CN1663573B - A stable and safe microecological formulation, its preparation and usage - Google Patents

A stable and safe microecological formulation, its preparation and usage Download PDF

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CN1663573B
CN1663573B CN2004100061251A CN200410006125A CN1663573B CN 1663573 B CN1663573 B CN 1663573B CN 2004100061251 A CN2004100061251 A CN 2004100061251A CN 200410006125 A CN200410006125 A CN 200410006125A CN 1663573 B CN1663573 B CN 1663573B
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supernatant fluid
fermented supernatant
preparation
lactobacillus
lactobacillus casei
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CN1663573A (en
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崔云龙
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Beijing Puerkang Medicine Technology Co ltd
Qingdao Eastsea Pharmaceutical Co ltd
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BEIJING PUERKANG MEDICAL HIGH TECHNOLOGY Co Ltd
QINGDAO EASTSEA PHARMACEUTICAL CO Ltd
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Abstract

The invention discloses one kind of stable and safe microecological preparation, which comprises one kind or more probiotics ferment supernatant or condensate of ferment supernatant and/or one kind or more probiotics element, wherein probiotics points to one kind or more bifidobacteria, lactic acid bacillus, clostridium bytyricum and condensational bacillus; ferment supernatant points to liquid or other condensate of probiotics ferment liquid get rid of thallus. The preparation of this invention can be made to liquid, solid, self-solid for taking orally or exterior formulation, used to treat or prevent diarrhea, constipation and other intestinal tract diseases and/or bacterial vaginopathy, infectious colpitis and other vaginopathy, characterized in safe, stable, overcomes the problems of unstable of viable bacteria preparation and potential unsafe.

Description

Microbial ecological agent of a kind of stability and safety and its production and use
Technical field
The present invention relates to microbial ecological agent of a kind of stability and safety and its production and use.
Background of invention
Microecology studies show that, is that the probiotic bacteria of representative has the important physical function to body with the bacillus bifidus, comprises the growth that suppresses pathogenic entero becteria, reduces cholesterol in serum content, activating immune system and effect such as anticancer.With the microbial ecological agent that probiotic bacteria is made, have the little ecological disturbance of the human body of adjustment, the effect of keeping ecological balance is to reach the purpose of preventing and curing diseases.Microbial ecological agent was considered to have only microorganism alive just can play the balanced action of little ecology when 20th century, rose the seventies, therefore assert that microbial ecological agent is an active bacteria formulation, even for some time, microbial ecological agent was just called " active bacteria formulation ".Yet it is reported that external next probiotics is difficult in field planting in the human body, ways of addressing this issue is exactly constantly to replenish viable bacteria.But active bacteria formulation also has many defectives: 1, the non-spore active bacteria formulation quality of selling in the market is extremely unstable, adopts 2～8 ℃ of low temperature storage.But the low temperature storage condition has limited the accumulating and the market sale of product.Bacterium was easily by gastric acid and bile deactivation when 2, active bacteria formulation was oral, and viable count can descend significantly before arriving intestinal.Have only the bacterium that is ingested about 1.5% to arrive ileum after most of lactobacilluss and bacillus bifidus goods are oral, thereby influence the therapeutic effect of active bacteria formulation.3, active bacteria formulation can not share with antibiotic, and oral share easily killed by antibiotic such as ampicillins.4, transmit drug resistance.For example can be transferred to bacillus subtilis and reservation from the isolated Zhi grain of bacillus cereus.5, potential morbific possibility.Once there was the report lactobacillus can cause endocarditis, pneumonia and pyemia.
Along with going deep into of scientific research, the continuous development of microbial ecological agent, great mass of data proves that dead thalline, thalline composition, metabolite also have the effect of adjusting little ecological disturbance.After for example Jiamosi Medical College dyes mice with the antibiotic decontamination, give clothes bifidobacteria viable bacteria, the dead bacterium of deactivation, antibacterial culturing supernatant respectively, the result takes three and forms the recovery of branch bacillus bifidus significantly than natural recovering group height, and three groups of no significant differences it seems that the bifidobacterium fermentation supernatant has the effect that promotes probiotic bacteria growth in the body equally.
Fermented supernatant fluid is a culture fluid of removing thalline after the probiotic's culture, include bacterial growth reproductive process abundant metabolite and a part of bacterial chip (composition), its effect is as follows: 1, contain the adhesion that the adhesin material can impel probiotics in the body in the probiotic's culture liquid; 2, excretory acidic materials of probiotic bacteria and bacteriocin etc. have antagonism, killing action to harmful bacterium, promote microecological balance in the body; 3, the aminoacid after the probiotic bacteria decomposition nutrition matter, and synthetic vitamin all in culture fluid, also comprises the excretory enzyme useful to human body of antibacterial; 4, some component in the inoculum also has immunologic enhancement to human body.Metabolite in the inoculum is significant to the effect of human body in a word.The research of relevant probiotic bacteria metabolite has both at home and abroad been carried out much also having applied for relevant patent: Japan Patent JP2247127 extracts a kind of angiotensin converting enzyme and is used for the treatment of hypertension from the suspension of lactobacillus; JP8198724 obtains hyaluronic acid from the lactobacillus culture supernatant and salt is used for beautifying skin; JP7010740 is used to suppress melanin with the immersion of bacillus bifidus culture fluid high speed membrane filtration and generates; JP2002191387 uses the lactobacillus ferment supernatant as the skin treating composition; WO9835014 from Lactobacillus salivarius isolating a kind of can be by E.C. 3.4.21.64 and the destructive antibiotic substance of pronase; EP0577903EP1000625 kills helicobacter pylori with the lactobacillus supernatant; US5578302 treats gastric ulcer with Lactobacillus johnsonii fermented supernatant fluid or fermentation liquid.Chinese patent has: CN00814779.5 treats intestinal giardia lamblia stiles disease with lactobacillus and bacillus bifidus supernatant.CN 95111991.5 is a kind of little ecological viable bacteria beverage, adds prebiotics as the liquid part with lactobacillus and bacillus bifidus supernatant, is solid constituent with two kinds of thalline lyophilized powders, two kinds of compositions is mixed take then.
