared in the step S6 for 20min to obtain the composition for preventing and treating infantile diarrhea.
Comparative example 11
In comparison with example 5, the difference is that no solid fine powder was added in step S5.
The method comprises the following steps:
s5, mixing superfine powder: montmorillonite powder is used as superfine powder.
Comparative example 12
The difference from example 5 is that montmorillonite powder was not added in step S5.
The method comprises the following steps:
s5, mixing superfine powder: and (3) drying the solid in the step (S3), crushing and sieving with a 700-mesh sieve to obtain solid fine powder which is superfine powder.
Comparative example 13
In comparison with example 5, the difference is that no ultrafine powder was added in step S7.
The method comprises the following steps:
s7, preparing a composition for preventing and treating infantile diarrhea: adding 13.5 parts by weight of the fermentation product prepared in the step S2 into 100 parts by weight of water, adding 8.5 parts by weight of sodium alginate, 4 parts by weight of sodium carboxymethylcellulose and 0.7 part by weight of lecithin, stirring and mixing for 20min, adding into fish oil, emulsifying by a rapid membrane, dropwise adding 20 parts by weight of 5wt% calcium chloride solution with the pore diameter of 3 microns, solidifying at normal temperature for 25min, freeze-drying to obtain fermentation product microspheres, and mixing 17 parts by weight of the fermentation product microspheres with 10 parts by weight of the Zn-Ca complex enzymolysis product prepared in the step S4 and 2.5 parts by weight of the prebiotics prepared in the step S6 for 20min to obtain the composition for preventing and treating infantile diarrhea.
Comparative example 14
In comparison with example 5, the difference is that no prebiotics are added in step S7.
The method comprises the following steps:
s7, preparing a composition for preventing and treating infantile diarrhea: adding 13.5 parts by weight of the fermentation product prepared in the step S2 into 100 parts by weight of water, adding 8.5 parts by weight of sodium alginate, 4 parts by weight of sodium carboxymethylcellulose and 0.7 part by weight of lecithin, stirring and mixing for 20min, adding into fish oil, emulsifying by a rapid membrane, dropwise adding 20 parts by weight of 5wt% calcium chloride solution with the pore diameter of 3 microns, solidifying at normal temperature for 25min, freeze-drying to obtain fermentation product microspheres, and mixing 17 parts by weight of the fermentation product microspheres with 10 parts by weight of the Zn-Ca complex enzymolysis product prepared in the step S4 and 13.5 parts by weight of the superfine powder prepared in the step S5 for 20min to obtain the composition for preventing and treating infantile diarrhea.
Comparative example 15
In comparison with example 5, the difference is that no embedding is performed in step S7, and the components are simply mixed.
The method comprises the following steps:
s7, preparing a composition for preventing and treating infantile diarrhea: and (3) mixing 17 parts by weight of the fermentation product with 10 parts by weight of the Zn-Ca complex enzymatic hydrolysate prepared in the step S4, 13.5 parts by weight of the superfine powder prepared in the step S5 and 2.5 parts by weight of the prebiotics prepared in the step S6 for 20min to prepare the composition for preventing and treating the infantile diarrhea.
Test example 2
1. Grouping and dosing animals:
healthy, clean-grade female BALB/C mice were randomly assigned to normal, model, examples 2-7, and comparative examples 1-15, 10 animals per group, after 3d of laboratory-adaptive rearing. The mice of examples 2 to 7 and comparative examples 1 to 15 were subjected to gastric administration with the corresponding prepared fungus powder or composition, 1 g/(day), and the mice of the normal group and the model group were respectively subjected to the administration of an equal amount of sterile water 2 times per day for continuous gastric administration for 7d.
2. And (3) molding:
in addition to the normal group, the mice of the other groups were intraperitoneally injected with 0.25 mL/10 after the end of the administration 7 cfu/mL of escherichia coli ATCC25922 bacterial suspension is used for observing the mental state, the fur, the diarrhea and other conditions of the mice, and modeling is successful when diarrhea occurs.
3. Diarrhea condition
The amount of solid feces, semisolid feces, liquid feces and the time of appearance of the initial semisolid feces within 6 hours after administration of the bacterial suspension were observed, and the diarrhea degree was judged by the following formula.
Emptying index = solid feces number x 1+ semisolid feces number x 2+ liquid feces number x 3
The results are shown in Table 2.
