CN103981169A - Method for preparing bifidobacterium microcapsules by using xanthan gum / chitosan / xanthan gum and application thereof - Google Patents
Method for preparing bifidobacterium microcapsules by using xanthan gum / chitosan / xanthan gum and application thereof Download PDFInfo
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Abstract
A method for preparing bifidobacterium microcapsules by using xanthan gum / chitosan / xanthan gum and application thereof. The method is as below: inoculating a bifidobacterium freeze-dried powder to an MRS broth, activating three times, collecting thalli to obtain bacterial sludge, and preparing the bacterial sludge into a bacterial suspension; mixing the bacterial suspension and xanthan gum, adding a deoxidant solution, mixing well to obtain a bifidobacterium glue solution, adding the bifidobacterium glue solution into a chitosan solution, and stirring to obtain the microcapsules; and after complete crosslinking of the microcapsules, filtering, mixing and stirring the microcapsules with a xanthan gum solution again, filtering and washing, so as to finally obtain a double-layer bifidobacterium microcapsules with viable count of 3.0-4.5*10<9> CFU / g. After addition of the prepared bifidobacterium microcapsules into homemade yogurt and storage for 4 weeks, the viable count is still greater than 10<6> CFU / g, and acidity and pH of the yogurt changed little, and the yogurt maintains unaffected quality and can effectively play its probiotic effect after drinking.
Description
Technical field
The invention belongs to probiotic composition preparing technical field, be specifically related to method and application that a kind of Huang of utilization Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum is prepared bifid bacterium microcapsule.
Background technology
Bifidus bacillus (Bifidobacteria) is by French doctor Tisser isolated a kind of anerobe from the ight soil of breast feeding babies.Find after deliberation, bifidus bacillus is a kind of important probiotic bacterium in human intestinal, and its quantity of surely growing in enteron aisle often exists relation with people's healthy state.Bifidus bacillus, as the typical probiotic bacterium of one, can be brought into play nourishing function by the balance of improving enteric microorganism flora, for example, have the function of double recuperation stomach and intestine, antitumous effect, immunoloregulation function etc.But it is very responsive to oxygen, poor to the resistibility of low pH value, and activity keeping is difficulty comparatively.
Microcapsulary has the release that prevents external environment factors disrupt core, effectively control core, the peculiar smell of covering core, changes the physics and chemistry character of core, makes it easy to the effects such as storage and transport.Can utilize microcapsule and technology to carry out double-layer embedment to probiotic bacterium, to probiotic bacterium is played to stronger provide protection.
At present, utilize the report of microcapsule embedded technical protection bifidus bacillus a lot, great majority are all to utilize the materials such as alginates, as embedding wall material, thalline is carried out to individual layer or double-layer embedment.For example, disclose the microcapsule of a kind of probiotic bacterium in CN1969889A, these microcapsule are taking sodium alginate and calcium chloride as wall material carries out individual layer embedding, then on alginate calcium, carry out secondary with chitosan coated; In CN10275589A, in disclosed bifidus bacillus embedding method, mention, first utilize pectin to carry out individual layer embedding to thalline, recycling sodium alginate carries out double-layer embedment; In CN1884513A, also disclose a kind of preparation method of double-deck bifid bacterium microcapsule, this wall material that for microcapsule, modified starch, gum arabic, polyoxyethylene glycol, sodium alginate, chitosan, calcium chloride form carries out double-layer embedment; Document " preparation of bifid bacterium microcapsule and stability study " discloses a kind of preparation method of double-deck bifid bacterium microcapsule; it first forms acidproof protective layer with the compound gelatin substance parcel such as gelatin, xanthan gum, pectin bifidus bacillus thalline, then with sodium alginate and the better bilayered microcapsule of chitosan crosslinked formation acid resistance.