More than all patents all be to have utilized a kind of composition of probiotics fermention supernatant as active substance, perhaps add that with fermented supernatant fluid viable bacteria and prebiotics make up as a preparation, be used for dermatosis or gastropathy or parasite the treatment or the health care, rather than add prebiotics as effective ingredient with the probiotics fermention supernatant, be used to suffer from diarrhoea, the treatment of intestinal tract disease such as constipation and/or vagina such as bacterial vaginosis, infective vaginitis.We add fermented supernatant fluid with fermented supernatant fluid, viable bacteria, the viable bacteria of probiotic bacterias such as bacillus bifidus, lactobacillus respectively and have carried out the extracorporeal bacteria inhibitor test and the animal test of pesticide effectiveness, prove that all fermented supernatant fluid effect and viable bacteria group is similar.Developed thus and added combination of prebiotics with probiotics fermention supernatant or probiotics fermention supernatant and become various preparations, be used for the treatment of intestinal tract disease and/or vagina such as bacterial vaginosis, infective vaginitis such as diarrhoea, constipation, the preparation safety that this method is made is reliable, and has novelty.Finished the present invention thus.
Summary of the invention
The microbial ecological agent that the purpose of this invention is to provide a kind of stability and safety.
Another object of the present invention provides the preparation method of this microbial ecological agent.
Another purpose of the present invention is the new purposes of this microbial ecological agent.
The microbial ecological agent of stability and safety of the present invention is made up of concentrate and/or one or more prebioticses of one or more probiotics fermention supernatant or probiotics fermention supernatant, and wherein probiotic bacteria is meant one or more in bacillus bifidus, lactobacillus, Bacillus coagulans, the clostridium butyricum; Fermented supernatant fluid is meant liquid or its concentrate of probiotics fermention liquid after removing thalline.Above-mentioned preparation can be made liquid, solid, semi-solid dosage form for oral or external as required, is used for the treatment of or prevents intestinal tract disease and/or vagina such as bacterial vaginosis, infective vaginitis such as diarrhoea, constipation.Be characterized in safety, stable, overcome active bacteria formulation quality instability, uppity shortcoming.
Used herein term " probiotics fermention supernatant " refers to: probiotics fermention is cultivated the back and is removed the supernatant liquid that thalline obtains or the concentrate of this supernatant liquid with centrifugal or filtering method.
Term used herein " prebiotics (prebiotics) " refers to: prebiotics is by optionally stimulating one or more at activity or the growth and breeding of the permanent bacterium of colonic, reach and improve host health, and composition in kind or the preparation that is digested by the host not, it comprises the extract of oligosaccharide class, polysaccharide and natural plants and Chinese herbal medicine.
Used herein term " dosage form " is meant that medicine all should make certain dosage form, is applied to treatment, prevents or diagnoses the illness with the form of preparation.The effectiveness of preparation, safety, reasonability and elaboration etc. have then reflected medical level, have determined the effect of medication.
Make all used percentage ratios or ratio all by weight or weight/capacity or capacity/volumeter herein, except as otherwise noted, and all herein measurements all are to carry out at 25 ℃, except as otherwise noted.
Summary of the invention
As the quality instability, there is potential insecurity at the existing shortcoming of present probiotics viable bacteria preparation in the inventor.Adopted probiotics fermention supernatant or probiotics fermention supernatant to add combination of prebiotics and made preparation, be used for the treatment of or prevent intestinal tract disease and/or vagina such as bacterial vaginosis, infective vaginitis such as diarrhoea, constipation as effective ingredient.Be characterized in safety, stable, overcome active bacteria formulation quality instability, had shortcoming such as potential insecurity.It below is detailed description of the present invention.
Be applicable to that Bifidobacterium of the present invention is polymorphic bacillus, the thalline shape is Y word, V word, bending, racket shape, club-like, spherical even club shape.Gram-positive, no spore, pod membrane and flagellum, no mobility, this bacterium is an obligate anaerobic when just generation separates, though grow in the culture medium that contains reducing substances such as cysteine behind the successive transfer culture, can not become aerobic fully.Compare with other gram positive bacterias, it is not strict to the requirement of nutrition, optimum pH 6.5-7.0, optimum growth temperature 37-42 ℃.This bacterium catalase, nitrate reduction, indole generation, gelatin liquefaction and arginine hydrolysis are all negative.The glucose of 1mol generates the acetic acid of 1.5mol and the lactic acid of 1.0mol through the fermentation of D-fructose-6-phosphoric acid approach, does not generate CO 2, butanoic acid and propanoic acid.Optimum growth temperature is 36～38 ℃.The G+C content molar fraction of DNA is 57～68%.
Bifidobacterium is one of main member of micropopulation in human and animal's (comprising insecticide) normal bowel.The bacillus bifidus Pseudomonas is used in probiotic bacteria at most, and as current the most popular probiotics preparation both at home and abroad, nearly all there is suitability for industrialized production in each advanced country, and multiple dosage form is arranged.This bacterium is an anaerobe, not anti-bile and gastric acid, and anti-adversity ability is poor, and the time-to-live is short, and preparation is preserved difficulty.The kind that relates among the present invention has: hebephrenictype bacillus bifidus (B.adolescentis), bifidobacterium bifidum (B.bifidum), bifidobacteria infantis (B.infantis), bifidobacterium longum (B.longum), bifidobacterium breve (B.breve), chainlet bacillus bifidus (B.catenulatum) and false chainlet bacillus bifidus (B.pseudo catenulatum) etc., being used for the most preferred strain of preparation of the present invention is bifidobacteria infantis, bifidobacterium breve and chainlet bacillus bifidus.
