Summary of the invention
The object of the present invention is to provide a kind of Bacillus licheniformis and herbal polysaccharide compositions and preparation thereof, further relate to said composition and can under oxygen consumption and anaerobic condition, significantly increase the propagation of probiotic bacteria, strengthen the ability of the bacterium that suppresses to cure the disease, digestive tract disease symphysis unit's compositions and preparation that more effective treatment and prevention alteration of intestinal flora cause.
The object of the present invention is achieved like this: a kind of Bacillus licheniformis and herbal polysaccharide compositions, comprise Bacillus licheniformis, herbal polysaccharide, adjuvant, Bacillus licheniformis is Bacillus licheniformis CMCC63516 viable bacteria powder, the mass ratio of Bacillus licheniformis CMCC63516 viable bacteria powder and Chinese herbal medicine extract is 1:1.25-25, and herbal polysaccharide is a kind of in lycium barbarum polysaccharide, lentinan, astragalus polysaccharides, Radix Codonopsis polysaccharide, Radix Et Caulis Acanthopanacis Senticosi polysaccharide; Polyoses content in described herbal polysaccharide is 70%-95%; Under oxygen consumption and anaerobic state, every kind of herbal polysaccharide all has proliferation function to Bacillus licheniformis, and every kind of herbal polysaccharide is relevant with the addition in seed culture medium to the proliferation function of Bacillus licheniformis, the composition of seed culture medium is: beef powder 0.5g/100ml, peptone 1.0g/100ml, NaCl0.5g/100ml, adjusts pH7.0-7.2; The percentage by weight that herbal polysaccharide addition accounts for seed culture medium is: lycium barbarum polysaccharide 0.01%-0.05%, preferably 0.01%; Lentinan 0.01%-0.05%, preferably 0.05%; Astragalus polysaccharides 0.01%-0.10%, preferably 0.10%; Radix Codonopsis polysaccharide 0.01%-0.10%, preferably 0.05%; Radix Et Caulis Acanthopanacis Senticosi polysaccharide 0.01%-0.05%; Every 1000 parts of each constituent contents of described Bacillus licheniformis and herbal polysaccharide composite preparation are: Bacillus licheniformis CMCC63516 viable bacteria powder is 25-50 part, herbal polysaccharide is 31-625 part, diluent 300-700 part, binding agent 20-200 part, lubricant 1-20 part, correctives 0.1-1 part; Said composition is prepared into oral formulations, according to common process, Bacillus licheniformis CMCC63516 viable bacteria powder, oligosaccharide class prebiotics and adjuvant is prepared into conventional tablet or enteric coated tablet, conventional capsule or enteric coated capsule, plain particles or enteric coated granule; In the adjuvant of described Bacillus licheniformis and herbal polysaccharide compositions, diluent refers to one or more in lactose, dextrin, cane sugar powder, mannitol, glucose, sorbitol, starch, microcrystalline Cellulose, dalcium biphosphate, calcium carbonate, magnesium carbonate; Adhesive refers to one or more in starch slurry, dextrin, pregelatinized Starch, methylcellulose, hydroxy methocel, hydroxypropyl emthylcellulose, HPMC, gelatin, arabic gum, sodium alginate; Disintegrating agent is one or more in alginic acid, ion exchange resin, Sodium Hydroxymethyl Stalcs, cross-linking sodium carboxymethyl cellulose, polyvinylpolypyrrolidone, carboxymethylcellulose calcium, low-substituted hydroxypropyl cellulose, part alphalysed starch; Lubricant refers to one or more in magnesium stearate, calcium stearate, wax class, micropowder silica gel, polyethylene glycols, sodium lauryl sulphate, polyoxyethylene monostearate, Brij30; Fluidizer refers to one or more in Pulvis Talci, micropowder silica gel, wheaten starch; Coating materials refers to one or more in hydroxypropyl methylcellulose acetate succinate, carboxymethylethylcellulose, titanium dioxide, II acrylic resin, III acrylic resin, polyvidone, poloxamer, cellulose acetate phthalate, polyvinyl acetate phthalic acid ester, Opadry series; Correctives comprises sweeting agent and aromatic, sweeting agent refers to one or more in sucrose, orange oil, sorbitol, mannitol, aspartame, saccharin sodium, radix asparagi sweet extract, protein sugar, steviol glycosides, cyclamate, and aromatic refers to one or more in Fructus Citri Limoniae, Oleum menthae, aqua methnae, aqua cinnamomi, apple essence, flavoring banana essence, flavoring pineapple essence, Fructus Chaenomelis essence.
