CN103222539B - Preparation method of microbial pre-fermentation coating multilayer microcapsule - Google Patents
Preparation method of microbial pre-fermentation coating multilayer microcapsule Download PDFInfo
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Abstract
The present invention relates to a scale preparation method of microbial pre-fermentation coating multilayer microcapsule, and belongs to the fields of microbial technology and biotechnology. The method takes sodium alginate, calcium chloride and modified chitosan as the coated materials of microcapsule, wherein the deacetylation degree of the modified chitosan is more than 90% and molecular weight is about 40000; the method comprises the following steps of (1) preparing single-layer microcapsule and (2) preparing multilayer microcapsule. When performing second coating, a film of low concentration chitosan is formed, the amount of chitosan is little, and almost all of chitosan molecules can react with calcium alginate gel for polyelectrolyte complexation reaction, therefore, the compact film with good toughness can be easily formed; the film is thickened and the mechanical strength is increased along the increase of the chitosan concentration. Furthermore, the process is more convenient to reuse a buffer solution for dissolving chitosan, so as to reduce production costs.
Description
Technical field
Before the present invention relates to a kind of fermentable, bag is by the large-scale preparation method of Multi-layer microcapsule, belongs to microbial technique and biological technical field.
Background technology
Probiotic bacterium is the microbial preparation of living, and can improve the acceptor of the competition intestinal mucosa of host and improve immunity of organisms to play effect.Probiotic bacterium can produce beta-galactosidase enzymes in enteron aisle, can alleviate the symptom of lactose intolerant patient.Probiotic bacterium also has antitumor and effect that is anti-mutation, can also improve immunity of organisms.So the survival of probiotic bacterium in host and multiplication capacity have material impact to prebiotic effect, the bacterium in probiotic composition should metabolic stability, and activity is comparatively strong, by having large number of viable after upper digestive tract, plays prebiotic effect after entering enteron aisle.But many research displays, probiotic bacterium is easy in inactivation in processing and transportation; After entering human or animal's digestive tube, great majority are difficult to stand the effect of the hydrochloric acid in gastric juice of low ph value, bile acide and pancreatic juice etc., are difficult to have enough quantity reach enteron aisle or be settled in enteron aisle and play a role.In order to solve the problems referred to above that probiotic bacterium exists in use, microcapsule embedded technical protection probiotic bacterium is utilized to become the focus of research both at home and abroad at present.
So-called microcapsulary, in fact refer to and utilize high-polymer encapsulated material that is natural or synthesis, by solid, liquid or even the small capsule nuclear substance of gas is coated is formed as diameter a kind of technology with the minigel of semipermeability or sealing cystic of (normally between 5-400 μm of size) within the scope of 1-5000 μm.Microcapsule embeddedly probiotic bacterium can be protected from the infringement of phage, improve its survival rate in freezing dry process, increase the stability in storage process.Be embedded in wall material by probiotic bacterium microcapsulary, also can reduce the damage of external environment to cell, what also can realize probiotic cell repeats multiple batches of utilization simultaneously.The microcapsule embedded technology being applied to probiotic bacterium can be divided into two classes: extrusion process and emulsion process.The survival rate of probiotic bacterium can be improved 80-95% by these two kinds of methods.The microbial preparation prepared by microcapsulary oneself be applied in each field such as agriculture forestry planting industry, animal husbandry and fishery aquaculture, medical and health health care, food and environment protection.
Chinese patent CN100563811C discloses a kind of method that microbe microcapsules is prepared in a large number, and the method for microcapsule capsulating material, carries out individual layer bag by film forming process to probiotics with sodium alginate, calcium chloride, obtains the microcapsule with individual layer coated film.But the stability of the Single-layer microcapsules formed by the method is bad, be that the affinity of sequestrant to calcium is stronger than alginate calcium, and now alginate calcium is very unstable because when there being sequestrant (as lactic acid, citric acid) to exist on the one hand; The microcapsule that individual layer bag is processed simultaneously still cannot meet the demands in the morphological stability of film.