TABLE 2
| Group of | Initial semi-solid time (min) | Emptying index |
| Normal group | / | / |
| Model group | 40.72±4.72 | 13.95±2.81 |
| Example 2 | 80.82±6.08* | 6.20±1.18* |
| Example 3 | 81.27±5.62* | 6.12±1.22* |
| Example 4 | 82.15±6.11* | 6.01±1.31* |
| Example 5 | 82.58±4.82* | 5.96±1.38* |
| Example 6 | 77.84±5.12 | 6.45±1.27 |
| Example 7 | 77.59±4.89 | 6.57±1.30 |
| Comparative example 1 | 73.84±4.77 | 7.18±1.24 |
| Comparative example 2 | 73.21±5.10 | 7.12±1.26 |
| Comparative example 3 | 74.19±5.05 | 7.05±1.33 |
| Comparative example 4 | 72.45±4.69 | 7.49±1.39 |
| Comparative example 5 | 72.82±4.72 | 7.44±1.41 |
| Comparative example 6 | 70.71±5.04 | 8.20±1.35 |
| Comparative example 7 | 75.92±4.82 | 6.94±1.44 |
| Comparative example 8 | 73.45±3.88 | 7.10±1.42 |
| Comparative example 9 | 74.27±3.92 | 7.03±1.36 |
| Comparative example 10 | 72.79±4.02 | 7.45±1.26 |
| Comparative example 11 | 73.17±4.17 | 7.14±1.29 |
| Comparative example 12 | 72.49±5.22 | 7.42±1.30 |
| Comparative example 13 | 71.56±4.56 | 7.87±1.21 |
| Comparative example 14 | 73.29±4.37 | 7.10±1.41 |
| Comparative example 15 | 71.10±4.82 | 8.12±1.39 |
Annotation: * P <0.05 for comparison with the model group.
As can be seen from the above table, the bacterial powder prepared in example 2 or the compositions for preventing and treating infantile diarrhea prepared in examples 3 to 5 of the present invention have excellent diarrhea treatment and prevention effects.
4. Index of inflammatory factor
Mice were sacrificed 24h after the end of dosing, and a portion of the small intestine was removed to prepare a small intestine tissue homogenate, and the amounts of inflammatory cytokines TNF-alpha and IL-6 in the supernatant were measured, and the operation method was performed according to ELISA kit instructions.
The results are shown in Table 3.
TABLE 3 Table 3
Annotation: * P <0.05 compared to the normal group; # is P <0.05 compared to model group.
As is clear from the table above, the bacterial powder prepared in example 2 or the compositions for preventing and treating infantile diarrhea prepared in examples 3-5 have good therapeutic effects on intestinal inflammation.
Examples 6 and 7 compare with example 5 in that the protease is a single trypsin or alkaline protease. Comparative example 7 in comparison with example 5, the enzymatic hydrolysis of step S3 was not performed, and the enzymatic hydrolysis product of step S4 was replaced by the solid in step S2. The effect of treating diarrhea is reduced. The invention uses protease enzymolysis to make the protein enzymolysis in the traditional Chinese medicine component produce a large amount of small molecular protein peptide, short peptide, amino acid and other active substances, has good chelation and fixation effects on metal zinc ions and calcium ions, and makes the composition contain abundant zinc elements and calcium elements, thereby playing a better effect.
In comparative examples 1, 2 and 3, as compared with example 5, no scutellaria baicalensis, coptis chinensis or yam was added in step S2. The effect of treating diarrhea is reduced, and the inflammatory factor index is improved. The Scutellariae radix contains baicalein, wogonin, benzoic acid, beta-sitosterol, protein, polysaccharide, etc., and has effects of inhibiting microorganism such as Bacillus dysenteriae, diphtheria bacillus, staphylococci, pseudomonas aeruginosa, streptococcus, etc., and has antiinflammatory and antiallergic effects, wogonin has spasmolytic effects, and baicalin has antiviral and tranquilizing effects. The rhizoma Coptidis contains berberine, coptisine, palmitoleine, phellodendrine, protein, polysaccharide, etc., and has antibacterial and acetylcholine antagonism effects, and also has antipyretic, antiviral, cholagogic, antidiuretic, local anesthetic, tranquilizing, and analgesic effects. The Chinese yam contains rich nutrients such as protein, fat, carbohydrate, cellulose, vitamin C, vitamin B6, magnesium, calcium and the like, also contains special mucin, has the effect of protecting gastric mucosa, can relieve gastrointestinal discomfort, contains Chinese yam saponin with good anti-inflammatory, antioxidant and antitumor effects, and has the effect of regulating immunity, enhancing immunity and improving organism resistance. Under the synergistic effect of the components, the health-care food has the effects of protecting gastric mucosa, resisting inflammation and oxidation, relieving spasm and fever, relieving gastrointestinal discomfort, providing rich protein sources and having an immunoregulation effect.
