CN101125151A - Bacteroides signal molecule microecological preparation and its preparation method - Google Patents
Bacteroides signal molecule microecological preparation and its preparation method Download PDFInfo
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Abstract
The present invention relates to a b.fragilis signal molecule microbial preparation, the b.fragilis bacterium are inoculated in a culture medium for anaerobic fermentation at 35 to 38 DEG C, after that, the b.fragilis bacteria concentrate is obtained by centrifugal concentration; the bacteria cell walls are broken by high pressure; further, the bacteria concentrate is treated with enzyme hydrolysis by trypsin, chymotrypsin and pepsin, and then the preparation raw powder is obtained by solvent extraction and decompression drying. The preparation raw powder is taken as the main ingredient, and other excipients are added to prepare the oral capsules, tablets, granules, oral liquor and other preparations. The b.fragilis signal molecule product of the present invention is proven to have no acute toxicity, no influence on the times of spontaneous activity, blood pressure and respiration of the animals and can not cause the allergic reactions of the animals by animal trials. The animal trials further prove that: the product has the effects of regulating blood-lipid metabolism, reducing fat accumulation and slimming; at the same time, the present invention can improve the immunity and the anti-tumor effect by adjusting the gene expression and regulating the immune functions.
Description
Technical field
The present invention relates to derive from the field of the pharmaceutical product of microbial material, particularly derive from the pharmaceutical products of antibacterial.
Background technology
The production technology of probiotic composition (Probiolics) has been passed through first generation product and two era developments of second filial generation product.First generation product is the viable bacteria goods, and second filial generation product is dead bacterium goods.These two epoch have been passed through semicentennial process.First generation product mainly is the viable bacteria that utilizes bacillus bifidus (Bifidobacterium) and lactobacillus (Lactic acid bacteri).Its mechanism of action is to adjust intestine dysbacteriosis (Dysbacteriosis), replenishes the harmful bacterium of probiotics inhibition and keeps microecological balance (Eubiosis) just to be beneficial to health.As a kind of microecologic regulator, the viable bacteria goods of probiotic bacteria have been widely used in human health care and prophylactic effective ways in worldwide.But this goods have strict requirement, at first enough viable counts will be arranged, and it is less important to have the work of certain hour to deposit the phase
(1)This has proposed strict requirement to producing and selling, thereby also limits the development of these class health product.Actually, production circle is in order to overcome above-mentioned two restriction viable counts, and the viable bacteria phase has proposed again with dead thalline as this based article effective ingredient, thereby has avoided the bottle neck effect of production and selling.Dead thalline is exactly a second filial generation product.Though second filial generation product has been avoided viable count and the phase of depositing two the bottleneck restrictions of living, and fatal shortcoming is also arranged.Here it is, and dead thalline is difficult to quantize, and is unfavorable for quality control, in fact is difficult to become qualified product.Second filial generation product occurs later, and domestic have only minority enterprise declaration second filial generation product, because quality is wayward, national approving authority is still among considering.
First and second in generation product, its curative effect is sure.Through the practice of nearly half a century, all at this type of microecologic regulator of widespread usage,, be universally recognized reality as the functional factor of Yoghourt fermentation bacteria beverage, health food and medicine (mainly in China) in Europe, North America, Japan and China.But, because first generation product viable count and the restriction of the phase of depositing of living and second filial generation control of product quality (dead thalline self-dissolving) are difficult to implement.This just causes arising at the historic moment of third generation product.
Third generation product is with the cellular lysate of probiotic bacteria or fragmentation, uses the thalline important component as detecting index, helps the application and the popularization of qualitative, quantitative quality control and product.Find that through the newest research results over nearly 5 years the effect of probiotic bacteria is the signaling molecule (Signal molecular) that is contained in its thalline.The sort signal molecule is the important component part in contemporary intercellular communication (the cell to cellcammunication) system
(2)
Bacteroid is the class that the class bar belongs to 3 subclass in (Bacteroides).This class bacterium is called bacteroides fragilis (Bacteroidesflagilis, veillon and zuber).Following minute one of them kind of 5 subspecies of bacteroides fragilis is multiform bacteroides fragilis (B.flagilissubsp.thitaiotamicron).The multiform bacteroides fragilis is that Gram-negative is polymorphic or form is consistent bacillus does not move or uses limit flagellar movement, non-living spore type bacillus, obligate anaerobe.This bacterium mainly is present in the animal intestine intracavity.This bacterium can produce succinic acid, acetic acid, formic acid, lactic acid, propanoic acid, butanoic acid or folic acid from protein, glucose fermentation.Growth is the suitableeest under pH7.0, the 37 ℃ of conditions.Reference culture is that its G+C percentage ratio of ATCC8492 is 39.6-45%
(3)
Bacteroid is maximum probiotic bacterias of the mankind or mammal enteral.Bacteroid holds pride of place, and dyspepsiacoccus accounts for second, and bacillus bifidus accounts for the 3rd class.Secular facts have proved, bacillus bifidus and lactobacillus are useful to people and animals, and bacteroid is not put into the production strain of probiotics preparation, it is sorry that academia is felt always, because bacteroid usually becomes one " pathogen " in many abdominal infections, this pathogen does not drop into, but the result who infects.Though in focus, can check out bacteroid, this bacterium and avirulence.Have report to point out, infecting has two-phase, and infecting beginning is aerobe (escherichia coli, staphylococcus etc.), and aerobe then consumes oxygen, has created anaerobic environment, is beneficial to the passive growth of anaerobic bacteroid, because bacteroid has comparative advantage in the intestinal.We think that in fact bacteroid there is no the effect that causes infection, is misread for no other reason than that enter focus passively.Be that this is when redressing now.In the past half a century, be not put into probiotic bacteria, should be corrected because it easily causes " infection ".In eighties of last century nineties, domestic just have human bacteroid viable bacteria to make probiotic composition
(4), its effect is well, and is safe, but has stopped production because of not obtaining national approval about administration section.