Be applicable to that lactobacillus cell form of the present invention is various, to crooked shape and quarter butt, clavate, club shape often arranged also, generally form chain from elongated rod-shaped; Usually do not move, the sporter then has peritrichous; No spore; Gram-positive.Lactobacillus is little aerobic.When on solid medium, cultivating, common anaerobic condition or reduce oxygen to press and be filled with volume fraction be that 5～10% carbon dioxide can increase the superficial growth thing.Lactobacillus obligate ground decomposes sugar, and half is a lactate at least in the end-product; Common azymic lactate, by-product may be acetic acid, ethanol, carbon dioxide, formates or succinate.Lactobacillus does not produce the volatile acid more than 2 carbon atoms; Seldom see the reaction of nitrate reduction, have only pH value finally to be equilibrated at 6.0 and could reduce nitrate when above.Lactobacillus is liquefy gelatin not, does not decompose casein, but most of bacterial strain can produce a spot of soluble nitrogen, does not produce indole and hydrogen sulfide.It does not produce catalase, acellular color element; The only a few bacterial strain is with false catalase decompose hydroperoxide; The benzidine reaction feminine gender; It is rare to produce the pigment person, then is yellow or orange red or brick-red to rust if any pigment.Lactobacillus nutritional requirement complexity needs aminoacid, peptide, salt, fatty acid or lipid and fermentable carbohydrate.Its optimum growth temperature is 30～40 ℃; Acidproof, optimum pH is generally 5.5～6.2, generally pH be 5 or lower situation under can grow; Under neutral or initial alkaline pH condition, can reduce its growth rate usually.
The molar fraction of the G+G content of lactobacillus DNA is 32～53%.
Lactobacillus is in close relations with human life, lactobacillus is used as functional food and medicine, the feed additive of animal and the bio-feritlizer of plant both at home and abroad.The kind that relates among the present invention is bacillus acidophilus (L.acidophilus), lactobacillus casei (L.casei), lactobacillus rhamnosus (L.rhamnosus), Lactobacillus plantarum (L.plantarum), Lactobacillus fermenti (L.fermentum), Lactobacillus reuteri (L.reuteri), lactobacillus helveticus (L.helveticus), Deshi Lactobacillus (L.dellrueckii), Lactobacillus bulgaricus (L.bulgaricus), Lactobacillus salivarius (L.salivarius), Lactobacillus gasseri (L.gasseri), Lactobacillus crispatus (L.crispatus), Lactobacillus brevis (L.brevis), Lactobacillus Jensenii (L.jensenii).Spore lactobacillus (B.coagulans) and clostridium butyricum (Clostridium butyricum) etc. are arranged in addition in addition.The most preferred strain that is used for preparation of the present invention is lactobacillus casei, Deshi Lactobacillus, Lactobacillus gasseri and Lactobacillus crispatus.Above-mentioned any strain is commercially available, and also the chamber is cultivated and obtained by experiment.
The culture medium that is applicable to population of Bifidobacteria among the present invention is PTY GCulture medium, it is 10 that the final viable count counting in cultivation back should reach concentration 8～10 10Cfu/ml, the culture medium that is applicable to lactobacillus colony is the MRS culture medium, it is 10 that the final count plate in cultivation back should reach concentration 8～10 10Cfu/ml.This is that thalli growth is vigorous because in this concentration range, and its metabolite is also fully discharged, and active component increases in the fermentation liquid.These active component are the product of antibacterial in metabolic process, and as short-chain fatty acid-acetic acid of producing in the fermentation, lactic acid etc., these acid can make the intestinal pH value reduce, and cause sour environment, are unfavorable for the growth and breeding of pathogenic bacterium; As if bacteriocin (polypeptide or protein) is the bactericidal substance of the synthetic protein type of antibacterial, though they only have the bacteriostatic activity of narrow spectrum, promptly suppresses identical or the bacterial strain activity of the strain that is closely related, can cause replacing or suppressing the growth of other antibacterials; Peptidoglycan energy activating macrophage and β lymphocyte produce interleukin, r-interferon etc., and the adjusting immunization is arranged; Some lactobacillus such as Deshi Lactobacillus, lactobacillus casei etc. can also produce hydrogen peroxide to suppress the propagation of various noxious bacteria.By the synthetic vitamin of antibacterial, aminoacid, some useful enzyme also is present in the fermented supernatant fluid in addition.
The fermented supernatant fluid that uses above-mentioned strain to make, it can be the blended fermented supernatant fluid of bacillus bifidus and/or lactobacillus, the preparation method of bacillus bifidus and lactobacillus ferment supernatant is after probiotic's culture is arrived the corresponding time among the present invention, take out fermentation liquid, remove by filter thalline by centrifugal (8000r/min) and/or microporous membrane (0.2 μ m) and obtain fermented supernatant fluid (through the no viable bacteria growth of antibacterial culturing checking), directly make liquid preparation or become dry powder through vacuum lyophilization, add suitable prebiotics and make solid preparation, for clinical practice.
The multiple prebiotics that relates among the present invention has only the oligosaccharide (oligosaccharides) that is not digested by the host to can be used as prebiotics at present, and it is to be formed with straight chain or branched structure by 2～10 monosaccharide.Because oligosaccharide is a kind of not by the digestibility saccharide, can only be utilized (as bacillus bifidus or lactobacillus) by a few antibacterial in the human body, can play the effect same with probiotic bacteria.
All oligosaccharide goods are developed in countries in the world, particularly Japan in recent years.Oligosaccharide extensively is present in the various natural foods, as fruit, vegetable, milk, Mel etc.In more than ten years, oligosaccharide is widely used as the low heat value sweeting agent in the past, especially uses more general in Japan, Europe.Japanese government was " food of special healthy purposes (FOSHU) " legislation in 1991, and oligofructose, oligomeric galactose, soybean oligo saccharide and palatinose are listed in wherein, have the same year 450 kinds of products to use oligosaccharide.Ratified 58 kinds of food again to FOSHU in 1996, had 34 kinds of oligosaccharide to be listed in functional food additives, lactulose, lactulose, oligomeric xylose, dextrinose have been arranged.Oligosaccharide makes bacillus bifidus propagation in the intestinal, keeps good intestinal environment, is a kind of dietary fiber simultaneously.U.S. FDA has approved that fruit oligose is safe food composition, is one of main component as clinical nasal feeding material nutrient liquid.Studies confirm that oligosaccharide can promote the absorption of calcium, treatment osteoporosis, bone density improving.Japanese food the expert point out, oligosaccharide is a product the most rising in the functional food.Japan produces the topmost country of oligosaccharide, and the use of oligosaccharide then spreads all over all over the world.The oligosaccharide of using as prebiotics both at home and abroad has tens of kinds at present.Selected oligosaccharide has following several among the present invention: oligofructose (Fructo-oligosaccharide), oligomeric galactose (Galacto-oligosaccharide), soybean oligo saccharide (Soybean oligosaccharide), oligomeric xylose (Xylo-oligosaccharide), oligomeric lactulose (Lactosucrose), lactulose (Lactulose), oligomeric isomaltose (Isomalto-oligosaccharide), stachyose (Stachyose), cottonseed sugar (Rafinose), trehalose (Trehalose).Polysaccharide has starch (Starch), grifolan (Grifolan), astragalus polysaccharides (Astraglan), pachyman (Pachymose).
