CN1969889A - Enteric-coated multilayer encapsulated probiotic microcapsule and preparation method thereof - Google Patents
Enteric-coated multilayer encapsulated probiotic microcapsule and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an enteric-solubility multilayer encysted bacterium microcapsule and making method in the biological agent technical domain, which is characterized by the following: adopting sodium alginate, calcium chloride and chitose as clad material of microcapsule; making lactic acid bacteria or bifidobacteria as core-clad material; proceeding ionic exchange for sodium alginate and calcium chloride; forming second clad on the surface of calcium alginate through different isoelectric points of calcium alginate and chitose; freezing at low temperature to obtain the product.
Description
One, technical field
The present invention relates to a kind of microorganism formulation, be specifically related to a kind of enteric-coated multilayer encapsulated probiotic microcapsule and preparation method thereof.
Two, background technology
Microorganism formulation is as nontoxic, the no Drug resistance of a kind of natural biologically active agents, noresidue, and have enhance immunity, promote growth, improve the effect of efficiency of feed utilization, be widely used in human health care's food, and can be used as feed additive and be applied in the breed of livestock-raising and aquatic animal.Referring to disclosed Chinese patent application 200510045358.7 on July 12nd, 2006, " multifunctional probiotic preparation and preparation method thereof ".
Probiotics preparation mainly is an active bacteria formulation, is obligate anaerobe mostly, and lactobacillus wherein and bacillus bifidus are a class anaerobe, is difficult to survival in air, under drying or the room temperature condition, and the shelf-life of its product is shortened.After lactobacillus and bacillus bifidus are eaten by the human or animal simultaneously, can not tolerate stomach sour environment (pH value 1～2), cause probiotic bacteria death can not reach required benefit and come into force really at human or animal's gastric.
CN1613455 discloses a kind of targeting probiotic microcapsule, this microcapsule is made up of probiotic bacteria and/or protective agent and three-layer protection layer, and wherein the ground floor protective layer forms primary microcapsules for the protein after adopting T-5398 crosslinked with the probiotic bacteria embedding; Second layer protective layer be with primary microcapsules with after the suitable dilution agent mixes, coat at 30-40 ℃ hydrogenated oil and fat with fusing point and to form secondary microcapsule; The three-layer protection layer is a release controlling coating material.The weak point of this method is: bag is by complex process, and the operating time is long; In operating process, probiotic bacteria is long-time contact with air or operating process in temperature drift or change too fastly, cause the easy inactivation of thalline; And the part capsulating material has certain influence to the thalline activity, is difficult for long preservation.
Three, summary of the invention
At the deficiencies in the prior art, the invention provides a kind of enteric-coated multilayer encapsulated probiotic microcapsule and preparation method thereof.
Enteric-coated multilayer encapsulated probiotic microcapsule of the present invention, with lactobacillus or bacillus bifidus is core-clad material, sodium alginate, calcium chloride and chitosan are the microcapsule capsulating material, alginate is by β-1 in the capsulating material, 4-D type mannuronic acid (M) and α-1,4-L type guluronic acid (G) copolymerization forms.At Ca2
+Forming gel in the solution utilizes sodium alginate and calcium chloride generation ion exchange to form calcium alginate bag quilt, it forms one deck semipermeable membrane on every side at alginate (electronegative), utilize the different chitosans that make with the chitosan isoelectric point, IP of calcium alginate to form secondary bag quilt on the calcium alginate surface, frozen drying is made.
Above-mentioned probiotic microcapsule particle diameter is at 10～100 μ m.
Calcium alginate has good acid resistance in the capsulating material of the present invention, chitosan is a kind of positively charged polymer, can interosculate so have good intestinal adsorptivity with electronegative intestinal epithelial cell, the gained coated multilayer encapsulated probiotic microcapsule can long preservation also can be located the effect that discharges at intestinal.The health food or the animal feed additive that can be used for the people.