Chinese patent CN100537759C discloses a kind of enteric-coated multilayer encapsulated probiotic microcapsule and preparation method thereof, the method with sodium alginate, calcium chloride and chitosan for microcapsule capsulating material, with milk-acid bacteria or bifidus bacillus for capsule core material prepares probiotic microcapsule, its principle utilizes sodium alginate and calcium chloride to there occurs ion-exchange to form alginate calcium bag quilt, and the recycling alginate calcium chitosan that makes different from chitosan iso-electric point forms the derma of secondary on alginate calcium surface.But find in practical application, because the method is applicable to carry out bag quilt after thalline fermentation, that a large amount of and bag of highdensity cell after fermentation is processed, but some are needed to carry out wrapping processed demand for seed liquor before fermentation, lower owing to treating the density of coated cell, cause the supportive of microorganism cells to the mechanical property of capsule itself poor, make to adopt the method and the microcapsule that formed still exist some shortcomings in the toughness of film forming properties especially film, the morphological stability of the microcapsule not only formed is inadequate, the transport of its subsequent product and retention is caused to be a greater impact, namely the even damaged phenomenon of modification is there is after part that What is more there will be fermentative processing, more be not suitable for large-scale industrialization transport and utilize.
Summary of the invention
Technical problem to be solved by this invention is not suitable for the processed problem of the front microorganism bag of fermentation to the method for coating of microorganism after fermentation in prior art and then provides a kind of processing machinery intensity high, and before the fermentable of good stability, bag is by the method for Multi-layer microcapsule.
Second technical problem to be solved by this invention be to provide that a kind of deformation of fermenting is little, physical strength is high, be easy to the microorganism Multi-layer microcapsule of preserving.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
(1) preparation of Single-layer microcapsules: aseptically, gets the sodium alginate soln after aseptically process, adds and waits to wrap processed microorganism seed liquid, mix; And mixed solution obtained above is mixed in the calcium chloride solution of aseptically process, stir and solidify, leaching supernatant liquor, obtaining required Single-layer microcapsules, and with sterile water wash;
(2) preparation of Multi-layer microcapsule: the described Single-layer microcapsules obtained in step (1) is added in chitosan acidic buffer and carries out film formation reaction, and make the concentration of described chitosan solution constantly increase by feed supplement, leach supernatant liquor subsequently, obtain required Multi-layer microcapsule, and use sterile water wash.
Method of the present invention is applicable to the use of the bag quilt of microorganism nearly all in prior art, and the process of preferred seed liquid is to the bacterial classification of logarithmic phase.
Method of the present invention is applicable to the probiotic bacterium related in various prior art, specifically can include but not limited to the prebiotic bacterial classification such as milk-acid bacteria, yeast.
In described step (2), the concentration of described chitosan solution rises with the velocity gradient of per minute 0.1-0.2g/L.Whether gradient described herein rises and not only terminates in rising at the uniform velocity, in fact shows, as long as ensure that the concentration of described chitosan solution rises with the speed of per minute 0.1-0.2g/L, no matter at the uniform velocity rise, also little on the impact of its mechanical property and toughness.
The starting point concentration of described chitosan acidic buffer is that 1-2g/L rises to final concentration for 5-6g/L with the velocity gradient of per minute 0.1-0.2g/L.
As the implementation that can convert, described in make chitosan concentration increase step can also be realized by the mode of other feed supplements, specifically can include but not limited to:
The starting point concentration of described chitosan acidic buffer is 1-2g/L, and described feed supplement refers to that filling into the concentration identical with original interpolation volume is the chitosan solution of 10-12g/L;
The starting point concentration of described chitosan acidic buffer is 1-2g/L, and described feed supplement refers to and fills into the flow velocity of 10-20ml/min the chitosan solution that concentration is 20-30g/L continuously.
In fact, in scheme of the present invention, make the concentration of described chitosan solution constantly increase by the mode of feed supplement and namely can realize the object that the present invention is enhanced to film properties, the above-mentioned three kinds of feasible schemes provided are only technique effect preferably mode in research, and any exercisable feed profile well known to those skilled in the art all can realize object of the present invention.
Described chitosan is that deacetylation is greater than 90%, molecular weight about 40000 modified chitosan.
In view of the water solubility of chitosan is lower, chitosan is dissolved in preparation in the damping fluid such as low-concentration hcl, phosphoric acid and obtains by described chitosan acidic buffer.