Oligofructose (Fructo-oligosaccharide)
Fructose and glucose connect by β-2,1 glycosidic bond, 4.8 of average fructosyls.The food that contains the oligofructose composition has Bulbus Allii, Bulbus Allii Cepae, Fructus Arctii etc.The first method of oligofructose production is to adopt microbial enzyme method production, do substrate with sucrose, the industrial saccharase that utilizes Aspergillus niger to produce changes the fructosyl effect, combining with 1～3 fructose molecule on β (1 → 2) glycosidic bond on the sucrose molecule, ketose (GF2), C24H42 O21 (GF3), the Fructofuranosyl nystose (GF4) that forms belongs to the straight chain oligosaccharide of fructose and sucrose formation, also have fructose, glucose and unreacted substrate-sucrose completely in the product that forms, the employing chromatography removes monosaccharide and disaccharidase makes highly purified oligofructose.The second method of producing oligofructose is the inulin that extracts from Herba Cichorii, does substrate hydrolysis with inulin.Oligofructose is not digested by any mammal.Experimental results show that in the body and take 15 gram oligofructoses every day, bacillus bifidus quantity is raised to 22% by 6% after 2 weeks, and bacteroid drops to 0.4% by 25%, and the enteral corrupt substance obviously reduces.
Oligomeric galactose (Galacto-oligosaccharide)
Connect through β-1,6 key by several galactosyls and a Fructus Vitis viniferae base.By lactose is beta galactosidase (EC3.2.1.23) Production by Enzymes that raw material produces through aspergillosis.Oligomeric galactose is heavy carbohydrate, and it is carbon source that nearly all human body bacillus bifidus and lactobacillus all utilize it, and is not utilized by most harmful intestinal tract bacteria.It is a kind of good bacillus bifidus multiplicaiton factor.Absorb 10 grams every day, bacillus bifidus improves greatly in the week back feces, and intestinal Putrefying bacteria and reduce by the cancerigenic factor time bile acid that Putrefying bacteria produces, the activity of glycuronidase and nitroreductase also obviously descends.
Soybean oligo saccharide (Soybean oligosaccharide)
Soybean oligo saccharide is the general name that is present in the solubility sugar in the Semen sojae atricolor, is made of Semen sojae atricolor defat supernatant extracting solution, and main component is stachyose and Raffinose.The assorted oligosaccharide of the side chain that stachyose and Raffinose all are made up of galactose, glucose and fructose, be glucose residue one side at sucrose molecule with C (1,6) keyed jointing on one or two galactose form.These two kinds of sugar all are indigestibility sugar.Bacillus bifidus is the fermented soybean oligosaccharide effectively, and effect surpasses oligofructose, and bacillus aerogenes capsulatus, clostridium difficile, bacillus septicus, coliform, this sugar of Escherichia azymic.Adult's food every day is got syrup 10 grams (including stachyose, Raffinose 3 grams).Bacillus bifidus obviously increases in 3 all hindguts, and harmful bacterium significantly reduces.Soybean oligo saccharide is widely used in the production of various Food ﹠ Drink.
Oligomeric xylose (Xylo-oligosaccharide)
Form straight chain sugar by 2～6 xyloses through β-1,4 chain combination.Industrial generally is to be raw material with the plant resources such as corn cob, bagasse, cotton seed hulls, paddy and wheat, bamboo and the wheat bran etc. that are rich in xylan (Xylan), the internal cutting type xylanase endo-1.4-β xylanase (EC3.2.1.8) that produces by aspergillus niger (Astergillus niger) or ball hair shell enzyme (Chaetomium globosum) carries out enzymolysis, then, make oligomeric xylose through separating, purifying.Oligomeric xylose is difficult to the human consumption enzyme system and decomposes, and with saliva, gastric juice, pancreatic juice and intestinal fluid oligomeric xylose is tested respectively, and the result shows that nearly all Digestive system all can not decompose oligomeric xylose.Oligomeric xylose is a kind of efficient bifidus factor, and except that bacillus bifidus, most of intestinal bacteria are all relatively poor to the utilization of oligomeric xylose.Human body edible 0.7 gram oligomeric xylose every day, after 17 days, after the bacillus bifidus proportion was brought up to 17.9%, 21 day by 8.5%, the bacillus bifidus proportion brought up to 26.2%.Oligomeric xylose also has the function of the constipation of preventing and promotes the effect of digesting and assimilating of calcium.
Oligomeric lactulose (Lactosucrose)
Oligomeric lactulose is to be raw material with lactose and sucrose (1: 1), under saccharase (EC3.2.1.26) catalytic action that arthrobacterium (Arthrobacter) produces, the fructosyl that sucrose decomposition is produced is transferred on the C1 position hydroxyl of lactose reducing end under neutral, and generating galactosyl sucrose (trisaccharide) is oligomeric lactulose.The oligosaccharide product of commercially producing comprises 37% oligomeric lactulose, 28% sucrose, 13% lactose, glucose and fructose and 5% other sugar of 17%, and sugariness is about 70% of sucrose.In Japan, this product was introduced to the market in 1991.Oligomeric lactulose is absorbed by human consumption hardly, can not cause the fluctuation of interior blood sugar level of body and blood insulin level after the absorption, can supply diabetes patient.This sugar also is the bacillus bifidus multiplicaiton factor, takes in 5 grams every day, brings up to 32.6% from 10.5% before bacillus bifidus number ratio is taken in the feces after the week.If with physical qualification preferably the man be subjects, then the bacillus bifidus increase accounts for more than 50% at least.Compare with the oligomeric galactose that is both the bacillus bifidus multiplicaiton factor, oligomeric isomaltose etc., the bacillus bifidus proliferation activity of oligomeric lactulose is higher, and sweet taste characteristic also more approaches sucrose.This sugar is at day herbal classic acute toxinology experiment and cause prominent test and wait and confirm safety non-toxic.