Bacteroid is the maximum normal flora member in people, the animal intestinal, and the every gram intestinal of its quantity Dissolve things inside is 10
12More than, contacting closely with intestinal mucosa, the area of intestinal mucosa has the so big (260-300m of a basketball court as launching a plane
2), and directly contact with intestinal epithelial cell, its physiological action is the most tangible.
By germfree mouse inoculation bacteroides thetaiotaomicron, bacillus bifidus and colibacillary comparison, confirmed that the bacteroides thetaiotaomicron physiological effect is the strongest.Bacteroides thetaiotaomicron causes the Na in the ileum
+Glucose covalency albumen (Co.transpor, SGLT-1) the mRNA level improves, a plurality of signals (Signal) in host's lipoid mechanism of absorption increase, and comprise the increase of lipase albumen 2 (PLRP-2), colipase (Co-lipase), fat fat acid conjugated protein (L-FABP) and apolipoproteins (Apolipoprotein)
(5)
Colipase is that the outer pancreas secretory cell of pancreas produces, and colipase also has expression in mice ileum crypts epithelial cell, and behind the inoculation bacteroides thetaiotaomicron, its expression has improved 10 times.Colipase plays a crucial role in lipid metabolism by the activity of stimulating pancreas triglyceride enzyme and pancreas lipase associated protein-2 (PRLP-2).
Fat metabolic disturbance is the basic reason of obesity.Because antibiotic application destroys the intestinal flora balance, the quantity that has reduced bacteroid is likely one of popular reason of obesity over nearly 60 years.
Comprise that bacteroid normal bowel flora has specific part (signaling molecule) and combines with specific receptor (Receptor) on the epithelial cell and can produce the regulating action of a series of cell physiologicals.Receptor on host's epithelial cell all contains the outer ligand binding domain of born of the same parents, have and hydrophobicly stride the film district and have three zones of tyrosine kinase activity cytoplasmic region.Wherein the EGFR family receptors can combine with the multiple EGF of comprising, TGF-α and HB-EGF parts such as (the heparin desmin factors) and trigger the signal cascade reaction in the born of the same parents, signal is passed to cause a series of biologicallies adjusting cells physiological mechanism in the nuclear.The known signal conducting system has cyclic nucleotide system, calmodulin, CaM system, inositol phosphate system and tyrosine-kinase enzyme system at present.The biologically of cell shows the aspects such as mitosis, apoptosis, mobility, vascularization and differentiation adjusting of cell.Therefore conditioning signal transduction path as new means adjusting intestinal flora, will become the focus of the physiological action of third generation probiotic bacteria with some the crucial signaling molecules in the signal transduction.
In recent years to Toll-like Receptor, (TLR) receptor research is verified, gram-positive LTA (lipoteichoic acid) and gram-negative LPS (lipopolysaccharide) are various antibacterial signaling molecules, can cause intracellular signal emission flow process (Signalling cascade), regulate cellular physiological activity.TLRs is the bacterial receptor of high degree of specificity, to the reaction of pathogenic bacterium, has done deep research, but the research of physiological antibacterial is still just begun now
(6,7)Here it is the important opportunity of signaling molecule as the rake point of beneficial preparation.
Discovered in recent years folic acid is playing an important role aspect the signaling molecule transmission.Folic acid is one of vitamin B group, by pteridine, amino benzoic Acid and one or glutamic acid be combined into
[8]
Folic acid is the important factor that the intracellular signal in place transmits, and it can regulate the hereditary information transduction, regulates the normal operation of DNA or RNA.Height contains CPG bigeminy nucleoside district and is called CPG island (CPG islands) near the structural gene 5 ' end, and the contained CPG transcription factor complex of gene regulation element causes and methylates.Dna methylation and gene silencing (gene silence) interrelate, and non-genomic methylates (non methglation) and gene activation interrelates.Therefore, folic acid particularly tetrahydrofolic acid be that regulator gene transduction and signal are launched flow process important relationship is arranged
(9)
Folic acid has food source and intestinal flora source, and the latter has higher biologic activity and utilizability.Bacteroid is important folate source.
In sum, although the experimentation of the various physiological regulation information of transmission and theoretic discussion have had very big progress and obtained really effectively achievement between signaling molecule that normal intestinal flora is contained and the somatic cell, Shang Weiyou can provide effective product of practical application to occur.
List of references
(1) Kang Bai chief editor: " microecology principle " 206-215 of Dalian publishing house, 2002.
(2) Kang Bai " Chinese microecology magazine " 18[1 that " meets the arrival of microecology intercellular communication New Times "]: 1-5,2002.
(3) R.E. Buchanan, NE Ji Bensibaijie " Bacteria Identification handbook, the 535-536 of Science Press, 1984.
