CN104887647A - Probiotics double-layered microcapsule and manufacturing method thereof - Google Patents
Probiotics double-layered microcapsule and manufacturing method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and particularly relates to a probiotics double-layered microcapsule and a manufacturing method thereof. The probiotics double-layered microcapsule and the manufacturing method are improved according to defects in the prior art. A high-pressure homogeneous emulsification method is adopted; sodium alginate and chitosan serve as capsule wall materials and are cross-linked with calcium chloride to form a double-layer film; bifidobacterium is taken as an experimental subject; an L9(34) orthogonal experiment is designed, and the double-layered microcapsule is manufactured. The double-layered microcapsule is high in encapsulation rate, the problem that excessive water loss is likely to happen to a single-layer capsule and accordingly activated probiotics is dead can be solved, and resistance to outside bad environments of probiotics can be improved. The method is easy to operate. Cost of raw materials is low. The survival rate of probiotics bodies can be obviously increased. Cost performance is high.
Description
Technical field
The invention belongs to biological technical field, be specifically related to probiotic double layer microcapsule and preparation method thereof.
Background technology
At present, the health-care effect of various probiotic bacteria gains universal acceptance.Wait in gastroenteropathy in treatment diarrhoea, probiotics preparation widely uses, and serves important auxiliary therapeutic action.According to practical application display, this kind of preparation has strict demand to storage condition, especially need seal cold preservation, user once ignore described in condition of storage, easily make loss of drug potency.Research display, probiotic bacteria is very responsive to oxygen, poor to the resistance of the low pH (as gastric acid etc.) in upper digestive tract, therefore long-time bad mode is stored, again after oral, can be few with the bacterium quantity of activated state field planting intestinal, its product also also exists keeping quality difference, sharply descends degradation shortcoming with holding time prolongation viable count, and effective application of probiotic bacteria is greatly limited.
Microcapsule utilizes macromolecular material (being referred to as capsule material) that is natural or synthesis, capsule core material embedded into the capsule of small densification.Conventional microcapsule wall material mainly contains the carbohydrate material such as alginate.Microcapsule embedded technology can significantly improve probiotic bacteria oxytolerant and anti-gastric acid ability, makes it arrive intestinal with activated state, and then plays corresponding effect.Research display, even with traditional sodium alginate monolayer capsule embedding probiotic bacteria, because the eggbox structure of its microcosmic contains large water gaging, but cannot prevent moisture from evaporating, therefore, still there is the shortcoming that distortion after easily excessively dehydration causes storage is for a long time stiff, extend viable count decline with the holding time, etc.
Present inventor intends providing probiotic double layer microcapsule and preparation method thereof, makes the good acid resistance of generation and enteric solubility, and has better bin stability than conventional monolayers capsule.
Summary of the invention
Be to provide a kind of probiotic double layer microcapsule order of the present invention.
Be to provide the preparation method of probiotic double layer microcapsule further order of the present invention.
The defect that the present invention is directed to prior art existence is improved, in view of most of probiotic bacteria is to O
2and low pH is comparatively responsive in upper digestive tract, for improving the tolerance degree of probiotic bacteria poor environment to external world, and improving the problem that the easy dehydration of conventional monolayers capsule causes thalline death, the present invention increases by the second tunic to conventional monolayers capsule embedding probiotic bacteria and prevents dehydration, obtained probiotic double layer microcapsule.
The present invention adopts high pressure homogenize emulsion process, take bacillus bifidus as experimental subject, is first cross-linked with sodium alginate and calcium chloride and is wrapped to form acidproof protective layer, then be cross-linked to form the bilayered microcapsule of anti-dehydration with chitosan and calcium chloride; Result shows, obtained bilayered microcapsule has good acid resistance and enteric solubility, has better bin stability than conventional monolayers capsule.
Probiotic double layer microcapsule of the present invention; probiotic double layer microcapsule; comprise active component and capsule wall; it is characterized in that; described capsule wall comprises protective layer and anti-dehydration layer; described protective layer adopts sodium alginate and calcium chloride to be cross-linked to be wrapped to form acidproof protective layer, and described anti-dehydration layer adopts chitosan and calcium chloride to be cross-linked to form anti-dehydration layer.