Lactulose (Lactulose)
Also claim lactulose, lactulose, isomerized lactose.The output maximum of lactulose is the same with oligomeric galactose in all oligosaccharide, and lactulose also is to be raw material with the lactose.Lactose gets lactulose after soda lime is handled, lactose is made up of galactose and glucose, and lactulose then is to have galactose and fructose to combine the disaccharidase that forms with β (1 → 4) glycoside bond.Lactulose is not is not digested and assimilated in small intestinal, arrives large intestine and is utilized by bacillus bifidus.Breast-feeding infant is that with artificial feeding infant's an outstanding difference bacillus bifidus number in the former feces is more much more than the latter, but if give the artificially feeding infant an amount of lactulose of feeding simultaneously, then situation is different, the multiplication rate that can be observed bacillus bifidus greatly improves even reaches the level of breast-feeding infant, and the bacillus bifidus number increases and other putrefaction bacteria minimizings in its feces.Test shows takes in behind the lactulose that glucose does not have the rising phenomenon in the human plasma.In addition, lactulose is listed in low grade fever sweeting agent and functional food additives.Lactulose is except that being used as food additive, also in the encephalopathy that pharmaceutically is used for the treatment of constipation and portal system.Slvay (Germany) is maximum in the world lactulose maker, produces 10,000 tons of lactulose every year approximately, and 90% of its product is used for medicine.
Oligomeric isomaltose (Isomalto-oligosaccharide)
Oligomeric isomaltose is that the alpha-D-glucose base is with the combination of α (1 → 6) key glycosidic bond.In the mixture of oligomeric isomaltose, both contained glucosyl group with the bonded oligosaccharide of α (1 → 6) glycosidic bond, contained the Fructus Vitis viniferae base again with the bonded oligosaccharide of α (1 → 4) glycosidic bond, as panose.The enzyme process synthetic reaction of oligomeric isomaltose in two steps, the first step is that α-Dian Fenmei (EC3.2.1.1) is with starch liquefacation, second step was hydrolyzed into maltose by beta amylase (EC3.2.1.2) with the starch that liquefies, and alpha-glucosidase (EC3.2.1.20) is translated into oligomeric isomaltose again.The enteral fermenting experiment is except that bifidobacterium bifidum, and most of human body bacillus bifiduss all can utilize oligomeric isomaltose to be carbon source, and escherichia coli does not utilize this sugar, and bacteroides fragilis, enterococcus faecalis and part clostridium utilize this sugar.Oligomeric isomaltose slurry (15 gram/sky) is taken in human body and evidence, and intestinal microbial population is had obvious regulating action.
Stachyose (Stachyose)
Be to be polymerized by glucose, galactose, galactose, four monosaccharide of fructose.It has facilitation to bacillus bifidus, Lactobacillus acidophilus, Bulgarian Lactobacillus, and escherichia coli is had facilitation slightly, and it is not utilized by human body, and the energy selectivity promotes the growth of intestinal physiology antibacterial.Stachyose mainly extracts behind enzyme fermentation from Semen sojae atricolor.
Cottonseed sugar (Rafinose)
The employing chromatography extracts from beet molasses and makes with extra care and gets.Sugariness contains 98% oligosaccharide more than 20%, is only crystallization oligosaccharide that can moisture absorption in the oligosaccharide.All very stable to heat and acid, be mainly used in confection, cake powder or the lamellar health food.
Trehalose (Trehalose)
Trehalose be glucose with Q, Q-1, the synthetic irreducibility disaccharidase of 1 bond extensively is present in animals and plants and microbial cell; Past extracts from yeast (content 10～20%), can utilize oligomeric Fructus Hordei Germinatus base trehalose synthetase and oligomeric Fructus Hordei Germinatus base trehalose hydrolytic enzyme to act on the trehalose that glucidtemns can obtain 82% yield now.Trehalose is a kind of functional oligose, has effects such as low heat value, anti-caries, but is not bifidus factor.Trehalose is digestible oligosaccharide, and sugariness is 50% of a sucrose, and trehalose is hot and cold stable down; Its special physiological function is to improve the ability of poor environments such as animal and plant cells opposing drying, high temperature resistance and cold.When microbial freezing drying is preserved,,, widely utilized as the freeze drying protectant of microorganism if an amount of trehalose that adds can reduce death.
Being used for the most preferred oligosaccharide of preparation of the present invention is soybean oligo saccharide, oligomeric isomaltose, oligomeric lactulose, can any one or multiple mixing in the combination of preparation.
Polysaccharide
Following several polysaccharide can be selected one or more mixing arbitrarily for use according to the combination of preparation among the present invention.
Starch (Starch)
Starch is to make polysaccharide in the tuber of the caryopsis of grass Semen Maydis ZeamaysL. or the big plant Maninot esculenta crantz. Manihotutilissima Pohl of prestige section.Be commonly used for the adjuvant that solid drugs is made.
Grifolan (Grifolan)
Lentinan system makes in the hot water extraction liquid of Agaricaceae mushroom genus Lentinus Edodes Lentnus edodes (Berk) S.Ito.et Imai, by β-(1-3) is main chain, β-(1 → 3) has the hepatic tissue reparation of dirty tumor and acceleration damaged to play hepatoprotective effect for the macromolecule glucose of side chain is an immunostimulant.
Astragalus polysaccharides (Astraglan)
Astragalus polysaccharides comes from leguminous plant Radix Astagali Astragalus membranaceus (Fish) Bge.Var.morgholicus (Beg) Hsiao.The extract of root is a glucosan, mainly is that the condensation key of α-(1 → 4)-D-glucose is formed by connecting, and is heteropolysaccharide, has immunological enhancement and blood sugar regulation effect.