In described step (1), described mixed solution is sprayed in described calcium chloride solution through shower nozzle by high pressure sterile air.
Described mixed solution sprays into before described calcium chloride solution through shower nozzle, also comprises the step of filtering through sterilised membrane filter.
The aperture of described filter membrane is less than 0.22 μm.
The flow velocity of described high-pressure air is 4-8m
3/ h.
Described shower nozzle is hollow circular drop-wise shower nozzle.
The concentration of described sodium alginate soln is 1-3g/L.
The concentration of described calcium chloride solution is 0.1-0.3mol/L.
Present invention also offers the Multi-layer microcapsule that the above-mentioned preparation method of application prepares.
Chitosan has another name called chitosan, soluble chitin and chitosan etc., and chemical name is (Isosorbide-5-Nitrae)-2-amino-2-deoxy-D-Glucose, and molecular formula is (C
6h
11nO
4)
n.Chitin (β-(Isosorbide-5-Nitrae)-2-acetylaminohydroxyphenylarsonic acid 2-deoxidation-D-dextran) is very abundant in natural resource, and being extensively present in the lower animal such as crab, shrimp and the lower plant such as algae, fungi, is a kind of natural polymer.Chitin is heated in the basic conditions, can generating chitosan after sloughing N-ethanoyl.
Chitosan is a kind of alkaline polysaccharide only in organic sphere, and it is white, unformed, translucent, slightly nacreous solid, dissolves in mineral acid and most of organic acids such as rare hydrochloric acid, nitric acid, acetic acid, but is insoluble to dilute sulphuric acid and dilute phosphoric acid.The principal element affecting chitosan dissolving has the kind etc. of the relative molecular mass of deacetylation, chitosan, acid.In view of the limitation of chitosan self performance, people have carried out study on the modification to it, introduce other groups change its physico-chemical property by control reaction conditions on chitosan.The present invention's deacetylation used is greater than 90%, and molecular weight is modified chitosan at the chitosan of about 40000, and its solvability is better than unmodified chitosan.
Modified chitosan of the present invention can operate by method of modifying disclosed in prior art, modified chitosan described in following embodiment adopts the molten method of alcohol carry out de-acetyl modification to chitosan and obtain, modification procedure is as follows: take chitosan 5.0g in Erlenmeyer flask, the NaOH adding 45% now joins in solution, adds dehydrated alcohol (volume ratio of ethanol and NaOH is 1:1).Erlenmeyer flask is placed in water bath with thermostatic control, reacts 2h at 90 DEG C, water rinses to neutral, then with absolute alcohol dehydration, and kept dry.
The Multi-layer microcapsule performance prepared is studied, according to the relation that cubical expansivity Sw and the film toughness of microcapsule before and after fermentation are inversely proportional to, adopts Sw to characterize the film toughness of microcapsule, following equation calculating:
S
W=[（D
f/D
o）
3-1]×100%
Wherein, Sw (%) is microcapsule expansion rate; D
ofor microcapsule diameter before microencapsulation saccharomycetes to make fermentation; D
ffor microcapsule diameter after microencapsulation saccharomycetes to make fermentation.