Except above-mentioned embedded material, the blended gel that can also utilize the polyelectrolyte complex effect of chitosan and xanthan gum to form first carries out embedding to bifidus bacillus, then carries out two-step film forming in xanthan gum, obtains the bifid bacterium microcapsule of double-layer embedment.The probiotic microcapsule that this method obtains can be applied with fruit grain orange and wait in liquid beverage, develops novel liquid-food.Chitosan and and xanthan gum be all natural biomacromolecule, good biocompatibility, has no side effect.Chitosan is dissolved in acidic aqueous solution positively charged, can with electronegative anionic polysaccharide xanthan gum form by polyelectrolyte complex effect cogelled, thereby use as embedding wall material.At present also do not utilize the preparation of Huang Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum bifid bacterium microcapsule and the patent of invention of application, for this reason, a kind of method that provides Huang Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum to prepare bifid bacterium microcapsule and application is necessary.
Summary of the invention
The object of the invention is to overcome the shortcoming existing in above-mentioned prior art, provide a kind of Huang of utilization Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum to prepare method and the application of bifid bacterium microcapsule, there is embedding productive rate and the embedding viable count that can improve thalline, add fruit grain orange to wait in beverage the bifid bacterium microcapsule of preparing, can obtain the advantage of novel liquid-food.
For achieving the above object, a kind of Huang of utilization Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum is prepared the method for bifid bacterium microcapsule, comprises the following steps:
1) freeze-dried bifidobacteria powder is inoculated in MRS meat soup, MRS activation three times under 37 DEG C of culture condition, front twice with the switching of 3～5% inoculum size, and cultivate 22～24h under the condition of 37 DEG C, for the third time according to the switching of 2～3% inoculum size, and cultivate 18h under the condition of 37 DEG C, after carry out centrifugal treating and collect thalline, abandoning supernatant obtains bacterium mud, and the stroke-physiological saline solution that is 0.85～0.9% by bacterium mud and concentration is hybridly prepared into 1.2～1.3 × 10
9the bacteria suspension of CFU/mL is for subsequent use;
2) getting concentration is 0.5～1.1% and 0.1% xanthan gum solution, for subsequent use after sterilizing and centrifugal degassing processing;
3) getting concentration is 0.4～1.3% chitosan solution, regulates pH to 5～5.9 for subsequent use;
4) preparation oxygen scavenger solution, oxygen scavenger is SODIUM ISOVITAMIN C or cysteine hydrochloride, filtration sterilization is in order to being used;
5) by step 1) bacteria suspension and the step 2 prepared) concentration the prepared xanthan gum solution that is 0.5～1.1% is according to 1:(3～10) and mass ratio add step 4 after mixing) the oxygen scavenger solution that makes, after fully mixing, obtain bifidus bacillus bacterium glue, the addition of oxygen scavenger adds according to 0.03～0.07% of bacterium glue total amount, bifidus bacillus bacterium glue is added to the step 3 on magnetic stirring apparatus) stir in the chitosan solution prepared, bacterium glue and chitosan ratio are 1:(3～6), add rear stirring, obtain microcapsule;
6) after microcapsule are completely crosslinked, filter, by microcapsule and step 2 after filtering) 0.1% the xanthan gum solution prepared is with 1g:(20～40mL) mix and blend, after microcapsule are filtered, and washing, finally obtain double-deck bifid bacterium microcapsule, the viable count of microcapsule is 3.0～4.5 × 10
9cFU/g.
Described step 1) in rotating speed when centrifugal be 7000～10000r/min, centrifugation time is 10～15min.
Described step 2) in the temperature of sterilising treatment be: 80～110 DEG C, the time is 10～15min; The rotating speed of centrifugal degassing processing is: 5000～7000r/min, the time is 10～15min.
Described step 3) in, the NaOH solution that the reagent using while regulating pH value is 1N for concentration.
Described step 5) in, by the bifidus bacillus bacterium glue mode in the chitosan solution on magnetic stirring apparatus that adds be: to use needle gauge be 0.45～0.7mm asepsis injector is squeezed into bifidus bacillus bacterium glue in the chitosan solution on magnetic stirring apparatus in mode drop by drop; And churning time is 30～60min.
Described step 6) in, 160～200 object filtered through gauze when filtration, used; When washing, use physiological saline washing several times; Churning time is 20～40min.
Utilize the poly-sugared ∕ xanthan gum of yellow former glue ∕ shell to prepare an application for bifid bacterium microcapsule, in Yoghourt, with the add-on of 0.5%～1% (w/w), the Bifidobacterium capsule of preparation is added, obtain the liquid Yoghourt containing probiotic microcapsule.