Main points of the present invention are the compositions that Bacillus licheniformis viable bacteria powder and herbal polysaccharide form.Its principle is under this ratio, and herbal polysaccharide can significantly promote Bacillus licheniformis propagation under oxygen consumption and anaerobic condition, strengthens it and suppresses pathogenic bacterium ability, rapidly regulating intestinal canal colony balance.
Bacillus licheniformis and herbal polysaccharide compositions and preparation thereof are compared with prior art, there is Bacillus licheniformis and herbal polysaccharide compositions and under oxygen consumption and anaerobic condition, can both promote that Bacillus licheniformis significantly breeds, strengthen it and recover intestinal microbial population balanced capacity, improve rapidly intestinal field planting drag, compositions onset when the intestinal tract diseases such as treatment diarrhoea is faster, the course for the treatment of is shorter, can also avoid CMCC63516 to take in a large number the side effect of rear constipation, probiotic bacteria performance physiological action is had to the positive advantages such as meaning, will be widely used in microbial technology field.
Below in conjunction with embodiment, the present invention is described in detail.
The specific embodiment
Below to further illustrate of the present invention, rather than limitation of the present invention.
Embodiment mono-
The lycium barbarum polysaccharide, lentinan, astragalus polysaccharides, Radix Codonopsis polysaccharide or the Radix Et Caulis Acanthopanacis Senticosi polysaccharide that prove effective additive capacity have remarkable facilitation to Bacillus licheniformis CMCC63516 propagation under oxygen consumption condition, extend Bacillus licheniformis exponential phase, shorten the exponential phase Bacillus licheniformis antibacterial doubling time, reduce thalline self-dissolving rate.
Strain: Bacillus licheniformis CMCC63516 (strain number at Chinese medicine antibacterial preservation center)
Seed culture medium (g/100ml): beef powder 0.5, peptone 1.0, NaCl0.5, pH7.0-7.2.
Basal medium: same seed culture medium.
Herbal polysaccharide culture medium (g/100ml): beef powder 0.5, peptone 1.0, NaCl0.5, pH7.0-7.2, adds variable concentrations herbal polysaccharide (0.01,0.05,0.10,0.50,1.00,5.00).
Method
[1]: with reference to patent CN101537020A.
Variable concentrations herbal polysaccharide culture medium preparation: take respectively a certain amount of lycium barbarum polysaccharide, lentinan, astragalus polysaccharides, Radix Codonopsis polysaccharide, Radix Et Caulis Acanthopanacis Senticosi polysaccharide dry powder according to variable concentrations ratio and be placed in 250ml conical flask, through 121 ℃ of sterilizing 15min, after cooling, respectively at adding the aseptic basal medium of 50ml in each 250ml conical flask, mix and both obtain herbal polysaccharide culture medium.
Control medium preparation: according to the conclusion of patent CN101537020A, choose 5.00% soybean oligo saccharide culture medium in contrast, preparation method is the same.
Seed preparation: inoculate a ring inclined-plane seed under gnotobasis in seed culture medium, 37 ℃, 180rpm cultivates 15hr.
The growth effect of variable concentrations herbal polysaccharide to Bacillus licheniformis: adopt same inoculum concentration to be inoculated in respectively basal medium and control medium, be placed in 37 ℃, 180rpm shaken cultivation, gets 17hr and 24hr bacterium liquid and utilizes plate count method mensuration bacterium dense, the results are shown in Table 1.
Under table 1 oxygen consumption condition, herbal polysaccharide is to the short multiplication capacity result of the test of CMCC63516
(viable count unit: 10
8cFU/ml)
As can be seen from Table 1, while cultivating 17hr and 24hr, the herbal polysaccharide of different additive amount is to the proliferation function of Bacillus licheniformis BL63516 difference to some extent.In culture medium, add 0.01%-0.05% and 1.00%-5.00% lycium barbarum polysaccharide, optimum concentration is 0.05%, and the rate of increase is 2.359 times; 0.01%-0.05% and 1.00%-5.00% lentinan, optimum concentration is 0.01%, the rate of increase is 2.882 times; 0.01%-0.05% and 1.00% astragalus polysaccharides, optimum concentration is 0.05%, the rate of increase is 1.913 times; 0.01%-0.05% and 5.00% Radix Codonopsis polysaccharide, optimum concentration is 5.00%, the rate of increase is 1.754 times; 0.10% and 1.00% Radix Et Caulis Acanthopanacis Senticosi polysaccharide, optimum concentration is 1.00%, the rate of increase is 2.258 times.Wherein lentinan to the propagation degree of Bacillus licheniformis higher than soybean oligo saccharide.