(4) the preparation method ZL 90,102,847 1,995 09 27 of Zhang Jijie bacteroid microbial ecological agent.(5)Hooper?LV，Melissa，Hand?Hanson?L.：Molecular?Analysis?of?commensal?Host-microbial?Relashionshipsin?the?Intestinem，Science?291，5505(2001)881-884，2001。
(6)Yarden?Y，Sliwski?MX；untangling?the?ErbB?signalling?network，Nat?RER，Mol，Cell?Biol，2(2)：127-137，2001。
(7)O’Nell，LA，Fuzgerald，KA?and?Dowic，AG，Trends?Immaol，24(6)：286-290，2003。
(8)Thomas，T，Weeler?and?kyline?AH：the?Mammalian?Innate?Immune?System：PotentialTargets?for?drug?Development，Immune?Endocrine?and?Mefabolic?Disorder，5：237-247?2005。
(9)Geiman，TM，Robertson?KD：Chromatin?remodeling?histone?Modification?and?DNAMethylation?how?does?it?all?fit?cogether？J?Cell?Biochem?87(2)117-125?2002。
Summary of the invention
Defective at prior art exists not only has novelty to Bacteroides signal molecule as third generation probiotic composition, and has sufficient theory and experimental basis.This invention is intended to develop new Bacteroides signal molecule goods and preparation technology (Probiotics of Bacteroides signal mole-sular).A bacteroid molecule micro-ecological preparations and its preparation process are proposed, so that make the research and development of Bacteroides signal molecule microecological preparation enter practical stage.
The present invention researches and develops bacteroid as third generation probiotic composition.Be with the bacteroid thalline through cell wall breaking, enzymolysis, slightly carry, drying and pulverizing form because with bacteroid body lysate as the signaling molecule carrier, can not cause so-called " infection ".WPG wherein, DNA contain multiple Bacteroides signal molecule.
The preparation technology of Bacteroides signal molecule goods of the present invention comprises:
1, bacteroid its thalline that ferments concentrates: the weight ratio that contains that the bacteroid seed liquor is inserted after having sterilized is: in the culture medium matrix of peptone 1.5～2%, soy peptone 0.5%, glucose 0.5%, sodium hydrogen phosphate 0.4%, potassium dihydrogen phosphate 0.1%, Carnis Bovis seu Bubali cream 0.2%, yeast extract 1%, sodium chloride 0.3%, soluble starch 0.5%, behind 35～38 ℃ of anaerobic fermentations, promptly obtain bacteroid thalline concentrate through centrifugal concentrating again.
2, cell wall breaking:, use about high pressure cell disintegrating machine operating pressure 100MPa with the broken somatic cells wall of high pressure with this bacteroid thalline concentrate adding distil water or normal saline (1: 3～1: 5).
3, enzymolysis: add respectively trypsin 1～2mg/ml), Chymotrypsin (1～2mg/ml) and pepsin (1～2mg/ml) and hydrochloric acid (making its concentration is 0.01M/l), hydrolysis 12～24 hours, centrifugal, washing obtains the cell wall crude extract.
4, drying: cell extract is in 40～70 ℃ of drying under reduced pressure.Or these goods are carried out lyophilization.The dry thing that obtains promptly gets the former powder of bacteroid molecular signal molecular preparation through grinding, sieving.Preparation raw powder is white～pale powder, can dissolve fast in water, and is stable down with refrigerated condition at normal temperatures.The test item of this Bacteroides signal molecule goods and index feature are: 1) cell wall sugar inspection takes by weighing former powder 0.01g and adds 15% n-butanol aqueous solution and respectively be mixed with 10mg/ml solution.0.5ml is in test tube in absorption, drips 0.05% anthrone sulphate reagent 1.0ml, shakes up promptly to be aeruginous.2) cell wall meso meso diaminopimelic acid is checked.Adopt ply of paper to analyse-development process, should show distinctive olive-green speckle, back flavescence color fades.3) outward appearance should be white powder.4) loss on drying inspection, subtracting weight loss should not be higher than 5%.
The former powder of multiform bacteroides fragilis molecule micro-ecological preparations adds corresponding auxiliary material can make various dosage forms: for example get the former powder 50～200g of said preparation, add glucomannan or other oligosaccharide, lactose 870g, magnesium stearate 30g divides encapsulated, make health product capsule, orally use; The same lactose 400g that adds, magnesium stearate 3g, hyprolose 40g, microcrystalline Cellulose 150g, citric acid 2g, 10% polyethylene pyrrole irons alkane ketone 80g, makes bacteroid molecule micro-ecological preparations granule, tablet through mixing, pelletize or tabletting; Same Squalene, mannitol, polysorbate80 and the sterilized water etc. of adding add up to 100 liters, become uniform white suspension with ultrasonic emulsification, for fill, make bacillus bifidus molecule micro-ecological preparations oral liquid after aseptic filtration.
Bacteroides signal molecule goods of the present invention have carried out about animal acute toxicity test, the influence to the spontaneous activity in mice number of times, the influence to rat blood pressure and breathing, automatic allergic experiment animal, confirm no acute toxicity, nothing influence to spontaneous activity number of times, blood pressure and the breathing of animal can not cause allergic reaction to animal.Zoopery also proves: these goods have the blood lipid metabolism of adjustment, reduce accumulation of fat, the effect of fat-reducing; By adjusting gene expression, regulate immunologic function simultaneously, play and improve immunity and antitumor action.