The present invention adopts high pressure homogenize emulsion process, and being cross-linked into duplicature with sodium alginate, chitosan for capsule material and calcium chloride, take bacillus bifidus as experimental subject, design L
9(3
4) orthogonal experiment, obtained best double deck type microcapsule.
More specifically, the invention provides the method preparing probiotic double layer microcapsule, it is characterized in that, it comprises step:
1, the horizontal extent of each factor is determined by experiment of single factor,
2, then pass through L
9(3
4) orthogonal experiment, screen best double deck type microcapsule;
3, carry out character Comprehensive Experiment after obtaining optimum formula, comprise simulation stomach juice-resistant, enteric, storage experiment, and contrast, confirm its character.
In the present invention, adopt mycopowder to mix with wall material raw material, add deionized water, with 200Bar high pressure homogenize emulsifying 10min.Gained emulsion instills CaCl fast
2solution (consolidation liquid) leaves standstill solidification 20min.Chitosan and CaCl is implanted after collecting washing capsule
2solution film forming 20min, again collects and washs and obtain double deck type microcapsule microcapsule;
Wherein, the mensuration of microcapsule embedded productive rate: embedding productive rate=microcapsule bacterial number/add bacterium number × 100%;
Microcapsule Counting alive microbial: microcapsule is dissolved in the shell-broken liquid of 2.0Mol/L phosphate buffer (pH=7.0), counts until completely dissolved.
In the present invention, carry out experiment of single factor prepared by microcapsule:
Experiment measures different sodium alginate concentration (1.5%, 2.5%, 3.5%), CaCl by experiment of single factor
2: concentration (1%, 2%, 3%), lactose concn (4%, 5%, 6%), chitosan concentration (0.1%, 0.2%, the 0.3%) impact on microcapsule embedded productive rate, to determine orthogonal experiment horizontal extent.
In the present invention, carry out orthogonal experiment prepared by microcapsule:
On the basis of experiment of single factor result, adjust according to the level of above-mentioned experiment of single factor result to some factors, adopt L
9(3
4) orthogonal test, with the embedding productive rate of microcapsule for evaluation index, select optimum proportioning.Factor level is as shown in table 1.
Table 1: factor level table
In the present invention, carry out the Comprehensive Experiment of bilayered microcapsule character:
After orthogonal experiment terminates, the optimum proportioning microcapsule filtered out carries out character research, and for ensureing that experimental data is accurate, experiment all in an aseptic environment operation is carried out;
(1) Microscopic observation of bilayer vesicles: get microcapsule and be placed in microscope slide, Microscopic observation is also taken pictures.
(2) acid resistance of bilayer vesicles: get microcapsule and be placed in and fill simulated gastric fluid pH1.2, under 37 DEG C of conditions, 180r/min vibrates 1h, and every 20min sampling weighs dissolves Mass Calculation and to open one's purse rate, and calculates thalline survival rate; Manually to open one's purse after 1h count plate, and make blank with common bacterium liquid;
To open one's purse rate=existing quality/initial quality × 100%;
Viable count × 100% before viable count/embedding after viable bacteria rate=open one's purse.
(3.) the enteric performance of bilayer vesicles: get microcapsule and be placed in simulated intestinal fluid pH8.0, on shaking table 37 DEG C, 180r/min carries out disintegrate, every 20min sampling weighs dissolves Mass Calculation and to open one's purse rate, and count plate after manually opening one's purse, calculates viable bacteria rate.
(4) single bilayer vesicles storage control experiment: get microcapsule and insert in vial, open type manually open one's purse after being placed in superclean bench 48h after count plate, calculate viable bacteria rate.And make blank with monolayer sodium alginate micro gel capsule and common bacterium liquid.