Pachyman (Pachymose)
Pachyman comes from the dry sclerotia extract of Polyporaceae fungus Poria Poria cocos (schn) holf, is the main constituent in the Poria, and content accounts for 84.2%.For β-(1 → 6) Glucopyranose. is β (1 → 3) the glucose polysaccharide of side chain.Has immunomodulating, the liver protecting and ALT lowering, inducement interferon and antitumor action.
Above-mentioned oligosaccharide and polysaccharide mainly are to buy from the market among the present invention.
In addition, the present invention can make and contain in the tablet of lyophilization or cold drying preparation.Lyophilization or cold drying can be by forming exsiccant preparation the disintegrate that open substrate network structure helps preparation.In most applications, can cause the quick infiltration of liquid medium like this, promote the timely release transmission of product.Overall freeze-drying method is well known and commonly used.Any suitable normal freeze-drying method all can be used.One of method is that to be dried to final water content then be about 3% to about 5% to the quick freezing preparation preferably.Suitable lyophilization and production method can be referring to the United States Patent (USP)s 4642903 at Dacies on the 17th in February, 1987 of this reference, August nineteen ninety Blank on the 7th etc. United States Patent (USP) 4946684, December in 1981 15 days and February nineteen eighty-three Gregory on the 1st etc. United States Patent (USP) 4303502 and 4371516 and 1993 on February 23, Hes etc. United States Patent (USP) 5188825.
Equally, preparation of the present invention also can vacuum drying, and suitable vacuum drying method can be referring to the United States Patent (USP) 5298261 at Pebley on the 29th etc. in March, 1994 of this reference.
Preparation of the present invention can also comprise other optional components that pharmaceutical industry is known, corresponding content separately also is known, this class component for example is used to the natural or artificial sweetener that makes final finished good to eat and attractive in appearance, flavoring agent, pigment, essence, buffer agent etc., be used to prolong storage life as the BHA antioxidant with as antiseptic such as methyl parahydroxybenzoate or propyl ester, potassium sorbate or sodium benzoate.
Other suitable components, diluent and dosage form will be recognized or determine to persons skilled in the art very soon by normal experiment.And the administration of various preparations can be undertaken by the standard method that persons skilled in the art are known.
Preparation of the present invention can also comprise coating material, and the coating material that is applicable to preparation of the present invention can be water miscible and non-water-soluble.
The water solublity coating that is applicable to preparation of the present invention comprises sugar or Organic substance coating.The sweet tablet that is used for preparation of the present invention can be a syrup, wherein contains the polymer of monosaccharide or two or more sugar units.The Organic substance coating also can be used for preparation of the present invention.Spendable organic polymer or copolymer comprise those with a plurality of carboxylic acids or ester group.These groups mainly are responsible for effectively being hidden the flavor performance with active component generation physics or chemical interaction.Better polymerization thing or copolymer contain vinyl and acrylic acid and/or ester group, perhaps carboxylic acid and/or ester group, synthesizing and semi-synthetic material of for example ethyl cellulose, cellulose ethanoate, cellulosic phthalic acetate, acetic acid and/or the acid of phthalic acid ethylethylene, poly-acetic acid and/or O-phthalic vinyl acetate, ethyl methacrylate and/or methyl ester, esters and copolymer, hydroxy alkyl cellulose acetas and/or phthalic acid ester and so on, also can use the mixture of sugar and Organic substance coating substance.
Sugar of the present invention and/or Organic substance coating can utilize conventional method to implement, and comprising mechanical means, for example pan coating method, air suspension technology, porous centrifugation technique and spray drying technology, and physico-chemical method for example condense-be separated.In " practical drug preparation technique " 125-169 of this paper reference, the coating process there is more detailed argumentation.
Can also contain buffer agent in the preparation of the present invention,, pH can be buffered to acidity and improve taste for the mouth preparation.For the product that is used for vagina, be suitable as buffer agent in the preparation of the present invention and be those that to safeguard about 3.0 to about 5.5 reproductive system pH value.The pharmaceutically acceptable acid of any gentleness, suitable acid comprises boric acid, or organic acid, for example propanoic acid, malic acid, sorbic acid, benzoic acid, lactic acid, vitamin C, citric acid or acetic acid, and its corresponding sodium salt or other pharmaceutically acceptable salt combination (until reaching required pH).When using buffer agent, preparation of the present invention preferably is buffered to pH about 3.5 to about 5.0, about 3.7 to about 4.7 better, and preferably uses lactic acid and sodium lactate, perhaps lactic acid/sodium lactate and benzoic combination, the perhaps combination of lactic acid/sodium lactate and propanoic acid.
Preparation of the present invention can be added in many different carriers, and carrier depends primarily on the final use of preparation.These carriers are pharmaceutically acceptable, comprise the oral preparation that uses with the part.They can be inert fully, perhaps comprise or be exactly other active component, but carrier must be compatible with preparation disclosed herein." compatibility " refers at this, and carrier component can be mixed each other mutually with component of the present invention and carrier component, mix not take place obviously to reduce the activity of preparation or the interactional mode of vitality under general operating position.Certainly, carrier mass must have sufficiently high purity and enough low toxicity, makes them be suitable for being accepted the people of treatment.The present invention's combination can be used one or more carrier mass.
Mouth is a carrier of the present invention with dosage form, contains suitable compatibility solid or the liquid fillers diluent of using to population in these dosage forms.
Can comprise mouthful with liquid dosage form preparation of the present invention is dissolved or suspended in the drinkable liquid, for example in sterilized water or the clean water.Suitable form comprises solution, suspension, syrup, wherein contains this area inert diluent commonly used, for example pure water, sugar, polysaccharide, gelatin or alcohol.These inert diluents do not play an active part in therapeutic effect of the present invention.This class preparation can also contain wetting agent, emulsifying agent, suspending agent and other therapeutic active component.About the more detailed explanation of liquid and liquid-like dosage form can be referring to " the practical drug preparation technique " the 1st edition in this reference, People's Health Publisher (1999), 1519-1544 page or leaf.
Solid dosage forms comprises tablet and capsule, and tablet can be compressed tablet, molded tablet, chewable tablet, buccal tablet, casing sheet, coated tablet, film garment piece or multilayer tablet, wherein contains suitable bonding, lubricant, diluent, disintegrating agent and drain agent.