Compared with prior art, 1), the effect main body of using bacteroid molecule micro-ecological preparations of the present invention as bacteroid cell wall content (signaling molecule) preparation is host cell the present invention is characterised in that:, Bacteroides signal molecule just provides health signal to host cell, makes host cell produce corresponding physiological action and healthy performance.Signaling molecule causes the host cell gene expression, at the synthetic function corresponding enzyme of ribosome (Ribosome), brings into play its physiological action by functional enzyme.2) the bacteroid viable bacteria also is restricted at present as probiotics preparation, and its cellular content can be eliminated people may cause anaerobic infection to the bacteroid active bacteria formulation misgivings as product development.3) the viable bacteria body preparation is because the viable bacteria body is subjected to the restriction of temperature, oxygen and genetic development own, and goods are preservation at low temperatures, usually, thalline mortality rate height, and the product shelf life is very short; And dead bacteria preparation has its efficacy factor indeterminate, is difficult to identify and detect, and is rarely found on this based article market.Its efficacy factor of bacteroid molecule micro-ecological preparations of the present invention is clear and definite, is easy to detect, and goods can carry out suitability for industrialized production.Product is preservation at normal temperatures, has characteristics such as product is stable, shelf life is long.
The specific embodiment
Preparation technology is as follows for embodiment one, Bacteroides signal molecule goods:
(B.flagili, the weight ratio that contains after subsp.Thitaiotamicron) access of bacterial strain seed liquor has been sterilized is: peptone 1.5～2%, soy peptone 0.5%, glucose 0.5%, sodium hydrogen phosphate 0.4%, potassium dihydrogen phosphate 0.1%, Carnis Bovis seu Bubali cream 0.2%, yeast extract 1%, sodium chloride 0.3%, soluble starch 0.5% with multiform bacteroides fragilis DM9808.Culture medium matrix obtains bacteroid thalline concentrate through centrifugal after concentrated behind 35～38 ℃ of anaerobic fermentations.This bacteroid thalline concentrate is added normal saline, with high pressure crush method somatic cells wall.Add respectively trypsin 1～2mg/ml), Chymotrypsin (1～2mg/ml) and pepsin (1～2mg/ml) and hydrochloric acid (making its concentration is 0.01M/l), hydrolysis 12～24 hours, centrifugal, washing obtains the cell wall crude extract.These goods are carried out freezing drying.The dry thing that obtains in mortar porphyrize, sieve, promptly get the former powder of bacteroid molecule micro-ecological preparations.
The test item and the index feature of this bacteroides fragilis signaling molecule goods are: 1) cell wall sugar inspection takes by weighing former powder 0.01g and adds 15% n-butanol aqueous solution and respectively be mixed with 10mg/ml solution.0.5ml is in test tube in absorption, drips 0.05% anthrone sulphate reagent 1.0ml, shakes up promptly to be aeruginous.2) cell wall meso meso diaminopimelic acid is checked.Adopt ply of paper to analyse-development process, should show distinctive olive-green speckle, back flavescence color fades.3) outward appearance should be white powder.4) loss on drying inspection, subtracting weight loss should not be higher than 5%.
Embodiment two, (B.flagili, the weight ratio that contains after subsp.vulgatus) the bacterial strain seed liquor inserts and to have sterilized is: peptone 1.5～2%, soy peptone 0.5%, glucose 0.5%, sodium hydrogen phosphate 0.4%, potassium dihydrogen phosphate 0.1%, Carnis Bovis seu Bubali cream 0.2%, yeast extract 1%, sodium chloride 0.3%, soluble starch 0.5% with the common subspecies DM9707 of multiform bacteroides fragilis.Culture medium matrix obtains bacteroid thalline concentrate through centrifugal after concentrated behind 35～38 ℃ of anaerobic fermentations.This bacteroid thalline concentrate is added normal saline, with high pressure crush method somatic cells wall.Add respectively trypsin 1～2mg/ml), Chymotrypsin (1～2mg/ml) and pepsin (1～2mg/ml) and hydrochloric acid (making its concentration is 0.01M/l), hydrolysis 12～24 hours, centrifugal, washing obtains the cell wall crude extract.These goods are carried out freezing drying.The dry thing that obtains in mortar porphyrize, sieve, promptly get the former powder of bacteroid molecule micro-ecological preparations.
The test item and the index feature of this bacteroides fragilis signaling molecule goods are: 1) cell wall sugar inspection takes by weighing former powder 0.01g and adds 15% n-butanol aqueous solution and respectively be mixed with 10mg/ml solution.0.5ml is in test tube in absorption, drips 0.05% anthrone sulphate reagent 1.0ml, shakes up promptly to be aeruginous.2) cell wall meso meso diaminopimelic acid is checked.Adopt ply of paper to analyse-development process, should show distinctive olive-green speckle, back flavescence color fades.3) outward appearance should be white powder.4) loss on drying inspection, subtracting weight loss should not be higher than 5%.
Embodiment three, the weight ratio that contains that bacteroides melanogenicus (B.flagilis melanninogenicus) bacterial strain seed liquor is inserted after having sterilized are: peptone 1.5～2%, soy peptone 0.5%, glucose 0.5%, sodium hydrogen phosphate 0.4%, potassium dihydrogen phosphate 0.1%, Carnis Bovis seu Bubali cream 0.2%, yeast extract 1%, sodium chloride 0.3%, soluble starch 0.5%.Culture medium matrix obtains bacteroid thalline concentrate through centrifugal after concentrated behind 35～38 ℃ of anaerobic fermentations.This bacteroid thalline concentrate is added normal saline, with high pressure crush method somatic cells wall.Add respectively trypsin 1～2mg/ml), Chymotrypsin (1～2mg/ml and pepsin (and 1～2mg/ml) and hydrochloric acid (making its concentration is 0.01M/l), hydrolysis 12～24 hours, centrifugal, washing obtains the cell wall crude extract.These goods are carried out freezing drying.The dry thing that obtains in mortar porphyrize, sieve, promptly get the former powder of bacteroid molecule micro-ecological preparations.