Result shows, and the present invention is directed to the problem that traditional easy dehydration of monolayer capsule causes capsule deformation, thalline death, increases chitosan film prevent dehydration from making stability that bilayer vesicles can improve probiotic bacteria on monolayer capsule basis.Adopt high pressure homogenize emulsion process, be 3% at sodium alginate concentration, calcium chloride concentration is 4%, when lactose concn is 3%, chitosan concentration is under the condition of 0.2%, embedding productive rate is the highest, and reach 91.2%, viable bacteria rate reaches 89.7%, gained bilayer vesicles has good acid resistance and enteric solubility, process 1h in simulation 37 DEG C of gastric acid after, viable bacteria rate still reaches 78.3%, discharges complete in intestinal juice in 20min, ensure that probiotic bacteria is with activated state field planting intestinal.
The bilayer vesicles that the present invention obtains and monolayer capsule are stored control experiment result and are shown, and have good storage performance, and bilayer vesicles reaches 84.6% and 79.6% respectively in the viable bacteria rate of 48h and 15d, and monolayer capsule only has 76.2% and 59.7%, is almost 0 without embedding group.Relative to traditional monolayer sodium alginate micro gel capsule, bilayer vesicles of the present invention has significant anti-dehydration ability, can overcome the shortcoming because holding time prolongation thalline survival rate declines, breach this application restriction.The raw material that the microcapsule that this method obtains adopts is the raw material that food service industry generally uses, inanimate object toxicity.This experimental technique is simple to operate, and cost of material is low, and can significantly improve thalline survival rate, cost performance is higher.
Figure of description
Fig. 1: sodium alginate concentration is on the impact of embedding rate.
Fig. 2: chitosan concentration is on the impact of embedding rate.
Fig. 3: CaCl
2concentration is on the impact of embedding rate.
Fig. 4: lactose concn is on the impact of embedding rate.
Fig. 5: bilayered microcapsule outward appearance.
Fig. 6: the acidproof situation in gastric juice.
Fig. 7: the acidproof contrast of thalline.
Monolayer capsule after Fig. 8: 48h.
Bilayer vesicles after Fig. 9: 48h.
Detailed description of the invention
In the present embodiment, the main agents of employing: sodium alginate (chemical pure), anhydrous calcium chloride (analytical pure), commercially available Bright brand defatted milk powder, lactose (analytical pure), chitosan (chemical pure), pancreatin, gastric enzyme.Above reagent is domestic;
Key instrument: PanDa1000+ high pressure homogenizer, TOMYSX-700 high-pressure steam sterilizing pan, upper clean CA920 superclean bench, training English THZ-C constant temperature oscillator, OLYMPUSBX61, SZX12 microscope;
Main solution:
Simulated gastric fluid (pH=1.2): get the rare HC116.4ml of 0.1mol/L, add water about 800mL and pepsin 10g, adds water and be settled to 1000mL after mixing;
Simulated intestinal fluid (pH=8.0): get KH
2pO
46.8g add water 500mL dissolve, by the NaOH solution adjust ph to 8.0 of 0.4%; Separately get trypsin 10g, add water and make dissolving in right amount, after both are mixed, add water and be settled to 1000mL;
0.2MPBS buffer (pH=8.0): get Na
2hPO
4(0.2mol/L) 94.7mL, NaH
2pO
4(0.2mol/L) 5.3mL, mixing;
Bacterial strain: Shanghai Sine Pharmaceutical Co., Ltd.'s trade name: Birid Triple Viable common name: bacillus bifidus live triple product batch number: 20130134.