Also can use rigid gelatine capsule, as conventional capsule and enteric coated capsule.Be preferably, gelatin shell is transparent basically, can improve capsular outward appearance like this.Rigid gelatine capsule contains gelatin, plasticizer and water usually.
Technology and the prescription of making solid oral dosage form can be referring to " tablet and capsule " " the practical drug preparation technique " in this reference, the 1st edition, People's Health Publisher, 1-30 pages or leaves (1999).
Be fit to the local carrier that uses of preparation of the present invention and comprise that vaginal tablet or capsule, vaginal suppository, supp anal, lavation are with solution, aerosol.They are to utilize the conventional method of preparation semi-solid preparation to prepare, and the excipient of use is the derivant of synthetic glyceride, wax, lanoline, lanonol (lanolin alcohols), Pyrusussuriensis ester, aliphatic alcohol, liquid/solid Polyethylene Glycol, propylene glycol, polyethylene, starch, acrylamide, Methacrylamide, cellulose and the CVP Carbopol ETD2050 of vaseline, paraffin, vaseline oil, vegetable oil, animal oil, solid and liquid for example.
Vaginal suppository, general suppository, vaginal capsule or tablet and effervescent tablet also can be used in the local prevention use of the present invention.Vaginal suppository is similar with general suppository, be avette, the main excipient that uses is semisynthetic glyceride and Polyethylene Glycol, the emulsifying agent and the surfactant that also have washability are used for the tablet of vagina or lactose, starch, polyvinyl pyrrolidone, cellulose derivative, magnesium stearate, the ethylene glycol that capsule contains the main excipient of conduct.Effervescent tablet contains and produces the necessary chemical composition of carbon dioxide (being sodium bicarbonate adding citric acid or tartaric acid) for effervescent takes place.
Following examples further specify the embodiment in the scope of the invention.These embodiment and should not be understood as that it is limitation of the invention only for the purpose of description, are within above-described the spirit and scope of the present invention because have many changes yet.
The preparation of embodiment 1 bifidobacteria infantis fermented supernatant fluid
Preparation sterilization PTY GCulture medium 300ml inoculates bifidobacteria infantis (CGMCC313-2) with slant strains, and 37 ℃ of static cultivations of anaerobism 48 hours measure that viable count should reach 10 in the fermentation liquid 8～10 10Cfu/ml.Fermentation liquid centrifugal (8000r/min) removes thalline, obtains fermented supernatant fluid through thin film (0.2 μ m) filtration sterilization again, is used to make preparation of the present invention.
The preparation of embodiment 2 lactobacillus casei fermented supernatant fluids
Preparation sterilization MRS culture medium 300ml inoculates lactobacillus casei (YL13-1) with slant strains, and 37 ℃ of static cultivations of anaerobism 24 hours measure that viable count concentration should be 10 in the fermentation liquid 8～10 10Cfu/ml.Fermentation liquid centrifugal (8000r/min) removes thalline, obtains fermented supernatant fluid through thin film (0.2 μ m) filtration sterilization again, is used to make preparation of the present invention.
The bacteriostasis of embodiment 3 bifidobacteria infantiies, lactobacillus casei fermented supernatant fluid
Preparation nutrient broth agar culture medium, with the wet-hot steam sterilization, be cooled to 50 ℃ after, proportionally, add a certain amount of pathogenic bacterium, ultimate density is 2-4 * 10 7Cfu/ml shakes up the back pour plate, punches several with 9 millimeters card punch.Add fermented supernatant fluid respectively in each agar hole, living bacterial liquid, each 50ul of viable bacteria+fermented supernatant fluid cultivate after 24 hours for 30 ℃ and observe bacteriostatic diameter (mm).
The result is in table 1 expression, and the fermented supernatant fluid of bifidobacteria infantis and lactobacillus casei all has and the akin bacteriostasis of living bacterial liquid.
The bacteriostasis of table 1 bifidobacteria infantis, lactobacillus casei fermented supernatant fluid
Figure G2004100061251D00131
Embodiment 4 bifidobacteria infantiies, lactobacillus casei fermented supernatant fluid bring out mice (BALB/C) diarrheal preventive effect to ampicillin
Fasting is respectively organized mice after 12 hours, use fermented supernatant fluid, living bacterial liquid (10 last evening respectively 8Cfu/ml), viable bacteria+fermented supernatant fluid and normal saline irritate stomach 0.5ml/ only/time, repeat to irritate stomach again 1 the 2nd day morning, after 1 hour with ampicillin irritate stomach 0.2g/0.5ml/ only/time, gave again every 6 hours ampicillin 0.15g/ only/time.Observe the diarrhoea situation of mice then.
The result is in table 2 expression, and the bifidobacteria infantis fermented supernatant fluid had the diarrhoea effect of the prevention mice the same with viable bacteria after 40 hours, and mice is transferred to normal just granular by the rare soft stool of row; All row was just granular 11 hours mices for the lactobacillus casei fermented supernatant fluid.
Table 2 bifidobacteria infantis, lactobacillus casei fermented supernatant fluid bring out the preventive effect of diarrhea of mouse to ampicillin
Embodiment 5 bifidobacteria infantiies, lactobacillus casei fermented supernatant fluid bring out mice (BALB/C) diarrheal therapeutical effect to ampicillin
Respectively the organize mice of fasting after 12 hours irritated stomach with ampicillin 0.2g/0.5ml//time, only gave ampicillin 0.15g/0.5ml/ again, cause diarrhea of mouse, then through fermented supernatant fluid, living bacterial liquid (10 every 6 hours 8Cfu/ml), viable bacteria+fermented supernatant fluid and normal saline irritate stomach, every day 1 time, for three days on end, observes the diarrhoea situation of mice.
The result is in table 3 expression, and the bifidobacteria infantis fermented supernatant fluid had the treatment diarrhoea effect identical with living bacterial liquid in 72 hours; The lactobacillus casei fermented supernatant fluid made diarrhea mice in 48 hours all row is normal just granular.