Technique scheme of the present invention has the following advantages compared to existing technology:
(1) wrap by the preparation method of Multi-layer microcapsule before fermentable of the present invention, carry out secondary bag by time, the chitosan solution that working concentration increases gradually carries out bag quilt, above-mentioned process characteristic first adopts lower concentration chitosan film forming, now chitosan amount is few, nearly all chitosan molecule can both be reacted with calcium alginate gel generation polyelectrolyte complex, therefore, the arrangement of chitosan molecule is more clear to have levels, more easily formed careful and the good film of toughness, along with the increase of chitosan concentration, the film formed is from straight uniform then to thickness and mechanical property development, the film formed is more and more thicker, the physical strength of film also improves thereupon.In addition, another feature of this technique to realize the recycling that chitosan dissolves required damping fluid more easily, thus reduce production cost;
(2) research finds, when derma process, when the concentration of described chitosan solution rises with the velocity gradient of per minute 0.01-0.02g/L, and the better mechanical property of preparation-obtained microcapsule, deformation is less;
(3) when derma process, when the starting point concentration of described chitosan acidic buffer is 0.1-0.2g/L and rises through gradient, the performance of the film obtained is more excellent, but when chitosan final concentration rises to more than 5-6g/L, the mechanical property of the microcapsule obtained is best, deformation is minimum, along with the further rising of chitosan concentration, little on the impact change of film forming properties;
(4) before fermentable of the present invention, bag is by the preparation method of Multi-layer microcapsule, and the modified chitosan utilizing deacetylation to be greater than 90% carries out secondary bag quilt; The solvability of described modified chitosan is better, thus can with sodium alginate complexing better, improve intensity and the film forming properties of microcapsule further;
(5) method that the present invention adopts high pressure sterile air extrusion to spray into has with low cost, simple in equipment, is easy to and the advantage such as fermentation tank is combined, is therefore more suitable for large-scale production microcapsule in enormous quantities;
(6) chitosan material that the present invention is used has good prebiotic performance, fatty deposits amount can be controlled in poultry cultivation process, improve intestinal microflora environment, and in livestock culturing, promote that animal increases weight, the effect of the cholesterol level reduced in blood and muscle tissue, raising animal immunizing power etc.; The described microorganism Multi-layer microcapsule obtained can carry out follow-up fermentation and commercialization process, the wet product that after fermenting, filtering separation obtains has good mobility and dispersiveness, chitosan material used can promote fish growth, improve intestinal microflora environment and improve fish immunity power etc., being applicable to very much aquatic products field and robbing feeding fish raising; And the wet product obtained is after super-dry, the dryed product obtained may be used for feed manufacturing and livestock-raising industry, has satisfactory stability; The destruction of gastric juice can be prevented in body during application, thus make probiotic bacterium thalline as much as possible arrive enteron aisle, play the effect of health care and treatment;
(7) product that Multi-layer microcapsule of the present invention obtains after fermentation has good mobility and dispersiveness, can effectively prevent microorganism cells inactivation, the microcapsule toughness of gained is good simultaneously, and effectively can ensure the integrity in transport and use procedure, the stability of product is better.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below in conjunction with accompanying drawing, the present invention is further detailed explanation, wherein,
Fig. 1 is the schematic diagram before yeast bilayered microcapsule fermentation described in the embodiment of the present invention 2;
Fig. 2 is the schematic diagram after yeast bilayered microcapsule fermentation described in the embodiment of the present invention 2;
Fig. 3 is the schematic diagram before yeast bilayered microcapsule fermentation described in the embodiment of the present invention 10;
Fig. 4 is the schematic diagram after yeast bilayered microcapsule fermentation described in the embodiment of the present invention 10.
Embodiment
Method of the present invention is applicable to the use of the bag quilt of various microorganism, in following each embodiment all with the fermentation seed liquid of yeast strain for bag by seed liquor, specifically comprise:
Preparation YPD seed liquor: yeast extract paste 3.85g/L, peptone 3g/L, glucose 10g/L, 28 DEG C, 180rpm constant temperature culture 20-24h is to terminating during logarithmic phase to cultivate, for subsequent use, and seed liquor is processed without the need to directly can carry out bag through process.
The preparation of sodium alginate glue: be the concentration of 1-3g/L according to ultimate density under constantly stirring, add sodium alginate in batches and enter in deionized water, make transparent glue, sterilizing 20min at 115 DEG C, 4 DEG C save backup.
Calcium chloride solution is prepared: be made into 0.1-0.3mol/L solution with distilled water, sterilizing 20min at 115 DEG C, 4 DEG C save backup.
The preparation of chitosan initial soln: in batches described modified chitosan is added in acidic buffer solution according to the concentration of final concentration 1-2g/L and is constantly stirred to dissolving, for subsequent use.Described acidic buffer is settled to 1000ml with 12ml Glacial acetic acid and 9g sodium-acetate and obtains, natural pH.
The bilayer bag obtained of the present invention is applicable to the use of the fermented liquid cultivation of various feasibility by microcapsule, in order to set forth conveniently, by YPD substratum all identical with fermentation seed liquid for the substratum of the microcapsule fermentative processing of bilayer bag quilt obtained in following each embodiment.