The test item and the index feature of this bacteroides fragilis signaling molecule goods are: 1) cell wall sugar inspection takes by weighing former powder 0.01g and adds 15% n-butanol aqueous solution and respectively be mixed with 10mg/ml solution.0.5ml is in test tube in absorption, drips 0.05% anthrone sulphate reagent 1.0ml, shakes up promptly to be aeruginous.2) cell wall meso meso diaminopimelic acid is checked.Adopt ply of paper to analyse-development process, should show distinctive olive-green speckle, back flavescence color fades.3) outward appearance should be white powder.4) loss on drying inspection, subtracting weight loss should not be higher than 5%.
Embodiment four, the former powder manufacturing of multiform bacteroides fragilis DM9808 signaling molecule goods are with embodiment one.Get said preparation and carry out following experiment:
1, animal toxicity test: adopt Kunming mouse, totally 20, be divided into two groups, the suitable 5g/kg.d of test group injection volume, observe 3d after the administration continuously, compare with control group mice, feed, activity, stool, fur and various reflection are all no abnormal, two groups of none death of mice, more also no significant difference between the body weight gain group.Point out this medicine not have obvious acute toxicity effect, clinical plan consumption safety.
2, to the influence of spontaneous activity in mice number of times: Kunming mouse, body weight 20 ± 2g, the male and female dual-purpose, is divided into 5 dosage groups, 15 every group at random by totally 75.With mice difference injecting normal saline, 0.25% caffeine sodium benzoate, 0.075% chlorpromazine, 10mg/ml and the 40mg/ml bacteroid one's share of expenses for a joint undertaking microbial ecological agent of 5 experimental grouies, injected dose all is 0.1ml/10g.Observed and recorded spontaneous activity in mice number of times in 0-15 minute and 15-30 minute respectively.The results are shown in Table 1.
Table 1 Bacteroides signal molecule goods are to spontaneous activity in mice influence (* P＜0.01, * * P＜0.001)
| Medicine | Dosage | Number of animals | Spontaneous activity in mice number after the medication (X ± SD) | |
| 0-15min | 15-30min | |||
| Normal saline 0.25% caffeine sodium benzoate 0.075% chlorpromazine 10mg/ml bacteroid molecular preparation 30mg/ml bacteroid molecular preparation | 0.1ml/10g 0.1ml/10g 0.1ml/10g 0.1ml/10g 0.1ml/10g | 15 15 15 15 15 | 40.15±11.4 107.7±24.3** 11.9±6.4** 36.6±22.5 44.1±10.3 | 26.0±11.2 89.4±18.1** 1.7±0.8** 21.6±9.8 29.2±8.7 |
The result shows, compares with normal saline, and this does not have obviously influence to multiform bacteroides fragilis DM9808 molecule micro-ecological preparations to spontaneous activity in mice.
3, to the influence of rat blood pressure and breathing: adopt the Wistar rat, weight range 150g-200g, the male and female dual-purpose, is divided into 4 dosage groups, 12 every group at random by totally 48.With the existing lumbar injection 20% urethane 1g/kg anesthesia of the rat of 4 groups.After the anesthesia, fixing, cut off downrights, the medisection skin of neck is plugged the arterial cannula that filling in advance has heparin saline at sidepiece carotid artery, blood pressure normal value before the record medicine.After treating that blood pressure is steadily, difference intraperitoneal injection of saline, 10mg/ml multiform bacteroides fragilis molecule micro-ecological preparations; Normal saline, 30mg/ml multiform bacteroides fragilis molecule micro-ecological preparations, injected dose all is 0.2ml/100g.Injection back 5 minutes, 10 minutes and 15 minutes, difference observed and recorded mice blood pressure situation.Experimental result sees Table 2
Table 2 multiform bacteroides fragilis signaling molecule goods influence rat blood pressure
| Medicine | Dosage | Rat blood pressure after the medication (X ± SD) | ||
| 5min | 15min | 30min | ||
| Normal saline 10mg/ml bacteroid molecular preparation normal saline 30mg/ml bacteroid molecular preparation | 0.2ml/100g 0.2ml/100g 0.2ml/100g 0.2ml/100g | 95.1±2.9 97.8±3.0 96.4±3.4 95.4±3.6 | 95.4±3.6 96.2±4.4 95.2±1.8 95.3±4.2 | 96.2±2.7 94.1±4.1 97.3±0.9 96.7±2.5 |
The result shows: low dose of combination heavy dose is organized rat blood pressure and is breathed all without any influence. and prompting Bacteroides signal molecule goods do not have obvious adverse reaction in the injection of test dose angular vein to rat.
4, automatic allergic experiment: 5 of Cavia porcelluss are provided body weight 350 ± 50g, male and female dual-purpose by Dalian Medical Univ's Experimental Animal Center.This laboratory detect multiform bacteroides fragilis molecule micro-ecological preparations to Cavia porcellus through hypodermic active anaphylaxis.Diluting this preparation with physiology salt before the experiment is 30mg/ml.Percutaneous is injection sensitization down.Injection volume is 0.5ml/kg.Again through the heart injection, injection volume is 10ml/kg after 10 days.Observe animal and have or not anaphylaxis.The result shows that allergic symptom does not all appear in whole 5 animals, and is no abnormal after dissecting.Reach a conclusion thus, multiform bacteroides fragilis signaling molecule goods can not cause allergic reaction.