Table 3 bifidobacteria infantis, lactobacillus casei fermented supernatant fluid bring out the therapeutical effect of diarrhea of mouse to ampicillin
Figure G2004100061251D00141
The preparation of embodiment 6 bifidobacteria infantiies, lactobacillus casei fermented supernatant fluid oral liquid
Utilize the technology of preparing of conventional oral liquid (mixture) that following component is prepared oral liquid of the present invention (mixture)
Composition % (capacity)
Bifidobacteria infantis fermented supernatant fluid 20.00%
Lactobacillus casei fermented supernatant fluid 20.00%
Soybean oligo saccharide solution 22.00%
Dextrinosan solution 22.00%
Lentinan solution 10.00%
Potassium sorbate solution 5.00%
Flavoring orange essence 1.00%
Above-mentioned each component is stirred, utilize conventional oral liquid technology of preparing,, make oral liquid by the unit dose fill.
The preparation of more than 7 kind of probiotics fermention supernatant of embodiment tablet
Utilize conventional tabletting technology to mix following component and make tablet of the present invention
Composition % (capacity)
Bifidobacteria infantis fermented supernatant fluid lyophilized powder 15.00%
Lactobacillus casei fermented supernatant fluid lyophilized powder 15.00%
Soybean oligo saccharide 50.00%
Lentinan 5.00%
Starch 11.00%
Pulvis Talci 3.40%
Magnesium stearate 0.60%
1-5 component in the above-mentioned component is stirred, and the reuse alcoholic solution is made granule, crosses 12 mesh sieves, 70～75 ℃ of dryings, dried granule is crossed 20 mesh sieves, with granule and Pulvis Talci and magnesium stearate mixing, utilizes conventional pressed-disc technique to be pressed into tablet by unit dose then.
The preparation of more than 8 kind of probiotics fermention supernatant of embodiment capsule
Utilize conventional glue capsule technology to mix following component and make capsule of the present invention
Composition % (capacity)
Bifidobacteria infantis fermented supernatant fluid lyophilized powder 15.00%
Lactobacillus casei fermented supernatant fluid lyophilized powder 15.00%
Soybean oligo saccharide 40.00%
Grifolan 10.00%
Pachyman 5.00%
Starch 12.00%
Pulvis Talci 3.00%
With the abundant mix homogeneously of above-mentioned component, will pack in the sizeable hard capsule by the mixture that unit dose forms then.

Claims (9)

1. a stable and safe microbial ecological agent is characterized in that being grouped into by following one-tenth
Bifidobacteria infantis fermented supernatant fluid 20.00%
Lactobacillus casei fermented supernatant fluid 20.00%
Soybean oligo saccharide solution 22.00%
Dextrinosan solution 22.00%
Lentinan solution 10.00%
Potassium sorbate solution 5.00%
Flavoring orange essence 1.00%
Wherein said fermented supernatant fluid is with bifidobacteria infantis and lactobacillus casei difference single culture, takes out fermentation liquid, removes thalline by centrifugal and/or microporous membrane and obtains fermented supernatant fluid, through the no viable bacteria growth of antibacterial culturing checking; Its preparation method is that above-mentioned each component is stirred, and utilizes conventional oral liquid technology of preparing, by the unit dose fill, makes oral liquid.
2. a stable and safe microbial ecological agent is characterized in that being grouped into by following one-tenth
Bifidobacteria infantis fermented supernatant fluid lyophilized powder 15.00%
Lactobacillus casei fermented supernatant fluid lyophilized powder 15.00%
Soybean oligo saccharide 50.00%
Lentinan 5.00%
Starch 11.00%
Pulvis Talci 3.40%
Magnesium stearate 0.60%
Wherein said fermented supernatant fluid lyophilized powder is with bifidobacteria infantis and lactobacillus casei difference single culture, take out fermentation liquid, remove thalline by centrifugal and/or microporous membrane and obtain fermented supernatant fluid, through the no viable bacteria growth of antibacterial culturing checking, adopt concentrate under reduced pressure at low temperature, spraying or lyophilizing mode to obtain above-mentioned fermented supernatant fluid then; Its preparation method is that the first five kind component in the above-mentioned component is stirred, and the reuse alcoholic solution is made granule, crosses 12 mesh sieves, 70～75 ℃ of dryings, dried granule is crossed 20 mesh sieves, with granule and Pulvis Talci and magnesium stearate mixing, utilizes conventional pressed-disc technique to be pressed into tablet by unit dose then.
3. a stable and safe microbial ecological agent is characterized in that being grouped into by following one-tenth
Bifidobacteria infantis fermented supernatant fluid lyophilized powder 15.00%
Lactobacillus casei fermented supernatant fluid lyophilized powder 15.00%
Soybean oligo saccharide 40.00%
Grifolan 10.00%
Pachyman 5.00%
Starch 12.00%
Pulvis Talci 3.00%
Wherein said fermented supernatant fluid lyophilized powder is with bifidobacteria infantis and lactobacillus casei difference single culture, take out fermentation liquid, remove thalline by centrifugal and/or microporous membrane and obtain fermented supernatant fluid, through the no viable bacteria growth of antibacterial culturing checking, adopt concentrate under reduced pressure at low temperature, spraying or lyophilizing mode to obtain above-mentioned fermented supernatant fluid then; Its preparation method is with the abundant mix homogeneously of above-mentioned component, will pack in the sizeable hard capsule by the mixture that unit dose forms then.
4. according to claim 1,2 or 3 described microbial ecological agent, the culture medium that it is characterized in that bifidobacteria infantis is the PTYG culture medium, and it is 10 that the final viable count in cultivation back should reach concentration 8～10 10Cfu/ml.
5. according to claim 1,2 or 3 described microbial ecological agent, the culture medium that it is characterized in that lactobacillus casei is the MRS culture medium, and it is 10 that the final count plate in cultivation back should reach concentration 8～10 10Cfu/ml.
6. claim 1,2 or 3 described preparations are preparing the purposes for the treatment of in the intestinal tract disease medicine.
7. by the described purposes of claim 6, wherein said intestinal tract disease is acute and chronic diarrhoea or constipation.
8. claim 1,2 or 3 described preparations are preparing the purposes for the treatment of in the bacterial vaginosis medicine.
9. claim 1,2 or 3 described preparations are preparing the purposes for the treatment of in the infective vaginitis medicine.
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