The preparation method of enteric-coated multilayer encapsulated probiotic microcapsule of the present invention, step is as follows:
1. with lactobacillus or the centrifugal collection wet thallus of bifidus bacillus fermented liquid, wet thallus washed out with the phosphate buffer of normal saline or pH value 6.8 be bacteria suspension.
2. the bacteria suspension 1: 5 by volume～10 of gained wet thallus is joined mixing in the sodium alginate soln of 1～2%wt, sodium alginate thalline suspension.
3. sodium alginate thalline suspension is made little drop, join in the calcium chloride solution of 2～4%wt and stir, little drop and calcium chloride solution volume ratio 1: 1～2 obtain wrapping the microcapsule granule by calcium alginate.
4. centrifugal collection microcapsule granule joins in the chitosan solution of 1～2%wt and carries out secondary bag quilt.Microcapsule granule and chitosan solution volume ratio 1: 1～1.5.
5. centrifugal collection microcapsule granule, lyophilization gets enteric-coated multilayer encapsulated probiotic microcapsule.
Method of the present invention is carried out at normal temperatures, simple to operate, selected capsulating material all is the harmless natural organic substance of edible safety, and outermost layer capsulating material oligochitosan has the immunity of raising, suppresses the growth of cancerous protuberance cell, promote liver spleen antibody to form, promote the absorption of calcium and mineral, promote human body beneficial's flora propagation such as bacillus bifidus, lactobacillus, blood fat reducing, blood pressure lowering, blood sugar lowering, cholesterol regulating, fat-reducing, functions such as prevention adult disease.Method for coating of the present invention need not be regulated acid-base value, also needn't use fixative (as harmful substances such as formaldehyde).
Method operating time of the present invention is short, makes thalline open-assembly time in air short, and capsulating material is harmless and edible safety to thalline; Microcapsule behind the bag quilt carries out frozen drying, can prolong the holding time of probiotic bacteria, and the product after the lyophilization can be preserved under 2～8 ℃ condition 5～10 years, can preserve more than 2 years under the room temperature condition.
The present invention is wrapped in the microcapsule lactobacillus or bacillus bifidus by preparation enteric solubility microcapsule, and secluding air can tolerate the stomach sour environment simultaneously.Because the isoelectric point, IP of microencapsulated material is opposite with the isoelectric point, IP of intestinal epithelial cell, can make microcapsule be adsorbed in the enteric epithelium surface, and microcapsule dissolves in intestinal juice, reach the effect that the location discharges, make lactobacillus or bacillus bifidus can arrive the position growth and breeding of suitable field planting, play benefit and come into force really.
Four, the specific embodiment
The present invention will be further described below in conjunction with embodiment, but be not limited thereto.
Embodiment 1:
(1) sodium alginate is made into 2% solution 100ml, prepares 4% calcium chloride solution 100ml, preparation 1%wt chitosan solution 100ml.
(2) with the centrifugal collection wet thallus of 500ml streptococcus acidi lactici fermented solution, wet thallus is suspended in the 10ml normal saline.
(3) suspension with wet thallus adds mixing in the above-mentioned sodium alginate soln, gets sodium alginate thalline suspension.
(4) sodium alginate thalline suspension is made little drop, add in the above-mentioned calcium chloride solution and stir, obtain the sodium alginate microcapsule granule.
(5) centrifugal collection microcapsule granule joins and carries out secondary bag quilt in the above-mentioned 1%wt chitosan solution.
(6) microcapsule granule of centrifugal collection derma, lyophilization.
(7) dried microcapsule granule diameter is at 10～100 μ m.
Microcapsule to present embodiment is checked as follows:
1, gets two parts of the freeze dried microcapsule granules of 0.1g, carry out the test of gastric solubility test and enteric solubility respectively and count plate.