Embodiment five, the former powder manufacturing of multiform bacteroides fragilis DM9808 signaling molecule goods are with embodiment one.Get said preparation and carry out following experiment:
1, inhibition test: getting body weight is the 18-20gBALB/c mice, and each 10 of test group and matched groups extract the S180 ascites tumor, are diluted to 2 * 107 cells behind the counting, and this preparation is dissolved in physiological sodium chloride solution, are mixed with 2000ug/ml.Get 1.0ml oncocyte and 1.0ml N-CWS solution mix homogeneously, as the test group sample.The mixed liquor (with preparation identical concentration and ratio during semi-finished product) of getting 1.0ml Squalene, mannitol and polysorbate80 simultaneously mixes with the 1.0ml oncocyte organizes injection in contrast.Respectively to mouse subcutaneous injection, every 0.2ml (this preparation of 200ug/only), inoculate back 14 days and dissect animal, get tumor and claim tumor heavy, calculate tumour inhibiting rate.Computing formula is: tumour inhibiting rate (%)=(the average tumor of the average tumor weight-test group of matched group is heavy)/average tumor of matched group heavy * 100%.Tumour inhibiting rate is not less than 50%.
Embodiment six, the former powder manufacturing of multiform bacteroides fragilis signaling molecule goods are with embodiment one.Getting said preparation carries out as the experiment of downward modulation blood fat:
Get the secondary wistar male rat of body weight 150-200 gram, raise to contain 1% cholesterol, the high lipid food of 5% Adeps Sus domestica and 0.2% cholate, be divided into four groups, experiment component is senior middle school's low dose group, give that multiform bacteroides fragilis signaling molecule goods are respectively 2,1,0.5mg/kg.bw/d, adopt the normal saline dilution after, irritate stomach 2ml every day respectively.Weigh, get tail hematometry blood lipids index, measure serum cholesterol (TC), serum levels of triglyceride (TG) and serum high-density LP (HDL-C) with elisa kit.Experimental result shows: measured respectively on the the 15th, 30 day 1. that high, normal, basic dosage group is respectively 66.4 ± 6.8,62.1 ± 6.6,62.9 ± 4.8 in the rat blood serum cholesterol experimental group; 71.4 ± 4.4,67.2 ± 4.3,71.9 ± 4.7, matched group then is 51.4 ± 10.7,110.5 ± 274 (mg/dl).The experimental group serum cholesterol concentration is than matched group low (p＜0.05).And the experimental group group difference is not remarkable.2. serum TG concentration: 15th, measured the low dose group rat blood serum TG of senior middle school concentration in 30 days respectively and show, the interior senior middle school's low dose group group difference of experimental group and matched group and experimental group is not obvious.3. Serum HDL-C concentration: 15th, measured senior middle school's low dose group and control rats Serum HDL-C concentration in 30 days respectively and show, difference (p＞0.05) that there are no significant between each group.4. the experimental group animal is compared the body weight there was no significant difference (p＞0.05) of respectively organizing rat with matched group.This experimental result shows: compare with the control rats of raising with high lipid food, measured the low and matched group of Serum TC concentration respectively at the 15th, 30 day that tests, and significant difference (p＜0.05) is arranged.By calculate showing that the dosage group HDL/TC of senior middle school ratio is high low with matched group and significant difference (p＜0.05) is arranged with matched group, atherogenic index.According to the criterion of " the health food function assessment assessment process and the method for inspection ", can think that the Bacteroides signal molecule goods have the effect of regulating rat fat.
Embodiment seven, the former powder manufacturing of multiform bacteroides fragilis DM9808 signaling molecule goods are with embodiment one.Get said preparation and carry out Study immune regulation:
Sample adopts multiform bacteroides fragilis molecule micro-ecological preparations, presses the human body recommended amounts and is everyone 60mg every day (2,1,0.5mg/kg.bw/d).Prepare each dose concentration with normal saline.The female white mice of laboratory animal secondary Kunming kind, body weight 18-22 grams.Test method: respectively organize by above-mentioned dosage and to irritate stomach every day once, after continuous 30 days animal is carried out every immune indexes and measure, method is as follows:
1, mice delayed allergy (DTH): irritate stomach the 26th day and only inject 2% sheep red blood cell (SRBC) (SRBC) 0.2ML/ to mouse peritoneal, before the 4th day left back sufficient sole of the foot subcutaneous injection 2%SRBC of portion of mice after immunity (20UL/ only) attacks and attack measured the same position of the left back sufficient sole of the foot of mice thickness in back 24 hours, 48 hours respectively, calculate and attack forward and backward difference, the result carries out difference analysis.Result such as table 3 show: (24h at interval) sufficient sole of the foot thickness difference and matched group differences had significance (P＜0.05) before and after middle dosage group was attacked with SRBC, and high and low dose group mice difference does not have significance (P＞0.05) with matched group difference.
Table 3 Bacteroides signal molecule goods are to mouse DTH reaction P＜0.01 as a result
2, ConA inducing mouse splenocyte conversion test (MTT): to the aseptic spleen of getting of each group mice, the system splenocyte suspension, adjusting splenocyte concentration with the RPMT1640 complete culture solution is 2 * 10
6Individual/ml, carry out lymphproliferation response according to mtt assay in the program, survey its optical density value (OD) at 752-C ultraviolet-uisible spectrophotometer 570nm wavelength place at last, calculate the optical density value difference in ConA+ and each hole of ConA-, the result carries out variance analysis.Table 4 result shows: there are no significant (p＜0.05) for each dosage group mice ConA+ and ConA-OD difference matched group differences.