Embodiment 1
1) experimental technique
Determined the horizontal extent of each factor by experiment of single factor, then pass through L
9(3
4) orthogonal experiment, screen best double deck type microcapsule.Carry out character Comprehensive Experiment (simulation stomach juice-resistant, enteric, storage experiment) after obtaining optimum formula and contrast, going to push up its character;
Prepare microcapsule: mycopowder mixes with wall material raw material, adds deionized water, with 200Bar high pressure homogenize emulsifying 10min.Gained emulsion instills CaCl fast
2solution (consolidation liquid) leaves standstill solidification 20min.Chitosan and CaCl is implanted after collecting washing capsule
2solution film forming 20min, again collects and washs and obtain double deck type microcapsule;
(1) experiment of single factor prepared of microcapsule:
Experiment measures different sodium alginate concentration (1.5%, 2.5%, 3.5%), CaCl by experiment of single factor
2: concentration (1%, 2%, 3%), lactose concn (4%, 5%, 6%), chitosan concentration (0.1%, 0.2%, the 0.3%) impact on microcapsule embedded productive rate, to determine orthogonal experiment horizontal extent;
Experiment of single factor result shows:
Internal layer capsule sodium alginate wherein:
Sodium alginate is the natural polymer be present in Brown algae, there is good biocompatibility and film property, its totally nontoxic, more clean than other polymer substances, sodium alginate can not be decomposed by the microorganisms simultaneously, and when it runs into calcium ion, sodium ion and out replaced, generate water-fast calcium alginate gel, this gel is not soluble when pH≤6.0, be easy to when pH >=8.0 dissolve, be applicable to the preparation of animal intestine soluble micro-capsule; When sodium alginate concentration is too low, effectively can not embed thalline, and gained capsule quality is softer, not spherical in shape, drag shape of tail structure, even completely in flocculent deposit, discontented sufficient requirement of experiment; And when the concentration is too high, bacterium emulsion viscosity is too high, homogenizing difficulty, gained capsule quality is really up to the mark again, cannot open one's purse easily, experimentally, when designing orthogonal experiment, is: 2%, 2.5%, 3% by sodium alginate horizontal adjustment;
Outer softgel shell polysaccharide wherein:
Because chitosan is larger molecular organics, in regular solution, dissolubility is lower, and its concentration is little on embedding rate impact, and its variation tendency is not obvious, therefore when designing orthogonal experiment, does not adjust chitosan concentration, keeps the concentration of 0.1%, 0.2%, 0.3%.
Coagulant calcium chloride wherein:
Calcium chloride provides calcium ion, precipitates because sodium alginate and calcium ion can form water-fast calcium alginate, defines the protecting film of one deck densification, therefore can increase the intensity of microcapsule on microcapsule surface; When calcium chloride concentration increases, micro-sphere crosslinked more crypto set, decreases the loss of text message core; But calcium chloride concentration is excessive, can cause that the particle diameter of microcapsule increases, cyst wall is thickening, not easily breaking cellular wall; Work as CaCl
2during <1%, capsule quality is softer, without solid form, in cotton-shaped; CaCl
2when concentration is excessive, capsule quality is really up to the mark, normally cannot open one's purse, be shown by result, CaCl
2concentration is higher, and embedding efficiency is higher, this experiment, when designing orthogonal experiment, to CaCl
2concentration is adjusted to: 2%, 3%, 4%.
Emulsifying agent lactose wherein:
Because lactose is emulsifying agent, embedding productive rate is not significantly affected.But because lactose concn increase can cause system viscosity to increase, therefore to a certain extent, have impact on embedding efficiency.When designing orthogonal experiment, lactose concn not adjusted, still keeping 4%, 5%, 6%.
In the present invention, the mensuration of microcapsule embedded productive rate: embedding productive rate=microcapsule bacterial number/add bacterium number × 100%;
In the present invention, microcapsule Counting alive microbial: microcapsule is dissolved in the shell-broken liquid of 2.0Mol/L phosphate buffer (pH=7.0), counts until completely dissolved;
(2) orthogonal experiment prepared of microcapsule,
On the basis of experiment of single factor result, adjust according to the level of its result to some factors, adopt L
9(3
4) orthogonal test, with the embedding productive rate of microcapsule for evaluation index, select optimum proportioning; Factor level is as shown in table 1.
Table 1: factor level table
Result shows, adopts orthogonal test determination optimised process to be A
3b
3c
1d
2, namely sodium alginate concentration is 3%, and calcium chloride concentration is 4%, and lactose concn is 3%, and chitosan concentration is 0.2%; Under this proportioning, the embedding productive rate of microcapsule is the highest, is 91.24%.
(3) comprehensive study of bilayered microcapsule character
After orthogonal experiment terminates, the optimum proportioning microcapsule filtered out carries out character research, for ensureing that experimental data is accurate, below testing operation all in an aseptic environment and carrying out;
The Microscopic observation of bilayer vesicles: get microcapsule and be placed in microscope slide, Microscopic observation is also taken pictures; Result shows, and capsule is full particle, and thalline is uniformly distributed in capsule, and due to sodium alginate, comparatively chitosan phase specific concentration is comparatively large, therefore light tight, and the present invention will be sent out by air-flow coating and reduce microcapsule to 50 μm, prepare the microcapsule that diameter is less.