The present invention has following beneficial effect: the prior art of comparing, most microcapsule embedded wall material is to use alginates, and alginates is in the environment that has phosphoric acid, lactic acid plasma to exist, calcium ion in gel is substituted, can cause the unstable of gel, this just limits its application in leavened food.In addition, the edible gum materials such as alginates, long-term taking can produce certain side effect to human body.The present invention utilizes Huang Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum to carry out double-layer embedment to bifidus bacillus, and this has just improved embedding rate and the viable count of probiotic bacterium, and this method is simple, strong operability.Chitosan and and xanthan gum be all natural biomacromolecule, good biocompatibility, has no side effect.Chitosan is dissolved in acidic aqueous solution positively charged, can with electronegative anionic polysaccharide xanthan gum form by polyelectrolyte complex effect cogelled, thereby use as embedding wall material.In addition, the bifid bacterium microcapsule of preparing is added in yoghourt, store after 4 weeks, its viable count is still greater than 10
6cFU/g, and the acidity of Yoghourt and pH change little, thereby can not impact the quality of Yoghourt, after drinking, can effectively bring into play its prebiotic effect.
Embodiment
Below in conjunction with embodiment, the present invention is elaborated.
Utilize Huang Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum to prepare a method for bifid bacterium microcapsule, comprise the following steps:
1) freeze-dried bifidobacteria powder is inoculated in MRS meat soup, MRS activation three times under 37 DEG C of culture condition, front twice with the switching of 3～5% inoculum size, and cultivate 22～24h under the condition of 37 DEG C, transfer according to 2～3% inoculum size for the third time, and cultivate 18h under the condition of 37 DEG C, after carry out centrifugal treating, centrifugal rotational speed is 7000～10000r/min, centrifugation time 10～15min, rear collection thalline, abandoning supernatant obtains bacterium mud, and the stroke-physiological saline solution that is 0.85～0.9% by bacterium mud and concentration is hybridly prepared into 1.2～1.3 × 10
9the bacteria suspension of CFU/mL is for subsequent use;
2) getting concentration is 0.5～1.1% and 0.1% xanthan gum solution, and sterilizing 10～15mim at 80～110 DEG C, carries out centrifugal degasification by the xanthan gum solution after sterilizing, centrifugal rotational speed 5000～7000r/min, and centrifugation time 10～15min, rear for subsequent use;
3) getting concentration is 0.4～1.3% chitosan solution,, prepares against and uses chitosan solution pH regulator to 5～5.9 with 1N NaOH solution;
4) preparation oxygen scavenger solution, oxygen scavenger is SODIUM ISOVITAMIN C or cysteine hydrochloride, filtration sterilization is in order to being used;
5) by step 1) bacteria suspension and the step 2 prepared) concentration the prepared xanthan gum solution that is 0.5～1.1% is according to 1:(3～10) and mass ratio add step 4 after mixing) the oxygen scavenger solution that makes, after fully mixing, obtain bifidus bacillus bacterium glue, the addition of oxygen scavenger adds according to 0.03～0.07% of bacterium glue total amount, bifidus bacillus bacterium glue is squeezed into the step 3 on magnetic stirring apparatus in mode drop by drop) stir 30～60min in the chitosan solution prepared, bacterium glue and chitosan ratio are 1:(3～6), add rear stirring, obtain microcapsule,
6) after microcapsule are completely crosslinked by 160～200 object filtered through gauze, by microcapsule and step 2 after filtering) 0.1% the xanthan gum solution prepared is with 1g:(20～40mL) mix and blend, stir 20～40min, with 160～200 object gauzes, microcapsule are filtered afterwards, and wash several times with physiological saline, finally obtain double-deck bifid bacterium microcapsule, the viable count of microcapsule is 3.0～4.5 × 10
9cFU/g.
Utilize the poly-sugared ∕ xanthan gum of yellow former glue ∕ shell to prepare an application for bifid bacterium microcapsule, in Yoghourt, with the add-on of 0.5%～1% (w/w), the Bifidobacterium capsule of preparation is added, obtain the liquid Yoghourt containing probiotic microcapsule.