Table 4 Bacteroides signal molecule goods are to mouse spleen lymphocyte conversion test (MTT) result (p＜0.05)
3, Turnover of Mouse Peritoneal Macrophages is engulfed the chicken red blood cell test: only respectively organize the equal lumbar injection 20% chicken erythrocyte suspension 1ml/ of mice after last is tried thing, put to death mice at interval in 30 minutes, the abdominal cavity only injects normal saline 2ml/, abdominal cavity washing liquid film-making is got in jolting after one minute, 37 ℃ of incubations 30 minutes, rinsing, fixing dyeing microscopic examination.Counting is engulfed the macrophage number and the macrophage phagocytic chicken red blood cell number of chicken red blood cell, carries out variance analysis with phagocytic rate and phagocytic index.Each dosage group mouse macrophage of table 5 demonstration is engulfed chicken red blood cell phagocytic rate, phagocytic index and matched group differences, and there are no significant (p＜0.05)
Table 5 Bacteroides signal molecule goods are engulfed the chicken red blood cell result of the test to Turnover of Mouse Peritoneal Macrophages
4, antibody-producting cell detects (PFC): in to mouse peritoneal injection 2%SRBC0.2ml/ only irritating stomach the 25th day, after immunity, put to death in the 5th day, spleen system cell suspension is got in dissection, adjusting cell concentration with the RPMI1640 complete culture solution is 5 * 106/ml, the follow procedure method prepares the agarose slide, incubation added complement after 1.5 hours in CO2 gas incubator, and incubation is 1.5 hours again. and count the hemolysis plaque number that forms on every thin agar layer plate, the result carries out variance analysis.Table 6 result shows that each dosage group hemolysis plaque number average is higher than matched group, wherein high dose group mice hemolysis plaque number and matched group differences have significance (p＜0.05), in, there are no significant (p＞0.05) for low dose group mice hemolysis plaque number and matched group differences.
Table 6 Bacteroides signal molecule goods are to mouse antibodies cellulation result of the test
| 0.5 contrast | 10 10 | 813.0±331.4 698.2±306.5 |
5, serum hemolysin is measured: in irritating stomach the 25th day to mouse peritoneal injection 2%SRBC (0.2ml/ only), plucked eyeball on the 5th day and get blood after immunity, separation of serum is also measured at the clear hemolysin of the enterprising promoting the circulation of blood of Microhemagglutination plate.Incubate the region between the heart and the diaphragm after 3 hours for 37 ℃, statistics hemagglutination degree calculates the corresponding antibodies product, and the result carries out variance analysis.Table 7 shows: high, middle dosage group antibody product and matched group differences all have highly significant (p＜0.01).
Table 7 Bacteroides signal molecule goods are to mice serum hemolysin measurement result
The immunomodulating experiment shows: the tardy allergy of cellular immune function assay mice (DTH) result and ConA inducing mouse splenocyte conversion test (MTT) are all positive; Humoral immune function is measured: antibody-producting cell detection (PFC) result and serum hemolysin measurement result are all positive; Monokaryon-macrophage function is measured: it is negative that Turnover of Mouse Peritoneal Macrophages is engulfed the chicken red blood cell result of the test.This laboratory observation is all positive to mouse cell immunity and humoral immune function result of the test to the Bacteroides signal molecule goods, illustrates that the Bacteroides signal molecule preparation has immunoregulation effect.
Embodiment eight, the former powder manufacturing of multiform bacteroides fragilis molecule micro-ecological preparations are with embodiment one.Get said preparation 50-200g adding glucomannan or other oligosaccharide, lactose 870g, magnesium stearate 30g, No. 0 capsule of packing is made health food, orally uses.
Embodiment nine, the former powder manufacturing of multiform bacteroides fragilis molecule micro-ecological preparations are with embodiment one.Get former lyophilized powder 50-200g, add lactose 400g, magnesium stearate 3g, hyprolose 40g, microcrystalline Cellulose 150g, citric acid 2g, 10% polyethylene pyrrole irons alkane ketone 80g, mixes, and granulates, and makes bacteroid molecule micro-ecological preparations granule.Or tabletting, make bacteroid molecule micro-ecological preparations tablet.
Embodiment ten, the former powder manufacturing of multiform bacteroides fragilis molecule micro-ecological preparations are with embodiment one.Get said preparation 50-200g and add 100 liters of totals such as Squalene, mannitol, polysorbate80 and sterilized water, become uniform white suspension with ultrasonic emulsification, for fill, it is oral to make the bacillus bifidus molecule micro-ecological preparations after aseptic filtration.
Claims (6)
1. Bacteroides signal molecule microecological preparation, it is characterized in that with bacteroid through anaerobic fermentation, cell wall breaking, enzymolysis, slightly carry, dry and pulverize the preparation raw powder that forms; It is white ~ pale powder, can dissolve fast in water, and stable down with refrigerated condition at normal temperatures, WPG wherein, DNA contain multiple Bacteroides signal molecule; Product feature is: 1) in the cell wall sugar inspection it is mixed with and is aeruginous when 10mg/ml 15% n-butanol aqueous solution drips 0.05% anthrone sulphate reagent 1.0ml; 2) cell wall meso meso diaminopimelic acid is checked: adopt ply of paper to analyse-development process, should show distinctive olive-green speckle, back flavescence color fades; 3) outward appearance should be white powder; 4) loss on drying inspection, subtracting weight loss should not be higher than 5%;
Described bacteroid is a bacteroides fragilis.
2. according to the described Bacteroides signal molecule microecological preparation of claim 1, it is characterized in that described microbial ecological agent is an oral capsule, wherein contain the former powder 50 ~ 200g of microbial ecological agent, add glucomannan or other oligosaccharide 870g, magnesium stearate 30g, No. 0 capsule of packing.