The acid resistance of bilayer vesicles: get microcapsule and be placed in and fill simulated gastric fluid pH1.2, under 37 DEG C of conditions, 180r/min vibrates 1h, and every 20min sampling weighs dissolves Mass Calculation and to open one's purse rate, and calculates thalline survival rate.Manually to open one's purse after 1h count plate, and make blank with common bacterium liquid;
To open one's purse rate=existing quality/initial quality × 100%;
Viable count × 100% before viable count/embedding after viable bacteria rate=open one's purse;
Result shows, alginic acid and derivant thereof derive from the hydrophilic colloidal state polysaccharide of Brown algae, by β-(1, 4)-D-MANNOSE aldehydic acid (M) and α (1, 4) linear polymer that forms of-L-guluronic acid (G), carboxyl and hydroxyl is freely contained in the macromole of each alginic acid and derivant thereof, can react with many kinds of metal ions and form gel, as sodium alginate is added in the medium of calcium ions, part hydrion in displacement sodium alginate macromole and sodium ion are converted into calcium alginate molecule by calcium ion, therefore there is NaAlg in gelinite simultaneously, HAlg and Ca (Alg)
2, the interlaced connection of these 3 kinds of structures, because respective adhesion is different, the spatial joins power of whole calcium alginate gel macromolecular structure is also unbalanced, creates larger space, therefore can hold a large amount of hydrones, the content of moisture is relevant with the component of gel with the size of adhesion, and Ca
2+play a leading role in whole system, contribute to molecule to link together, this structure is called as " egg-box " structure, and this structure can not suffer H
+destruction, very stable, therefore there is good acid resistance, H can be contacted in a large number by available protecting thalline
+and it is dead, and,
Emulsifying agent is not only by lactose, also has multiple hydroxyl, can with the combined with radical of phage surface, thalline is avoided to expose in media as well, and lactose easily and water molecules, decrease the content of free water and add the stickiness of solution, thus reaching the object of protection thalline;
The protection of double deck type microcapsule owing to there being above two kinds of materials prepared by this method, therefore process 1h in acid after, still have the viable bacteria rate up to 78.3%, and only have 2.5% without the blank group viable bacteria rate of embedding under the same terms.
The enteric performance of bilayer vesicles:
Get microcapsule and be placed in simulated intestinal fluid pH8.0, on shaking table 37 DEG C, 180r/min carries out disintegrate, every 20min sampling weighs dissolves Mass Calculation and to open one's purse rate; Count plate after manually opening one's purse, calculates viable bacteria rate;
Result shows, and because external capsule is made up of chitosan, chitosan is unstable in acid, therefore dissolves at gastric.Inner " egg-box " structure be made up of calcium alginate can be effectively acidproof, but can be destroyed rapidly when high concentration strong electrolytic solution or environment pH >=8.0, and microcapsule expands dissolving immediately; Therefore double deck type capsule of the present invention has good enteric solubility, can rapid disintegrate in 20min.
(4) single bilayer vesicles storage control experiment: get microcapsule and insert in vial, open type manually open one's purse after being placed in superclean bench 48h after count plate, calculate viable bacteria rate; And make blank with monolayer sodium alginate micro gel capsule and common bacterium liquid, result shows:
" egg-box " structure effectively can prevent H
+corrosion, but cannot suppress the evaporation of moisture, chitosan is as external capsule, serve the ability preventing dehydration, the chitosan in the present invention extracts gained from various Crustacean, its complex structure, formed film is fine and close, and its substructure is asymmetric, and this structure can make gas molecules as O
2, CO
2pass through, but hydrone cannot pass through, excessive vaporization causes thalline dead because of long-time storage effectively to prevent the moisture of capsula interna; Though described adventitia can through O
2, but inner membrance can place O
2continue invasion thalline, both combine, and define stable shelf characteric.