Step 1) the MRS meat soup that uses in preparation process is from Qingdao high-tech park Hai Bo Bioisystech Co., Ltd.
Specific embodiment:
Embodiment 1:
(1) freeze-dried bifidobacteria powder is inoculated in MRS meat soup, under 37 DEG C of culture condition, adopt this MRS liquid nutrient medium activation three times, wherein front twice with the switching of 3% inoculum size, and cultivate 22h under the condition of 37 DEG C, for the third time according to 2% inoculum size switching, and cultivate 18h under the condition of 37 DEG C, then centrifugal, centrifugal rotational speed 7000r/min, centrifugation time 15min, rear abandoning supernatant obtains bacterium mud, and the stroke-physiological saline solution of bacterium mud and 0.85～0.9% is hybridly prepared into 1.2 × 10
9the bacteria suspension of CFU/mL is in order to using;
(2) compound concentration is 0.5% and 0.1% xanthan gum solution, 80 DEG C, and sterilizing 15mim.Xanthan gum solution after sterilizing is carried out to centrifugal degasification, centrifugal rotational speed 5000r/min, centrifugation time 15min, rear for subsequent use;
(3) compound concentration is 0.4% chitosan solution,, prepares against and uses chitosan solution pH regulator to 5 with 1N NaOH solution;
(4) be prepared as the oxygen scavenger of saccharosonic acid sodium solution, filtration sterilization;
(5) bacteria suspension is mixed according to the ratio of 1:3 with 0.5% xanthan gum solution, oxygen scavenger adds with 0.03% addition of bacterium glue total amount, shakes up afterwards;
(6) asepsis injector that is 0.45mm by needle gauge, by step 5) mixed solution that makes is drop by drop added to the step 3 on magnetic stirring apparatus with fashion of extrusion) in the chitosan solution prepared, and the lasting 30min that stirs, bacterium glue and chitosan ratio are 1:3, filter to obtain wet capsule afterwards with 160 object gauzes;
(7) microcapsule that prepare are mixed with the ratio of 1g:20mL with 0.1% xanthan gum, and constantly stir 20min, afterwards microcapsule are filtered with 160 object gauzes, and with normal saline flushing 3 times, obtain double-deck bifid bacterium microcapsule, the viable count of microcapsule is 3.0 × 10
9cFU/g;
(8) in homemade Yoghourt, with the add-on of 0.5% (w/w), the bifid bacterium microcapsule of preparation is added, obtain the liquid Yoghourt containing probiotic microcapsule.
Embodiment 2:
(1) freeze-dried bifidobacteria powder is inoculated in MRS meat soup, under 37 DEG C of culture condition, adopt this MRS liquid nutrient medium activation three times, wherein front twice with the switching of 4% inoculum size, and cultivate 23h under the condition of 37 DEG C, transfer according to 2.5% inoculum size for the third time, and cultivate 18h under the condition of 37 DEG C, then centrifugal, centrifugal rotational speed 8000r/min, centrifugation time 13min, rear abandoning supernatant obtains bacterium mud, and the stroke-physiological saline solution of bacterium mud and 0.85～0.9% is hybridly prepared into 1.25 × 10
9the bacteria suspension of CFU/mL is in order to using;
(2) compound concentration is 0.7% and 0.1% xanthan gum solution, 100 DEG C, and sterilizing 13mim.Xanthan gum solution after sterilizing is carried out to centrifugal degasification, centrifugal rotational speed 6000r/min, centrifugation time 13min, rear for subsequent use;
(3) compound concentration is 1% chitosan solution,, prepares against and uses chitosan solution pH regulator to 5.5 with 1N NaOH solution;
(4) be prepared as the oxygen scavenger of saccharosonic acid sodium solution, filtration sterilization;
(5) bacteria suspension is mixed according to the ratio of 1:7 with 0.7% xanthan gum solution, oxygen scavenger adds with 0.05% addition of bacterium glue total amount, shakes up afterwards;
(6) asepsis injector that is 0.6mm by needle gauge, by step 5) mixed solution that makes is drop by drop added to the step 3 on magnetic stirring apparatus with fashion of extrusion) in the chitosan solution prepared, and the lasting 40min that stirs, bacterium glue and chitosan ratio are 1:5, filter to obtain wet capsule afterwards with 180 object gauzes;
(7) microcapsule that prepare are mixed with the ratio of 1g:30mL with 0.1% xanthan gum, and constantly stir 30min, afterwards microcapsule are filtered with 180 object gauzes, and with normal saline flushing 4 times, obtain double-deck bifid bacterium microcapsule, the viable count of microcapsule is 3.6 × 10
9cFU/g;
(8) in homemade Yoghourt, with the add-on of 0.7% (w/w), the bifid bacterium microcapsule of preparation is added, obtain the liquid Yoghourt containing probiotic microcapsule.