3. according to the described Bacteroides signal molecule microecological preparation of claim 1; it is characterized in that described microbial ecological agent is oral granule or tablet; wherein contain the former powder 50 ~ 200g of microbial ecological agent, add lactose 400g, magnesium stearate 3g; hyprolose 40g; microcrystalline Cellulose 150g, citric acid 2g, 10% polyethylene pyrrole irons alkane ketone 80g; mix, granulate or tabletting.
4. according to the described microecological bifid bacterium signal molecule preparation of claim 1, it is characterized in that described microbial ecological agent is an oral liquid, contain the former powder 50～200g of bacteroid molecule micro-ecological preparations in 10 liters of oral liquids, also contain Squalene, mannitol, the polysorbate of common amount.
5. the method for making of Bacteroides signal molecule microecological preparation is characterized in that its processing step is:
1) bacteroid its thalline that ferments concentrates: the weight ratio that contains that bacteroid bacterium seed liquor is inserted after having sterilized is: in the culture medium matrix of peptone 1.5 ~ 2%, soy peptone 0.5%, glucose 0.5%, sodium hydrogen phosphate 0.4%, potassium dihydrogen phosphate 0.1%, Carnis Bovis seu Bubali cream 0.2%, yeast extract 1%, sodium chloride 0.3%, soluble starch 0.5%, behind 35 ~ 38 ℃ of anaerobic fermentations, obtain bacteroid thalline concentrate through centrifugal after concentrated again;
2) cell wall breaking: with this bacteroid thalline concentrate adding distil water or normal saline, bacteroid thalline concentrate: distilled water or normal saline=1: 3-1: 5, use the high pressure cell disintegrating machine with the broken somatic cells wall of 100MPa left and right sides high pressure;
3) enzymolysis: adding the Chymotrypsin of trypsin, 1～2mg/ml of 1～2mg/ml and 1～2mg/ml pepsin and hydrochloric acid-make its concentration respectively is 0.01M/l, hydrolysis 12～24 hours, and centrifugal, washing obtains the cell wall crude extract;
4) drying: cell extract in 40～70 ℃ of drying under reduced pressure till the solvent-free flavor.
6. according to the method for making of the described Bacteroides signal molecule microecological preparation of claim 5, it is characterized in that the drying steps in its processing step is: the trehalose that adds 100～200mM/L carries out freezing drying.
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| CN101953855A (en) * | 2009-07-21 | 2011-01-26 | 明博医药技术开发（上海）有限公司 | Preparation method and application for lactobacillus cell wall lysate |
| CN102947441A (en) * | 2010-06-01 | 2013-02-27 | 穆尔研究企业有限责任公司 | Cellular constituents from bacteroides, compositions thereof, and therapeutic methods employing bacteroides or cellular constituents thereof |
| WO2017020783A1 (en) * | 2015-07-31 | 2017-02-09 | 广州知易生物科技有限公司 | Application of bacteroides fragilis in prevention and/or treatment of meningitis |
| WO2017020786A1 (en) * | 2015-07-31 | 2017-02-09 | 广州知易生物科技有限公司 | Application of bacteroides fragilis in treating and/or preventing obesity or diabetes |
| CN106852938A (en) * | 2015-12-09 | 2017-06-16 | 深圳华大基因研究院 | Application of the bacteroid (Bacteroides) in obesity-related disease is treated and prevented |
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| WO2019056404A1 (en) * | 2017-09-22 | 2019-03-28 | 中山大学 | Use of bacteroides fragilis in preparation of medicament for treating and preventing tumor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101953855A (en) * | 2009-07-21 | 2011-01-26 | 明博医药技术开发（上海）有限公司 | Preparation method and application for lactobacillus cell wall lysate |
| CN102947441A (en) * | 2010-06-01 | 2013-02-27 | 穆尔研究企业有限责任公司 | Cellular constituents from bacteroides, compositions thereof, and therapeutic methods employing bacteroides or cellular constituents thereof |
| WO2017020783A1 (en) * | 2015-07-31 | 2017-02-09 | 广州知易生物科技有限公司 | Application of bacteroides fragilis in prevention and/or treatment of meningitis |
| WO2017020786A1 (en) * | 2015-07-31 | 2017-02-09 | 广州知易生物科技有限公司 | Application of bacteroides fragilis in treating and/or preventing obesity or diabetes |
| CN106389475A (en) * | 2015-07-31 | 2017-02-15 | 广州知易生物科技有限公司 | Applications of Bacteroides fragilis in prevention and/or treatment of meningitis |
| CN106389475B (en) * | 2015-07-31 | 2019-11-08 | 广州知易生物科技有限公司 | Bacteroides fragilis is preventing and/or is treating the application in meningitis |
| CN106852938A (en) * | 2015-12-09 | 2017-06-16 | 深圳华大基因研究院 | Application of the bacteroid (Bacteroides) in obesity-related disease is treated and prevented |
| WO2019056404A1 (en) * | 2017-09-22 | 2019-03-28 | 中山大学 | Use of bacteroides fragilis in preparation of medicament for treating and preventing tumor |
| CN108968025A (en) * | 2018-06-13 | 2018-12-11 | 江苏新申奥生物科技有限公司 | Adjust the high concentration lactic acid bacteria freeze drying powder of blood pressure and blood lipoid |
| CN111714522A (en) * | 2019-03-04 | 2020-09-29 | 中国科学院微生物研究所 | Bacteroides and application thereof |
| CN111714522B (en) * | 2019-03-04 | 2022-07-15 | 中国科学院微生物研究所 | Bacteroides and application thereof |
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