This experiment also added the defatted milk powder that concentration is 3% when preparing capsule; because lactalbumin can form albuminous coat outward at thalline; cell is protected; and dry enzyme can be fixed; prevent the intracellular organic matter caused due to cell wall damage from leaking, also can improve the viable bacteria rate after long-time storage to a certain extent.
The each wall material of embodiment 2 is tested the impact of embedding rate
1, set each wall material respectively and carry out experiment of single factor
In this experiment, the sodium alginate arranging internal layer capsule is invariant: sodium alginate 2%, CaCl
22%, de-
Fat milk powder 3%, lactose 4%, chitosan 0.2%;
Result shows: when sodium alginate concentration is too low, effectively can not embed thalline, and gained capsule quality is softer, not spherical in shape, has dragged shape of tail structure, even completely in flocculent deposit, and discontented sufficient requirement of experiment; And when the concentration is too high, bacterium emulsion viscosity is too high, homogenizing difficulty, gained capsule quality is really up to the mark again, cannot open one's purse easily, in the present invention during orthogonal experiment, by sodium alginate horizontal adjustment is: 2%, 2.5%, 3%;
Setting sodium alginate is variable: concentration is 1.5%, 2.5%, 3.5%, and its surplus keeps constant, i.e. CaCl
2under the condition of 2%, defatted milk powder 3%, lactose 4%, chitosan 0.2%, prepare microcapsule, the embedding productive rate measuring microcapsule after opening one's purse is respectively 29.3%, 51.7%, 62.1%.
The chitosan setting outer capsule in this experiment is variable: concentration is 0.1%, 0.2%, 0.3%, and its surplus keeps constant, i.e. sodium alginate 2%, CaCl
2under the condition of 2%, defatted milk powder 3%, lactose 4%, prepare microcapsule, the embedding productive rate measuring microcapsule after opening one's purse is respectively 70.0%, 58.3%, 65.0%.
Coagulant calcium chloride CaCl is set in this experiment
2for variable: concentration is 1%, 2%, 3%, its surplus keeps constant, i.e., under the condition of sodium alginate 2%, defatted milk powder 3%, lactose 4%, chitosan 0.2%, prepare microcapsule, the embedding productive rate measuring microcapsule after opening one's purse is respectively 38.8%, 50.0%, 55.5%.
Setting emulsifying agent lactose in this experiment is variable: concentration is 4%, 5%, 6%, and its surplus keeps constant, i.e. sodium alginate 2%, CaCl
2under the condition of 2%, defatted milk powder 3%, chitosan 0.2%, prepare microcapsule, the embedding productive rate measuring microcapsule after opening one's purse is respectively 71.6%, 68.3%, 65.0%.
2, orthogonal experiment
On the basis of single factor experiment result, with sodium alginate, CaCl
2, lactose, chitosan be object of study, adopts L
9(3
4) orthogonal test, to embed productive rate for evaluation index, select optimum proportioning.
Table 1 factor level table
Orthogonal experiments is as shown in table 2, and the primary and secondary order affecting embedding rate factor is A>B>C>D, and from meansigma methods, the strong and weak order of each factor level to Influence on test result is A
3>A
2>A
1, B
3>B
2>B
1, C
1>C
2>C
3, D
2>D
1>D
3, therefore determine that optimised process is A
3b
3c
1d
2, namely sodium alginate concentration is 3%, and calcium chloride concentration is 4%, and lactose concn is 3%, and chitosan concentration is 0.2%.Under this proportioning, the embedding productive rate of microcapsule is the highest, is 91.24%.
Table 2: Orthogonal experiment results
3, the comprehensive study of bilayered microcapsule character
1) the Microscopic observation result display of bilayer vesicles, the double deck type microcapsule that basis of microscopic observation adopts optimised process obtained, its outward appearance be subcircular diameter at about 1mm, slightly cave in internal layer sodium alginate capsule surface, sharpness of border, and densification is light tight.The thalline of energy available protecting embedding; External capsule is chitosan film, can make up the out-of-flatness of capsula interna, and experiment also shows the function that it has anti-dehydration, and external capsule is smooth, sharpness of border, fine and close printing opacity.Interior external capsule combines closely, bubble-free gap.