Embodiment 3:
(1) freeze-dried bifidobacteria powder is inoculated in MRS meat soup, under 37 DEG C of culture condition, adopt this MRS liquid nutrient medium activation three times, wherein front twice with the switching of 5% inoculum size, and cultivate 24h under the condition of 37 DEG C, transfer according to 3% inoculum size for the third time, and cultivate 18h under the condition of 37 DEG C, then centrifugal.Centrifugal rotational speed 10000r/min, centrifugation time 10min, rear abandoning supernatant obtains bacterium mud, and the stroke-physiological saline solution of bacterium mud and 0.85～0.9% is hybridly prepared into 1.3 × 10
9the bacteria suspension of CFU/mL is in order to using;
(2) compound concentration is 1.1% and 0.1% xanthan gum solution, and 110 DEG C, sterilizing 10mim, carries out centrifugal degasification by the xanthan gum solution after sterilizing, centrifugal rotational speed 7000r/min, and centrifugation time 10min, rear for subsequent use;
(3) compound concentration is 1.3% chitosan solution,, prepares against and uses chitosan solution pH regulator to 5.9 with 1N NaOH solution;
(4) be prepared as the oxygen scavenger of cysteine hydrochloride solution, filtration sterilization;
(5) bacteria suspension is mixed according to the ratio of 1:10 with 1.1% xanthan gum solution, oxygen scavenger adds with 0.07% addition of bacterium glue total amount, shakes up afterwards;
(6) asepsis injector that is 0.7mm by needle gauge, by step 5) mixed solution that makes drop by drop fashion of extrusion be added to the step 3 on magnetic stirring apparatus) in the chitosan solution prepared, and continue to stir 60min, bacterium glue and chitosan ratio are 1:6.Filter to obtain wet capsule with 200 object gauzes afterwards.
(7) microcapsule that prepare are mixed with the ratio of 1g:40mL with 0.1% xanthan gum, and constantly stir 40min, afterwards microcapsule are filtered with 200 object gauzes, and with normal saline flushing 5 times, obtain double-deck bifid bacterium microcapsule, the viable count of microcapsule is 4.0 × 10
9cFU/g;
(8) in homemade Yoghourt, with the add-on of 1% (w/w), the bifid bacterium microcapsule of preparation is added, obtain the liquid Yoghourt containing probiotic microcapsule.
In homemade Yoghourt, with the add-on of 0.5%～1% (w/w), the bifid bacterium microcapsule of preparation is added, under the condition of 4 DEG C, store, and to contain 10
7～10
8the Yoghourt of the free bifidus bacillus of CFU/mL in contrast.Store after 4 weeks, have decline in various degree though find to be added with viable count in the Yoghourt of microcapsule, its viable count is still greater than 10
6cFU/g, and the acidity of Yoghourt and pH change little, thereby can not impact the quality of Yoghourt.The number of active bifid bacteria of embedding does not acutely decline, and its viable count is less than 10
5cFU/mL, does not reach the viable count standard that probiotic food requirement reaches, and the acidity of Yoghourt and pH change greatly.So the bifid bacterium microcapsule that utilizes Huang Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum to prepare can strengthen the resistance to acid of thalline, brings into play the effect of its useful HUMAN HEALTH.