[1] Zheng Baishui, Li Xianlin, Su Xianying etc., the symphysis unit of Bacillus licheniformis and oligosaccharide prebiotics and compositions and preparation, [P] CN101537020A.
Embodiment bis-
Proof effective dose herbal polysaccharide is not identical to the proliferation of Bacillus licheniformis CMCC63516 under oxygen consumption condition and anaerobic condition.
The preparation of strain, seed culture medium, basal medium and herbal polysaccharide culture medium is all with embodiment mono-.
Method:
1, seed method of making and detection method are all with embodiment mono-.
2, detect herbal polysaccharide Bacillus licheniformis growth effect is cultivated under oxygen-free environment, gas composition is N
285%, H
210%, CO
25%, cultivation temperature and ventilation are with embodiment mono-.
Under table 2 anaerobic condition, herbal polysaccharide is to the short multiplication capacity result of the test of CMCC63516
(viable count unit: 10
8cFU/ml)
As can be seen from Table 2, various herbal polysaccharides have remarkable difference to the proliferation of Bacillus licheniformis CMCC63516 under oxygen consumption condition He under anaerobic condition.Under anaerobic, add 0.01%-0.05% lycium barbarum polysaccharide in culture medium, optimum concentration is 0.01%, and the rate of increase is 1.968; 0.01%-0.05% lentinan, optimum concentration is 0.05%, the rate of increase is 1.862; 0.01%-0.10% astragalus polysaccharides, optimum concentration is 0.10%, the rate of increase is 1.952; 0.01%-0.10% Radix Codonopsis polysaccharide, optimum concentration is 0.05%, the rate of increase is 1.850; 0.01%-0.05% Radix Et Caulis Acanthopanacis Senticosi polysaccharide, optimum concentration is 0.05%, the rate of increase is 1.753.Most herbal polysaccharides are under anaerobic better than soybean oligo saccharide to the proliferation function of Bacillus licheniformis.
Embodiment tri-
With shaking the test of pipe method
[2,3]confirm the under anaerobic impact of the pathogenic staphylococcus aureus ability of different herbal polysaccharides promotion Bacillus licheniformis CMCC63516 rejection condition.
[2] Cheng Xiufang, Liu Hong, Zhang Zhi's spark etc., the biocidal property of two probiotics and symphysis unit thereof are to test mice intestinal microbial population effect research [J] Shandong medicine thing, 2010,29 (4): 234-236.
[3] Wu Tielin, Ge Zhenghang. antifungal Miao Ethnomedicine is observed [J] traditional Chinese medical science journal, 2010,25 (146): 101-102 to the extracorporeal bacteria inhibitor test of Candida albicans strain.
Indicator bacteria: staphylococcus aureus CMCC (B) 26003 buys from Chinese medicine antibacterial preservation center.
Staphylococcus aureus culture medium and condition of culture: glucose 0.2%, peptone 1.0%, NaCl0.5%, Carnis Bovis seu Bubali cream 0.5%, pH7.0,37 ℃, 180r/min shaking table shaken cultivation 18h, by the dilution of exponential phase bacterium liquid, makes viable count approximately 10
5-10
6cFU/ml, standby.
Method:
1, plant liquid preparation: aseptic inoculation one encircles slant strains to fluid medium, 37 ℃, 180rpm shaking table shaken cultivation 16hr, 5 times of the trophophase culture fluid of taking the logarithm dilutions, detect OD
660between 0.2-0.3, bacterium is dense in 1.0-1.5 * 10
7between, standby.
2, antibacterial cultivation altogether: the Bacillus licheniformis kind liquid and the mixed bacterium of staphylococcus aureus kind liquid that inoculate same bacterium amount, in culture medium, add the herbal polysaccharide of variable concentrations, and in same culture medium, inoculate respectively Bacillus licheniformis kind liquid and staphylococcus aureus kind liquid, prebiotics with contrast variable concentrations affects the propagation of the two, cultivate altogether after 24hr, the bacterium that detects respectively two kinds of bacterium is dense.