2) acid resistance research
Configuration simulated gastric fluid, prepares 3 parts of microcapsules.3 parts of capsules are inserted gastric juice, puts into constant-temperature table, under 37 DEG C of conditions, 180r/min 1h, every 20min sampling of vibrating weighs and dissolves Mass Calculation and to open one's purse rate, and calculates thalline survival rate, and every increment product are all free diphtheria emulsion in contrast,
Result shows, and obtained microcapsule has good acid-fast ability, and open one's purse hardly, this microcapsule energy available protecting thalline, significantly promotes acid-resisting ability.
Table 3: the acidproof situation in gastric juice
Table 4: the acidproof contrast of thalline
3) enteric performance
Configuration simulated intestinal fluid, prepares 3 parts of microcapsules.3 parts of capsules are inserted intestinal juice, put into constant-temperature table, 180r/min vibration 1h under 37 DEG C of conditions, every 20min sampling weighs dissolves Mass Calculation and to open one's purse rate, and calculates thalline survival rate, and result shows, namely this microcapsule dissolves completely in 20min, have good enteric ability, count plate display after enteric, thalline survival reaches 88%.
4) control experiment is preserved
Prepare bilayer vesicles, monolayer capsule (removing two-step film forming step), blank bacterium liquid, after super-clean bench opens wide and places 48h, count plate of opening one's purse.Result shows, and in long-time preservation, bilayer vesicles has better performance; Table 5: storage performance synopsis
Use the single bilayer vesicles after microscopic examination 48h respectively, result shows, and monolayer capsule is done because excessive dehydration quality is hard, nonelastic, and profile is irregular; Bilayer vesicles quality is similar to rubber, flexible, and profile still keeps original, but becomes fuzzyyer due to membrane junction place inside and outside part dehydration; As shown in Figure 8 and Figure 9: chitosan has good anti-dehydration ability, monolayer capsule can be improved to a certain extent and easily excessively lose water problem.
Claims (9)
1. probiotic double layer microcapsule; comprise active component and capsule wall; it is characterized in that; described capsule wall comprises protective layer and anti-dehydration layer; described protective layer is cross-linked by sodium alginate and calcium chloride and is wrapped to form acidproof protective layer, and described anti-dehydration layer is cross-linked to form anti-dehydration layer by chitosan and calcium chloride.
2., by probiotic double layer microcapsule according to claim 1, it is characterized in that, in described capsule wall, add lactose.
3., by probiotic double layer microcapsule according to claim 2, it is characterized in that, in described capsule wall, the proportioning of each composition is: sodium alginate concentration is 3%, and calcium chloride concentration is 4%, and lactose concn is 3%, and chitosan concentration is 0.2%.
4. by probiotic double layer microcapsule according to claim 3, it is characterized in that, described bilayered microcapsule embedding productive rate is 91.24%.
5., by probiotic double layer microcapsule according to claim 3, it is characterized in that, the viable bacteria rate after described bilayered microcapsule acid treatment 1h is 78.3%.
6., by probiotic double layer microcapsule according to claim 3, it is characterized in that, the enteric solubility of described bilayered microcapsule is disintegrate in 20min.
7. by probiotic double layer microcapsule according to claim 3, it is characterized in that, after described bilayered microcapsule storage 48h, viable bacteria rate is 84.6%.
8. the preparation method of the probiotic double layer microcapsule of claim 1 or 2 or 3, is characterized in that, adopt high pressure homogenize emulsion process, being cross-linked into duplicature with sodium alginate, chitosan for capsule material and calcium chloride, take bacillus bifidus as experimental subject, design L
9(3
4) orthogonal experiment, obtained double deck type microcapsule; It comprises step:
1) horizontal extent of each factor is determined by experiment of single factor;
2) L is passed through
9(3
4) orthogonal experiment, screening double deck type microcapsule components proportioning;
3) carry out character Comprehensive Experiment, comprise simulation stomach juice-resistant, enteric, storage experiment, and contrast,
Confirm obtained double deck type microcapsule character.
9., by the method for claim 4, it is characterized in that, the determined sodium alginate concentration of orthogonal experiment is 3%, and calcium chloride concentration is 4%, and lactose concn is 3%, and chitosan concentration is 0.2%.
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