Embodiment 1
Yeast bilayered microcapsule described in the present embodiment prepares as follows:
(1) preparation of Single-layer microcapsules: aseptically, getting the concentration after aseptically process is 1g/L sodium alginate soln, adds the yeast starter liquid waiting to wrap processed logarithmic phase, mixes, to 10
6about cfu/ml density; And be that to filter rear flow velocity be 4m to the sterilised membrane filter being less than 0.22 μm by mixed solution diameter obtained above
3the concentration that the extruded hollow circular drop-wise shower nozzle of high-pressure air of/h sprays into through aseptically process is in 0.1mol/L calcium chloride solution, and low rate mixing evenly and solidify 20min, leaches supernatant liquor, obtains required Single-layer microcapsules, and with sterile water wash;
(2) preparation of bilayered microcapsule: the described Single-layer microcapsules obtained in step (1) being added starting point concentration is carry out film formation reaction in 1g/L chitosan acidic buffer, and carry out feed supplement with the same chitosan acidic solution of 10g/L, pass through concentration detection apparatus, the concentration of chitosan in on-line checkingi fermentor tank, and make the concentration of described chitosan solution increase with the velocity gradient of per minute 0.2g/L by feed supplement, until stop feed supplement when final concentration is 6g/L, continue curing reaction 5-10min, leach supernatant liquor subsequently, obtain the Multi-layer microcapsule that required diameter is 300-700 μm, and use sterile water wash.
Bag is by rear cultivation: access in aforementioned YPD fermented liquid by the bilayered microcapsule being surrounded by yeast cell described in obtaining, 28 DEG C, 150rpm about constant temperature culture 16h is to logarithmic growth latter stage, the wet product of the bilayered microcapsule after fermentation of filtering separation, the wet product obtained after this fermentation can directly as adding fertilizer for culture fishery.
Fluidised bed drying is carried out to the probiotic double layer microcapsule product that wets, obtains probiotic double layer microcapsule dryed product, can be used for feed manufacturing and livestock-raising industry.
Embodiment 2
Yeast bilayered microcapsule described in the present embodiment prepares as follows:
(1) preparation of Single-layer microcapsules: aseptically, getting the concentration after aseptically process is 3g/L sodium alginate soln, adds and waits to wrap processed yeast starter liquid, mix, to 10
6about cfu/ml density; And the rear flow velocity of sterilised membrane filter filtration mixed solution diameter obtained above being less than 0.22 μm is 6m
3the concentration that the high-pressure air of/h sprays into through aseptically process through hollow circular drop-wise shower nozzle is in 0.3mol/L calcium chloride solution, and low rate mixing evenly and solidify 20min, leaches supernatant liquor, obtains required Single-layer microcapsules, and with sterile water wash;
(2) preparation of bilayered microcapsule: the described Single-layer microcapsules obtained in step (1) being added starting point concentration is that 2g/L deacetylation is greater than 90%, molecular weight carries out film formation reaction in the modified chitosan acidic buffer of about 40000, and with the same chitosan acidic solution feed supplement of 8g/L, feed supplement is stopped when making the concentration of described modified chitosan solution rise to 5g/L with the velocity gradient of per minute 0.1g/L by on-line monitoring feed supplement, continue curing reaction 5-10min, leach supernatant liquor subsequently, obtain the bilayered microcapsule that required diameter is 300-700 μm, and use sterile water wash.
Bag is by rear cultivation: access in YPD fermented liquid by the described bilayered microcapsule obtained, 28 DEG C, 150rpm about constant temperature culture 16h is to logarithmic growth latter stage, filtering separation obtains the wet product of the bilayered microcapsule after fermenting, and by the metamorphosis of bilayered microcapsule described in microscopic examination before and after fermentation, specifically as illustrated in fig. 1 and 2.
The wet product obtained after fermentation, can be used for culture fishery.Fluidised bed drying is carried out to the probiotic double layer microcapsule product that wets, obtains probiotic double layer microcapsule dryed product, can be used for feed manufacturing and livestock-raising industry.