Comparative examples 4 and 5 in comparison with example 5, bifidobacterium breve FPHC4024 or lactobacillus bulgaricus was not inoculated in step S2. Comparative example 6 in comparison with example 5, no fermentation was performed in step S2, and extraction was performed by boiling with water for 4 hours. The effect of treating diarrhea is reduced, and the inflammatory factor index is improved. The probiotic composition of the invention breaks the cell wall of the traditional Chinese medicine preferentially through fermenting the traditional Chinese medicine composition, promotes the dissolution of active components in the baikal skullcap root, the coptis chinensis and the Chinese yam, improves the drug effect, simultaneously, in the fermentation process, greatly proliferates the probiotic, greatly generates beneficial products, improves the anti-inflammatory effect and the immunocompetence of patients, can maintain the balance of gastrointestinal tract strains of the patients after entering the bodies of the patients, improves the internal environment of the intestinal tract, repairs the intestinal mucosa, obviously improves diarrhea symptoms, controls the regeneration of pathogenic bacteria in the bodies of the patients, eliminates residual bacteria in the bodies, and shortens the recovery time and the disease course time of the patients. The bifidobacterium breve FPHC4024 has high gastric acid-resistant survival rate, can cause the absorption of excessive moisture in colon, has good effects of preventing, relieving and treating infantile diarrhea, promoting the proliferation and colonization of probiotics in vivo, inhibiting the growth of harmful bacteria, constructing a healthy biological barrier on the surface layer of gastrointestinal mucosa, preventing the invasion of the harmful bacteria, improving the intestinal environment of the infant, avoiding the continuous reproduction and growth of the harmful bacteria, finally improving the state of the intestinal flora of the infant, relieving diarrhea symptoms, reducing the incidence and severity of diarrhea, and has the advantages of good curative effect, quick response, safety, no side effect, short treatment period and the like; the lactobacillus bulgaricus can ferment sugar in food into lactic acid, reduce pH value in intestinal canal, inhibit growth of bad bacteria, decompose complex carbohydrate in food, provide nutrition for other beneficial bacteria in intestinal canal, increase number of beneficial bacteria in intestinal canal, reduce reproduction of harmful bacteria, improve balance of intestinal flora, simultaneously promote normal functions of immune system positively, enhance resistance streptococcus thermophilus of organism, ferment sugar to produce organic acid such as lactic acid, reduce environmental pH value, produce polysaccharide, bacteriocin and lactic acid, have anti-inflammatory and antioxidant effects, can help lactose intolerant people decompose lactose, help children decompose lactose, reduce diarrhea problem caused by lactose intolerance, have symbiotic effect with lactobacillus bulgaricus, synergistically produce more beneficial substances, and promote intestinal health. Under the synergistic effect of the single bifidobacterium breve FPHC4024 or the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus, the activity of the decomposition products is beneficial to gastrointestinal peristalsis, and the harmful substances immobilized by the montmorillonite powder can be discharged from the intestinal tract, meanwhile, the defect that constipation is easily caused by excessive montmorillonite powder is avoided, the immune dysfunction of children patients is improved, and the safety of the composition is improved. Meanwhile, the number of beneficial bacteria in the body can be increased, the probiotic composition can be combined with other anaerobic bacteria to construct a biological barrier on the gastrointestinal mucosa surface layer of the infant to prevent invasion of harmful bacteria, acetic acid and lactic acid can be secreted from the probiotic composition, the intestinal tract environment of the infant can be improved, continuous reproduction and growth of harmful bacteria are avoided, the gastrointestinal flora state of the infant is improved, and diarrhea symptoms are relieved.
In comparative examples 8 and 9, no zinc gluconate or no calcium gluconate was added in step S4, as compared with example 5. Comparative example 10 in comparison with example 5, step S4 was not performed, and the zn—ca complex enzymatic hydrolysate was replaced with the enzymatic hydrolysate in step S7. The effect of treating diarrhea is reduced. Zinc is a trace element, and can improve the growth and development effects of children, strengthen the immunity of human bodies and promote the absorption of vitamin A. The zinc element is lacking in the body, the damage to the growth of metabolic cells of the human body can be caused, the diarrhea condition of children is very easy to occur, meanwhile, zinc is also an important electrolyte, the zinc demand is relatively large in the growth process of infants, and a large amount of zinc element can be lost after diarrhea diseases are generated, so that diarrhea symptoms are more serious. The invention uses proteinase enzymolysis to make the protein enzymolysis in the traditional Chinese medicine component produce a large amount of active substances such as small molecular protein peptide, short peptide, amino acid and the like, has good chelating and fixing effects on metal zinc ions and calcium ions, so that the composition contains abundant zinc elements and calcium elements, the increase of the zinc elements can prevent and treat infantile diarrhea, the calcium elements contain not less than 100 enzymes and activating factors, and can be directly used for synthesizing nucleic acid and protein, can also play roles of oxidation reduction, energy metabolism, humoral and cellular immunity and the like, and is one of main components for forming small intestines. By supplementing zinc and calcium elements, the method can promote the improvement of human T helper cells and T suppressor cells, improve the immunity of human bodies, improve diarrhea sensitivity, strengthen cellular immunity, protect intestinal mucosa, inhibit harmful bacteria in intestinal tracts, ensure that local intestinal tracts have an immune function, and effectively remove bacteria.