Table 3 herbal polysaccharide affects result of the test to CMCC63516 bacteriostasis
As can be seen from Table 3, there were significant differences to the growth of staphylococcus aureus for the herbal polysaccharide of variety classes variable concentrations, and the impact that CMCC63516 is suppressed to staphylococcus aureus growth also has difference.Under experimental condition, 0.01%-0.05% lycium barbarum polysaccharide, 0.01%-0.05% lentinan, 0.01% astragalus polysaccharides, 0.01-0.05% Radix Codonopsis polysaccharide and 0.01-0.05% Radix Et Caulis Acanthopanacis Senticosi polysaccharide can both promote CMCC63516 to suppress staphylococcus aureus growth.Wherein 0.05% lentinan is that 2.54, Dui Jin Portugal suppression ratio is more than 95% to the short rate of increase of CMCC63516 in mixed bacterium cultivation.
Following examples are symphysis unit composite preparations that Bacillus licheniformis and herbal polysaccharide form, it is the further explanation to claim, rather than the restriction to claim, in embodiment, every gram of compositions is 10-20 hundred million containing Bacillus licheniformis viable count, and Bacillus licheniformis viable bacteria powder is 30,000,000,000/g.
Embodiment tetra-
Get Bacillus licheniformis viable bacteria powder 20kg, lycium barbarum polysaccharide 25kg, calcium hydrogen phosphate 180kg, lactose 54.6kg, aspartame 0.02kg, polyvinylpolypyrrolidone 9.6kg, pulverized 80 mesh sieves, mix homogeneously.Add 2% hydroxypropyl emthylcellulose soft material processed in right amount, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add magnesium stearate 1.2kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 19.7 hundred million.
Embodiment five
Get Bacillus licheniformis viable bacteria powder 20kg, lycium barbarum polysaccharide 150kg, microcrystalline Cellulose 185.5kg, sucrose 31kg, stevioside 0.02kg, cross-linking sodium carboxymethyl cellulose 10.5kg, pulverized 80 mesh sieves, mix homogeneously.Add 2% hydroxypropyl emthylcellulose soft material processed in right amount, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add micropowder silica gel 0.5kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 14.1 hundred million.
Embodiment six
Get Bacillus licheniformis viable bacteria powder 20kg, lentinan 45kg, pregelatinized Starch 160kg, lactose 85.8kg, saccharin sodium 0.015kg, low-substituted hydroxypropyl cellulose 6.2kg, pulverized 80 mesh sieves, mix homogeneously.Add 3% methylcellulose appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add Pulvis Talci 2kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 18.2 hundred million.
Embodiment seven
Get Bacillus licheniformis viable bacteria powder 10kg, lentinan 62.5kg, dextrin 72.5kg, lactose 50.3kg, aspartame 0.018kg, carboxymethyl starch sodium 5.5kg, pulverized 80 mesh sieves, mix homogeneously.Add 10%PE6400 appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add magnesium stearate 1kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 15.8 hundred million.
Embodiment eight
Get Bacillus licheniformis viable bacteria powder 10kg, astragalus polysaccharides 12.5kg, mannitol 112.3kg, starch 85kg, saccharin sodium 0.01kg, polyvinylpolypyrrolidone 8.2kg, pulverized 80 mesh sieves, mix homogeneously.Add 8% starch slurry appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add Pulvis Talci 0.71kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 13.6 hundred million.
Embodiment nine
Get Bacillus licheniformis viable bacteria powder 10kg, astragalus polysaccharides 125kg, sorbitol 60kg, microcrystalline Cellulose 21.3kg, stevioside 0.01kg, polyvinylpolypyrrolidone 5.8kg, pulverized 80 mesh sieves, mix homogeneously.Add 5% polyvinyl alcohol appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add micropowder silica gel 0.5kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 13.1 hundred million.
Embodiment ten
Get Bacillus licheniformis viable bacteria powder 10kg, Radix Codonopsis polysaccharide 12.5kg, microcrystalline Cellulose 105kg, lactose 42.5kg, aspartame 0.015kg, carboxymethyl starch sodium 2.8kg, pulverized 80 mesh sieves, mix homogeneously.Add 2% hydroxypropyl emthylcellulose appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add magnesium stearate 1.1kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 19.8 hundred million.
Embodiment 11
Get Bacillus licheniformis viable bacteria powder 10kg, Radix Codonopsis polysaccharide 125kg, calcium sulfate 103.3kg, sucrose 58kg, saccharin sodium 0.01kg, low-substituted hydroxypropyl cellulose 6.3kg, pulverized 80 mesh sieves, mix homogeneously.Add 10%PEG6000 appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add Pulvis Talci 1.6kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 18.2 hundred million.