Embodiment 3
Yeast bilayered microcapsule described in the present embodiment prepares as follows:
(1) preparation of Single-layer microcapsules: aseptically, getting the concentration after aseptically process is 1g/L sodium alginate soln, adds and waits to wrap processed yeast starter liquid, mix; And be that to filter rear flow velocity be 4m to the sterilised membrane filter being less than 0.22 μm by mixed solution diameter obtained above
3the concentration that the high-pressure air of/h sprays into through aseptically process through hollow circular drop-wise shower nozzle is in 0.1mol/L calcium chloride solution, stirs and solidifies, leaching supernatant liquor, obtaining required Single-layer microcapsules, and with sterile water wash;
(2) preparation of bilayered microcapsule: the described Single-layer microcapsules obtained in step (1) being added starting point concentration is carry out film formation reaction in 3g/L chitosan acidic buffer, and make the concentration of described chitosan solution rise to 4g/L with the velocity gradient of per minute 0.05g/L with the same chitosan acidic solution feed supplement of 9g/L, leach supernatant liquor subsequently, obtain required bilayered microcapsule, and use sterile water wash.
Bilayered microcapsule obtained above is fermented through aforementioned YPD substratum, obtains the wet product after fermenting, can be used for culture fishery.
Fluidised bed drying is carried out to the probiotic double layer microcapsule product that wets, obtains probiotic double layer microcapsule dryed product, can be used for feed manufacturing and livestock-raising industry.
Embodiment 4
Yeast bilayered microcapsule described in the present embodiment prepares as follows:
(1) preparation of Single-layer microcapsules: aseptically, getting the concentration after aseptically process is 1g/L sodium alginate soln, adds and waits to wrap processed yeast starter liquid, mix; And be that to filter rear flow velocity be 4m to the sterilised membrane filter being less than 0.22 μm by mixed solution diameter obtained above
3the concentration that the high-pressure air of/h sprays into through aseptically process through hollow circular drop-wise shower nozzle is in 0.1mol/L calcium chloride solution, stirs and solidifies, leaching supernatant liquor, obtaining required Single-layer microcapsules, and with sterile water wash;
(2) preparation of bilayered microcapsule: the described Single-layer microcapsules obtained in step (1) being added starting point concentration is carry out film formation reaction in the chitosan acidic buffer of 1g/L, and carry out feed supplement with the feed rate Continuous Flow of the 20ml/min same chitosan acidic solution that to add concentration be 20g/L, supernatant liquor is leached after curing reaction terminates, obtain required bilayered microcapsule, and use sterile water wash.
By bilayered microcapsule obtained above through the fermentation of YPD substratum, obtain the wet product after fermenting, can be used for culture fishery.
Fluidised bed drying is carried out to the probiotic double layer microcapsule product that wets, obtains probiotic double layer microcapsule dryed product, can be used for feed manufacturing and livestock-raising industry.
Embodiment 5
The preparation process of the present embodiment is with embodiment 4, its difference is only with step (2), the starting point concentration of described chitosan acidic solution is 2g/L, and carry out feed supplement with the feed rate Continuous Flow of the 10ml/min same chitosan acidic solution that to add concentration be 30g/L, obtain required Multi-layer microcapsule.
Embodiment 6
The preparation process of the present embodiment is with embodiment 4, its difference is only with step (2), the starting point concentration of described chitosan acidic solution is 0.7g/L, and carry out feed supplement with the feed rate Continuous Flow of the 30ml/min same chitosan acidic solution that to add concentration be 10g/L, obtain required Multi-layer microcapsule.
Embodiment 7
Yeast bilayered microcapsule described in the present embodiment prepares as follows:
(1) preparation of Single-layer microcapsules: aseptically, getting the concentration after aseptically process is 1g/L sodium alginate soln, adds and waits to wrap processed yeast starter liquid, mix; And be that to filter rear flow velocity be 4m to the sterilised membrane filter being less than 0.22 μm by mixed solution diameter obtained above
3the concentration that the high-pressure air of/h sprays into through aseptically process through hollow circular drop-wise shower nozzle is in 0.1mol/L calcium chloride solution, stirs and solidifies, leaching supernatant liquor, obtaining required Single-layer microcapsules, and with sterile water wash;
(2) preparation of bilayered microcapsule: the described Single-layer microcapsules obtained in step (1) to be added starting point concentration be the reaction volume of 1g/L is carry out film formation reaction in the chitosan acidic buffer of 2L, and be that the same chitosan acidic solution 2L of 12g/L carries out feed supplement and terminates to curing reaction with concentration in whole solidification process, leach supernatant liquor subsequently, obtain required bilayered microcapsule, and use sterile water wash.