In comparative examples 11 and 12, as compared with example 5, no solid fine powder or montmorillonite powder was added in step S5. In comparative example 13, no ultrafine powder was added in step S7, as compared with example 5. The effect of treating diarrhea is reduced. The montmorillonite powder has the effect of strengthening the covering capacity of the digestive tract mucosa, can effectively inhibit viruses, germs, toxins and the like in the digestive tract, so that the barrier defense function of the digestive tract mucosa of the infant is improved, and the gastrointestinal function recovery of the infant is promoted, but the problems of constipation and the like are easily caused, therefore, the invention dries and superfine pulverizes the solid residue after the fermentation and extraction of the traditional Chinese medicine to obtain the traditional Chinese medicine fine powder, uniformly mixes the traditional Chinese medicine fine powder with the montmorillonite powder, and adds the traditional Chinese medicine fine powder into the composition, can cooperate with the montmorillonite powder to strengthen the covering capacity of the digestive tract mucosa, and simultaneously, the traditional Chinese medicine fiber can promote intestinal peristalsis, relieve the constipation problem caused by the montmorillonite powder, and discharge the harmful substances fixed by the montmorillonite powder from the intestinal tract, thereby ensuring that the composition is safer and more effective.
Comparative example 14 in contrast to example 5, no prebiotics was added in step S7. The effect of treating diarrhea is reduced, and the inflammatory factor index is improved. The prebiotics can selectively stimulate the growth and proliferation of the added probiotic bacterial colony, reduce the production of inflammatory factors, improve the microecological environment of the gastrointestinal tract of the infant, reduce diarrhea, improve the immunity of the infant and improve the immunity of the organism.
Comparative example 15 in comparison with example 5, embedding was not performed in step S7, and the components were simply mixed. The effect of treating diarrhea is reduced, and the inflammatory factor index is improved. After embedding, the tolerance of probiotics to gastric acid can be improved, and the quantity of the probiotics reaching the intestinal tracts is increased, so that the gastrointestinal mucosa of the infant is repaired, the harmful bacteria resistance of the organism is enhanced, the gastrointestinal flora state of the infant is improved, the organism immunity of the infant is finally enhanced, the infant can be recovered in a short time, and the infant has a wide application prospect.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (4)
1. A method for preparing a composition for preventing and treating infantile diarrhea, comprising the steps of:
s1, activating strains: inoculating probiotics into a Gao's culture medium, and performing activation culture to obtain strain seed liquid;
s2, fermenting: cleaning radix Scutellariae, rhizoma Coptidis and rhizoma Dioscoreae, drying, pulverizing, adding into water, sterilizing, inoculating the probiotic strain seed liquid obtained in step S1, fermenting, culturing, filtering, retaining solid, and lyophilizing the filtrate to obtain fermented product;
S3, protease enzymolysis: adding protease into the solid obtained in the step S2 for enzymolysis, inactivating enzyme, filtering, reserving the solid, and freeze-drying the filtrate to obtain an enzymolysis product;
complexing of Zn-Ca: dissolving soluble zinc salt and soluble calcium salt in water, adding the enzymolysis product obtained in the step S3, stirring, mixing, reacting, and freeze drying to obtain Zn-Ca complex enzymolysis product;
s5, mixing superfine powder: drying, crushing and sieving the solid in the step S3 to obtain solid fine powder, and uniformly mixing the solid fine powder with montmorillonite powder to obtain superfine powder;
s6, preparation of prebiotics: uniformly mixing soybean oligosaccharide and isomaltooligosaccharide to prepare prebiotics;
s7, preparing a composition for preventing and treating infantile diarrhea: adding the fermentation product obtained in the step S2 into water, adding sodium alginate, sodium carboxymethylcellulose and an emulsifying agent, stirring and mixing uniformly, adding into fish oil, quickly emulsifying by a film, dripping a calcium chloride solution, solidifying at normal temperature, freeze-drying to obtain fermentation product microspheres, and mixing uniformly with the Zn-Ca complex enzymolysis product obtained in the step S4, the superfine powder obtained in the step S5 and the prebiotics obtained in the step S6 to obtain the composition for preventing and treating infantile diarrhea;
the probiotics are bifidobacterium breve FPHC4024, and the inoculation amount of the probiotic strain seed liquid is 2.5-3.5%;
The bifidobacterium breve FPHC4024 is classified and named as bifidobacterium breve Bifidobacterium breve, the preservation number is GDMCC No.62946, the preservation date is 2022, 11 months and 3 days, and the preservation unit is the microorganism strain preservation center in Guangdong province, address: building 5, guangzhou city, first, china, no. 100, university, no. 59, university of Guangdong university, institute of microorganisms, post code: 510070;
the 16S rRNA sequence of the bifidobacterium breve FPHC4024 is shown as SEQ ID No. 1;
the bifidobacterium breve FPHC4024 is a bacillus of gram positive bacteria, and bacterial colonies are round, regular in edge, milky white, convex, smooth in surface and regular in edge on an MRS culture medium.