Embodiment 12
Get Bacillus licheniformis viable bacteria powder 20kg, Radix Et Caulis Acanthopanacis Senticosi polysaccharide 25kg, starch 153kg, lactose 83.6kg, stevioside 0.012kg, cross-linking sodium carboxymethyl cellulose 10.7kg, pulverized 80 mesh sieves, mix homogeneously.Add 3% methylcellulose appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add micropowder silica gel 0.5kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 18.69 hundred million.
Embodiment 13
Get Bacillus licheniformis viable bacteria powder 5kg, Radix Et Caulis Acanthopanacis Senticosi polysaccharide 31.25kg, mannitol 42.3kg, pregelatinized Starch 67.5kg, aspartame 0.015kg, polyvinylpolypyrrolidone 3.5kg, pulverized 80 mesh sieves, mix homogeneously.Add 3% hydroxypropyl emthylcellulose appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate, add Pulvis Talci 1.2kg, after mix homogeneously, pack and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 10.3 hundred million.
Embodiment 14
Get Bacillus licheniformis powder 10kg, lycium barbarum polysaccharide 12.5kg, Icing Sugar 35.7kg, dextrin 50.5kg, starch 166.5kg, polyvinylpolypyrrolidone 8.7kg, pulverized 80 mesh sieve mix homogeneously, added 2% hydroxypropyl emthylcellulose soft material processed in right amount, 18 mesh sieve wet granular processed, 45 ℃ are dry, 18 mesh sieve granulate, and the acrylic resin RL100 of take carries out powder coating as coating solution, in granule, add micropowder silica gel 1.5kg,, cyclamate 0.06kg, after mix homogeneously, pack and get final product.In every gram of compositions, contain Bacillus licheniformis 11.6 hundred million.
Embodiment 15
Get Bacillus licheniformis powder 10kg, lentinan 40kg, sorbitol 82.3kg, dextrin 35kg, cross-linking sodium carboxymethyl cellulose 9.8kg, pulverized 80 mesh sieve mix homogeneously, add 2% carboxymethylcellulose calcium soft material processed in right amount, 18 mesh sieves mix wet granular processed, and 45 ℃ dry, 18 mesh sieve granulate, take hydroxypropyl level cellulose titanate esters as coating solution carries out powder coating, in granule, add magnesium stearate 1.6kg, stevioside 0.04kg, after mix homogeneously, pack and get final product.In every gram of compositions, contain Bacillus licheniformis 15.3 hundred million.
Embodiment 16
Get Bacillus licheniformis powder 10kg, astragalus polysaccharides 65kg, microcrystalline Cellulose 130.8kg, lactose 30.5kg, low-substituted hydroxypropyl level cellulose 7.0kg, pulverized 80 mesh sieve mix homogeneously, add 5% polyvinyl alcohol soft material processed in right amount, 18 mesh sieves mix wet granular processed, and 45 ℃ dry, 18 mesh sieve granulate, take acrylic resin RL100 as coating solution carries out powder coating, in granule, add Pulvis Talci 1.8kg, saccharin sodium 0.015kg, mix homogeneously, packs and get final product.In every gram of compositions, contain Bacillus licheniformis 12.9 hundred million.
Embodiment 17
Get Bacillus licheniformis powder 5kg, Radix Codonopsis polysaccharide 125kg, mannitol 20.5kg, carboxymethyl starch sodium 6.8kg, pulverized 80 mesh sieve mix homogeneously, added 10% starch slurry soft material processed in right amount, 18 mesh sieves mix wet granular processed, 45 ℃ are dry, 18 mesh sieve granulate, and the hydroxypropyl level cellulose titanate esters of take is carried out powder coating as coating solution, in granule, add micropowder silica gel 1.2kg, cyclamate 0.05kg, mix homogeneously, packs and get final product.In every gram of compositions, contain Bacillus licheniformis 10.1 hundred million.
Embodiment 18
Get Bacillus licheniformis viable bacteria powder 10kg, Radix Et Caulis Acanthopanacis Senticosi polysaccharide 50kg, calcium hydrogen phosphate 75.2kg, lactose 30.5kg, polyvinylpolypyrrolidone 7.5kg, pulverized 80 mesh sieves, mix homogeneously.Add 10%EG6000 appropriate, soft material processed, 18 mesh sieve wet granular processed, 45 ℃ are dry, and 18 mesh sieve granulate be take acrylic resin RL100 as coating solution carries out powder coating, in granule, add Pulvis Talci 1.8kg, and stevioside 0.05kg, after mix homogeneously, packs and get final product.Every gram of compositions is containing Bacillus licheniformis viable bacteria 16.8 hundred million.