By bilayered microcapsule obtained above through the fermentation of YPD substratum, obtain the wet product after fermenting, can be used for culture fishery.
Fluidised bed drying is carried out to the probiotic double layer microcapsule product that wets, obtains probiotic double layer microcapsule dryed product, can be used for feed manufacturing and livestock-raising industry.
Embodiment 8
The preparation process of the present embodiment is with embodiment 7, and its difference is only with step (2), and the starting point concentration of described chitosan acidic solution is 2g/L, and is that the same chitosan acidic solution of 10g/L carries out feed supplement with concentration, obtains required Multi-layer microcapsule.
Embodiment 9
The preparation process of the present embodiment is with embodiment 7, and its difference is only with step (2), and the starting point concentration of described chitosan acidic solution is 3g/L, and is that the same chitosan acidic solution of 8g/L carries out feed supplement with concentration, obtains required Multi-layer microcapsule.
Embodiment 10
Yeast bilayered microcapsule described in the present embodiment prepares as follows:
(1) preparation of Single-layer microcapsules: aseptically, getting the concentration after aseptically process is 2g/L sodium alginate soln, adds and waits to wrap processed microorganism seed liquid, mix; And the rear flow velocity of sterilised membrane filter filtration mixed solution diameter obtained above being less than 0.22 μm is 8m
3the concentration that the high-pressure air of/h sprays into through aseptically process through hollow circular drop-wise shower nozzle is in 0.2mol/L calcium chloride solution, stirs and solidifies, leaching supernatant liquor, obtaining required Single-layer microcapsules, and with sterile water wash;
(2) preparation of bilayered microcapsule: the described Single-layer microcapsules obtained in step (1) being added concentration is carry out film formation reaction 35-40min in 5.5g/L chitosan acidic buffer, leach supernatant liquor subsequently, obtain required bilayered microcapsule, and use sterile water wash.
To be surrounded by the bilayered microcapsule access fermented liquid of yeast cell, 28 DEG C, 150rpm about constant temperature culture 16h, the bilayered microcapsule after fermentation of filtering separation, and by the metamorphosis of bilayered microcapsule described in microscopic examination before and after fermentation, specifically as shown in Figures 3 and 4.
The performance study of embodiment 11 bilayered microcapsule
Measure respectively under the microscope obtain in above-described embodiment 1-5 bilayer bag by the capsule diameter of microcapsule before and after fermentation, and calculate the cubical expansivity of each microcapsule before and after fermentation, concrete data see the following form.
| Sequence number | D o（μm） | D f（μm） | Sw（%） |
| Embodiment 1 | 310 | 316 | 6.30 |
| Embodiment 2 | 308 | 314 | 5.96 |
| Embodiment 3 | 312 | 328 | 16.02 |
| Embodiment 4 | 320 | 327 | 7.07 |
| Embodiment 5 | 318 | 326 | 7.85 |
| Embodiment 6 | 322 | 338 | 15.98 |
| Embodiment 7 | 315 | 324 | 9.23 |
| Embodiment 8 | 310 | 319 | 9.36 |
| Embodiment 9 | 318 | 338 | 20.02 |
| Embodiment 10 | 316 | 357 | 44.2 |
Above-mentioned data can be found out, in embodiment 2, yeast bilayered microcapsule Sw is minimum, namely almost do not expand, and Fig. 1 and Fig. 2 also shows, and described bilayered microcapsule form before and after fermentation is almost unchanged, and does not occur capsules break phenomenon, as shown in Figure 2; In embodiment 1, yeast bilayered microcapsule Sw is lower, and before and after fermentation, form is almost unchanged, does not also occur video picture of breaking; In embodiment 3, yeast bilayered microcapsule there will be trickle expansion video picture, but microcapsule also do not occur Fragmentation Phenomena after fermentation;
Same, with the microcapsule that scheme of the present invention is obtained by the mode constantly increasing described chitosan solution concentration in film-forming process, the stability after its fermentation is all comparatively strong, as embodiment 1-9, all show good toughness, contribute to the stability of film.Wherein, the microcapsule that the scheme described in embodiment 1,2,4,5,7,8 obtains are even better in the toughness of film.