2. The preparation method according to claim 1, wherein the temperature of the activation culture in the step S1 is 36-38 ℃, the time is 18-24 hours, and the bacterial content of the bacterial seed liquid is 108-109cfu/mL; in the step S2, the mass ratio of the baical skullcap root to the golden thread to the Chinese yam to the water is 7-10:5-7:5-7:120-150, the fermentation culture condition is 36-38 ℃,50-70r/min, and the fermentation culture is carried out for 48-72h; the protease in the step S3 is a mixture of trypsin and alkaline protease, the mass ratio is 5-7:3, the enzymolysis temperature is 35-40 ℃ and the enzymolysis time is 2-3h; in the step S4, at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc acetate, zinc citrate and zinc gluconate is selected as the soluble zinc salt, at least one of calcium chloride, calcium gluconate, calcium nitrate and calcium bicarbonate is selected as the soluble calcium salt, the soluble calcium salt and enzymolysis products are in a mass ratio of 3-5:1-2:15-20, and the stirring and mixing reaction time is 20-30min; the mesh number of the screened screen mesh in the step S5 is 500-1000 meshes, and the mass ratio of the solid fine powder to the montmorillonite powder is 10-12:15-20; the mass ratio of the soybean oligosaccharide to the isomaltooligosaccharide in the step S6 is 3-5:7, preparing a base material; in the step S7, the mass ratio of the fermentation product, sodium alginate to sodium carboxymethyl cellulose and the emulsifier is 12-15:7-10:3-5:0.5-1, the emulsifier is at least one selected from lecithin, sodium stearoyl lactate, calcium stearoyl lactate, diacetyl tartaric acid monoglyceride, sucrose fatty ester and distilled monoglyceride, the pore diameter of the membrane of the rapid membrane is 1-5 microns, the normal temperature curing time is 20-30min, and the mass ratio of the fermentation product microsphere, zn-Ca complex enzymolysis product, superfine powder and prebiotic is 15-20:7-12:12-15:2-3.
3. The preparation method according to claim 1, wherein the probiotics are a composition of bifidobacterium breve FPHC4024, lactobacillus bulgaricus and streptococcus thermophilus, and the seed solution of the probiotics is inoculated with the following seed solution: the inoculum sizes of the bifidobacterium breve FPHC4024, the lactobacillus bulgaricus and the streptococcus thermophilus strain seed liquid are respectively 2-3%, 1-2% and 0.5-1.2%.
4. A composition for preventing and treating infantile diarrhea prepared by the preparation method of any one of claims 1 to 3.
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| WO2001085186A1 (en) * | 2000-04-26 | 2001-11-15 | Bingxin Wu | Preparation that contains the intestinal beneficial bacterium fermentation culture |
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| CN111996153A (en) * | 2020-09-18 | 2020-11-27 | 南京益瑞兰生物科技有限公司 | Bifidobacterium breve and application thereof |
| CN112877236A (en) * | 2021-01-28 | 2021-06-01 | 生合生物科技（扬州）有限公司 | Bifidobacterium breve and application thereof in preparing liver function beverage for preventing non-alcoholic fat |
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| CN111996153A (en) * | 2020-09-18 | 2020-11-27 | 南京益瑞兰生物科技有限公司 | Bifidobacterium breve and application thereof |
| CN113005049A (en) * | 2020-12-30 | 2021-06-22 | 江南大学 | Bifidobacterium breve capable of relieving diarrhea and application thereof |
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