2, preparation simulated gastric fluid: hydrochloric acid 16.4mL, pepsin 10g adds and is settled to 1000mL, pH=1.2 after water stirs evenly.Simulated intestinal fluid: potassium dihydrogen phosphate 6.8g, add water 500mL dissolving, the sodium hydroxide solution with 0.4% is regulated pH value to 6.8; Other gets pancreatin 10g and adds water and make its dissolving in right amount, after two liquid are mixed, adds water and is settled to 1000mL.
3, get microcapsule 0.1g and place the triangular flask that fills 50mL, pH1.2 simulated gastric fluid, placing (37 ± 1) ℃ constant temperature, rotating speed again is on the shaking table of 150r/min, the situation of change of microcapsule light transmittance in simulated gastric fluid.Microcapsule contacts 3h with gastric juice, and light transmittance remains unchanged substantially in the treatment fluid, therefore can prove that probiotic microcapsule can tolerate the destruction of gastric juice.
4, get microcapsule 0.1g and place the triangular flask that fills 50mL, pH6.8 simulated intestinal fluid, placing (37 ± 1) ℃ constant temperature, rotating speed again is on the shaking table of 150r/min, microcapsule disintegrate fully behind 45min judges that thus microcapsule can discharge probiotic bacteria in the 45min in simulation intestinal environment.
5, with after the disintegrate bacteria suspension carry out flat board and pour into the methods,anaerobic count plate, the microcapsule after the lyophilizing on average contains viable bacteria 1～2 * 10
10CFU/mL.
Embodiment 2:
With the centrifugal collection wet thallus of 500ml bifidus bacillus fermented liquid, be diluted to 10ml with the phosphate buffer of pH value 6.8.Gained wet thallus suspension is joined mixing in 2% sodium alginate soln of 100ml, sodium alginate thalline suspension.Sodium alginate thalline suspension is made little drop, join in the calcium chloride solution of 100ml 3% and stir, obtain wrapping microcapsule granule by calcium alginate.
The above-mentioned microcapsule granule of centrifugal collection joins in 2% the chitosan solution of 100ml again and carries out secondary bag quilt.
The above-mentioned microcapsule granule of centrifugal collection, lyophilization, dried microcapsule granule diameter is at 10～100 μ m.Get enteric-coated multilayer encapsulated probiotic microcapsule.
Claims (3)
1, enteric-coated multilayer encapsulated probiotic microcapsule, it is characterized in that with lactobacillus or bacillus bifidus be core-clad material, sodium alginate, calcium chloride and chitosan are the microcapsule capsulating material, utilize sodium alginate and calcium chloride generation ion exchange to form calcium alginate bag quilt, utilize the different chitosans that make with the chitosan isoelectric point, IP of calcium alginate to form secondary bag quilt on the calcium alginate surface again, frozen drying is made.
2, enteric-coated multilayer encapsulated probiotic microcapsule as claimed in claim 1 is characterized in that described microcapsule granule diameter is at 10～100 μ m.
3, a kind of preparation method of enteric-coated multilayer encapsulated probiotic microcapsule, step is as follows:
(1) the centrifugal collection wet thallus of lactobacillus or bifidus bacillus fermented liquid washes out with the phosphate buffer of normal saline or pH value 6.8 and to be bacteria suspension;
(2) bacteria suspension of gained wet thallus 1: 5 by volume～10 joins mixing in the sodium alginate soln of 1～2%wt, sodium alginate thalline suspension;
(3) sodium alginate thalline suspension is made little drop, join in the calcium chloride solution of 2～4%wt and stir, little drop and calcium chloride solution volume ratio 1: 1～2 obtain wrapping the microcapsule granule by calcium alginate;
(4) centrifugal collection microcapsule granule joins and carries out secondary bag quilt in above-mentioned 1～2%wt chitosan solution; Microcapsule granule and chitosan solution volume ratio 1: 1～1.5;
(5) centrifugal collection microcapsule granule, lyophilization gets enteric-coated multilayer encapsulated probiotic microcapsule.
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