And the microcapsule obtained in embodiment 10 after fermentation, occurred video picture of comparatively significantly expanding, and there is fracture phenomena in described microcapsule granule, contrasts as shown in Figure 3,4.
Visible; the step that form with gradient increase chitosan concentration of the present invention carries out the process of secondary derma only carries out wrapping processed technique with the chitosan of single concentration relative in prior art; preparation-obtained microcapsule have better toughness and morphological stability, have better effect to the vital preservation of cell and form protection.
More excellent; employing deacetylation of the present invention is greater than 90%; molecular weight carries out film formation reaction in the modified chitosan acidic buffer of about 40000; and make the concentration ever-increasing mode film forming properties of described modified chitosan solution better by the mode of feed supplement, and there are the potentiality of mass-producing fermentative production.
The wet product that after fermenting in the various embodiments described above of the present invention, filtering separation obtains has good mobility and dispersiveness, chitosan material used can promote fish growth, improve intestinal microflora environment and improve fish immunity power etc., being applicable to very much aquatic products field and robbing feeding fish raising; And the wet product obtained is after super-dry, the dryed product obtained has satisfactory stability, can prevent the destruction of gastric juice in body during application, thus makes probiotic bacterium thalline as much as possible arrive enteron aisle, plays the effect of health care and treatment.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (6)
1. before fermentable, bag, by a preparation method for Multi-layer microcapsule, is characterized in that, comprises the steps:
(1) preparation of Single-layer microcapsules: aseptically, gets the sodium alginate soln that the concentration after aseptically process is 1-3g/L, adds and waits to wrap processed microorganism seed liquid, mix; And the concentration be mixed into by mixed solution obtained above through aseptically process is in the calcium chloride solution of 0.1-0.3mol/L, stirs and solidify, leaching supernatant liquor, obtaining required Single-layer microcapsules, and with sterile water wash;
(2) preparation of Multi-layer microcapsule: the described Single-layer microcapsules obtained in step (1) is added in chitosan acidic buffer and carries out film formation reaction, and make the concentration of described chitosan solution constantly increase by feed supplement, supernatant liquor is leached after reaction terminates, obtain required Multi-layer microcapsule, cleaning, to obtain final product;
Wherein, described feed supplement refers to:
The starting point concentration controlling described chitosan acidic buffer is 1-2g/L, and rises to final concentration for 5-6g/L with the velocity gradient of per minute 0.1-0.2g/L; Or
The starting point concentration controlling described chitosan acidic buffer is 1-2g/L, and filling into the concentration identical with original interpolation volume is the chitosan solution of 10-12g/L; Or
The starting point concentration controlling described chitosan acidic buffer is 1-2g/L, fills into the flow velocity of 10-20ml/min the chitosan solution that concentration is 20-30g/L continuously.
2. before fermentable according to claim 1, bag is by the preparation method of Multi-layer microcapsule, and it is characterized in that, in described step (1), described mixed solution is sprayed in described calcium chloride solution through shower nozzle by high pressure sterile air.
3. before fermentable according to claim 2, bag is by the preparation method of Multi-layer microcapsule, and it is characterized in that, described mixed solution sprays into before described calcium chloride solution through shower nozzle, also comprises the step of filtering through sterilised membrane filter.
4. before fermentable according to claim 3, bag is by the preparation method of Multi-layer microcapsule, and it is characterized in that, the flow velocity of described high-pressure air is 4-8m
3/ h.
5. before fermentable according to claim 4, bag is by the preparation method of Multi-layer microcapsule, and it is characterized in that, described shower nozzle is hollow circular drop-wise shower nozzle.
6. according to the Multi-layer microcapsule that the arbitrary described method of claim 1-5 prepares.
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