Claims (7)
1. utilize the poly-sugared ∕ xanthan gum of yellow former glue ∕ shell to prepare a method for bifid bacterium microcapsule, it is characterized in that: comprise the following steps:
1) freeze-dried bifidobacteria powder is inoculated in MRS meat soup, MRS activation three times under 37 DEG C of culture condition, front twice with the switching of 3～5% inoculum size, and cultivate 22～24h under the condition of 37 DEG C, for the third time according to the switching of 2～3% inoculum size, and cultivate 18h under the condition of 37 DEG C, after carry out centrifugal treating and collect thalline, abandoning supernatant obtains bacterium mud, and the stroke-physiological saline solution that is 0.85～0.9% by bacterium mud and concentration is hybridly prepared into 1.2～1.3 × 10
9the bacteria suspension of CFU/mL is for subsequent use;
2) getting concentration is 0.5～1.1% and 0.1% xanthan gum solution, for subsequent use after sterilizing and centrifugal degassing processing;
3) getting concentration is 0.4～1.3% chitosan solution, regulates pH to 5～5.9 for subsequent use;
4) preparation oxygen scavenger solution, oxygen scavenger is SODIUM ISOVITAMIN C or cysteine hydrochloride, filtration sterilization is in order to being used;
5) by step 1) bacteria suspension and the step 2 prepared) concentration the prepared xanthan gum solution that is 0.5～1.1% is according to 1:(3～10) and mass ratio add step 4 after mixing) in the oxygen scavenger solution that makes, after fully mixing, obtain bifidus bacillus bacterium glue, the addition of oxygen scavenger adds according to 0.03～0.07% of bacterium glue total amount, bifidus bacillus bacterium glue is added to the step 3 on magnetic stirring apparatus) stir in the chitosan solution prepared, bacterium glue and chitosan ratio are 1:(3～6), add rear stirring, obtain microcapsule;
6) after microcapsule are completely crosslinked, filter, by microcapsule and step 2 after filtering) 0.1% the xanthan gum solution prepared is with 1g:(20～40mL) mix and blend, after microcapsule are filtered, and washing, finally obtain double-deck bifid bacterium microcapsule, the viable count of microcapsule is 3.0～4.5 × 10
9cFU/g.
2. a kind of Huang of utilization Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum according to claim 1 is prepared the method for bifid bacterium microcapsule, it is characterized in that: described step 1) in rotating speed when centrifugal be 7000～10000r/min, centrifugation time is 10～15min.
3. a kind of Huang of utilization Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum according to claim 1 is prepared the method for bifid bacterium microcapsule, it is characterized in that: described step 2) in the temperature of sterilising treatment be: 80～110 DEG C, the time is 10～15min; The rotating speed of centrifugal degassing processing is: 5000～7000r/min, the time is 10～15min.
4. a kind of Huang of utilization Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum according to claim 1 is prepared the method for bifid bacterium microcapsule, it is characterized in that: described step 3) in, the NaOH solution that the reagent using while regulating pH value is 1N for concentration.
5. a kind of Huang of utilization Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum according to claim 1 is prepared the method for bifid bacterium microcapsule, it is characterized in that: described step 5) in, by the bifidus bacillus bacterium glue mode in the chitosan solution on magnetic stirring apparatus that adds be: to use needle gauge be 0.45～0.7mm asepsis injector is squeezed into bifidus bacillus bacterium glue in the chitosan solution on magnetic stirring apparatus in mode drop by drop; And churning time is 30～60min, bacterium glue and chitosan ratio are 1:(3～6).
6. a kind of Huang of utilization Yuan Jiao ∕ Ke Ju Tang ∕ xanthan gum according to claim 1 is prepared the method for bifid bacterium microcapsule, it is characterized in that: described step 6) in, 160～200 object filtered through gauze when filtration, used; When washing, use physiological saline washing several times; Churning time is 20～40min.
7. an application that utilizes the poly-sugared ∕ xanthan gum of yellow former glue ∕ shell to prepare bifid bacterium microcapsule, it is characterized in that: in Yoghourt, add-on with 0.5%～1% (w/w) adds the Bifidobacterium capsule of preparation, obtains the liquid Yoghourt containing probiotic microcapsule.
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| Publication number | Priority date | Publication date | Assignee | Title |
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