CN101249109B - Lactobacillus reuteri hollow micro-capsule and its preparing method - Google Patents
Lactobacillus reuteri hollow micro-capsule and its preparing method Download PDFInfo
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- CN101249109B CN101249109B CN2008100707676A CN200810070767A CN101249109B CN 101249109 B CN101249109 B CN 101249109B CN 2008100707676 A CN2008100707676 A CN 2008100707676A CN 200810070767 A CN200810070767 A CN 200810070767A CN 101249109 B CN101249109 B CN 101249109B
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Abstract
The invention discloses a method for preparing a probiotic hollow microcapsule of Lactobacillus reuteri, which includes: firstly separating and purifying the Lactobacillus reuteri, then adding 5-20g of starch, 5-20g of carboxymethyl cellulose, 2-5g of sodium cellulose sulfate and 5-20g of calcium chloride into 1000ml of cultured seed extractive solution, preparing the hollow microcapsule through an extrusion method, then preparing a fermentation culture medium, fermenting and culturing for a period of time under nitrogenous atmosphere, finally applying the membrane on the capsule and drying, then obtaining the probiotic hollow microcapsule of Lactobacillus reuteri. The invention provides the method for preparing a hollow microcapsule preparation of the Lactobacillus reuteri, which prolongs the storage period of the Lactobacillus reuteri and is convenient to store, transport and use. The prepared probiotic hollow microcapsule of Lactobacillus reuteri has advantages of prolonging the survival time of probiotics at a room temperature and preventing the probiotcs from being killed by gastric acid after oral intake to allow the probiotics to enter the intestinal tract, targeted delivering and playing curative effect.
Description
Technical field
The present invention relates to a kind of method for preparing of lactobacillus reuteri hollow microcapsule.
Background technology
Lactobacillus reuteri (latin name: be in a kind of intestinal that inhabits humans and animals Lactobacillus reuteri), the lactic bacteria useful of tool intestinal epidermis adhesive capacity.Research shows materials such as the newborn element of the Luo Yishi of Lactobacillus reuteri metabolism generation, albumen, organic acid, can suppress the growth of gram positive bacteria, gram negative bacteria, yeast, fungus and protozoon etc. widely, has broad-spectrum antibacterial action.Lactobacillus reuteri can improve microbial population of animal intestinal tract and distribute as feed additive, suppresses the breeding of harmful bacterium, thereby avoids animal the disease of some intestinal classes to occur.The lactobacillus reuteri preparation also can be used for improving the problem that probiotics quantity descends in people's intestinal that body aging, intestinal tract disease, antibiotic abuse, environmental pollution etc. cause, promotes the propagation of intestinal beneficial bacterium, thereby promotes health.China's Ministry of Public Health was also ratified Lactobacillus reuteri as health food probiotic bacteria kind in 2003.
Probiotic composition mainly is an active bacteria formulation, and existing at present lactobacillus reuteri bacteria preparation occurs, and for guaranteeing the viable count in effect duration, generally adopts 2-8 ℃ cryopreservation, brings great inconvenience for storage, transportation and sale.Lactobacillus reuteri is a kind of obligate anaerobe, very easily receive Effect of Environmental such as oxygen, temperature, soda acid, so liquid bacterial agent is difficult to keep for a long time its high activity.Probiotic bacteria will produce beneficial effect to human body; At first it must and be grown on intestinal mucosa through esophagus, gastric environment arrival Di surely, but oral Lactobacillus reuteri liquid preparation, when passing through the stomach of humans and animals; Sour environment in the stomach (pH is about 2) and bile can kill Lactobacillus reuteri; Viable count is sharply reduced, cause Lactobacillus reuteri in intestinal, not grow surely in a large number, thereby influence the therapeuticing and health effect of the adjusting intestinal microbial population of Lactobacillus reuteri.It is reported, oral lyophilizing microbial inoculum, survival rate under one's belt is merely 1,000,000/about, survival rate is extremely low.
Summary of the invention
In order to address the above problem; The object of the present invention is to provide a kind of method for preparing of lactobacillus reuteri hollow microcapsule; Adopt the prepared lactobacillus reuteri hollow microcapsule of this method for preparing to have active substance, enzyme and the probiotic bacteria that to protect wherein,, receive killing of gastric acid when prolonging the probiotic bacteria time-to-live at ambient temperature and avoiding oral with the influence of opposing external environment; Make its intestinal that arrives safe and sound, the directed characteristics that discharge the performance curative effect.
For reaching above-mentioned purpose, the present invention adopts following technical scheme,
A kind of method for preparing of lactobacillus reuteri hollow microcapsule comprises the steps:
(1) separation of Lactobacillus reuteri, purification: comprise the preparation of strain separating culture medium and the separation screening of strain;
The strain separating culture medium
Beef protein powder 10g, flesh of fish juice 10g, Yeast diffusion juice powder 5g, glucose 10g, arabinose 5g, sucrose 5g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 80 0.1g, magnesium sulfate 0.58g, manganese sulfate 0.28g, agar 18g, distilled water 1000ml.Regulate pH to 5.0 with acetic acid.
The separation screening of strain
The above-mentioned culture medium Hunga t e test tube of packing into is prepared culture medium and rolls pipe.Get three months baby's Excretas it is dispersed in the sterilized water, shake up staticly, get supernatant, add Hungate and roll pipe, rolling is evenly distributed on supernatant to roll the pipe media surface.After 24-72 hour, white colony appears with 37 ℃ of following cultivations of 5%-12% CO2 gas incubator.Add sterilized water bacterium is dispersed in the water, after the taking-up, be coated with flat board.The Gram-positive that obtains and catalase negative bacteria are further carried out acidproof, bile tolerance, anti-high-copper and Chinese People's Anti-Japanese Military and Political College's enterobacteria screening.Bacterial strain to screening obtains is investigated the ability that its glycerine converting generates the Luo Yishi element, can determine that it is Lactobacillus reuteri.
(2) Lactobacillus reuteri seed culture: comprise the preparation of solid slant culture base, the preparation of seed culture medium, the inoculation that will be obtained by step (1) separation screening was then cultivated 24 hours down at 37 ℃ in the solid slant culture base.Then the inoculation on the inclined-plane was cultivated 12 hours to 37 ℃ of following anaerobism of liquid seed culture medium, then the seed culture fluid adding is contained in the 3L fermentation tank of 2L fermentation medium, cultivated 12 hours 37 ℃ of following anaerobism.
The solid slant culture base
Beef protein powder 10g, flesh of fish juice 10g, Yeast diffusion juice powder 5g, glucose 20g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 80 0.1g, magnesium sulfate 0.58g, manganese sulfate 0.28g, agar 18g, distilled water 1000ml.Regulate pH to 5.4 with acetic acid.
Seed culture medium
Beef protein powder 10g, flesh of fish juice 10g, Yeast diffusion juice powder 5g, glucose 20g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 80 0.1g, magnesium sulfate 0.58g, manganese sulfate 0.28g, distilled water 1000ml.Regulate pH to 5.4 with acetic acid.
Fermentation medium
Beef protein powder 10g, flesh of fish juice 10g, Yeast diffusion juice powder 5g, glucose 25g, glycerol 20g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 80 0.1g, magnesium sulfate 0.58, manganese sulfate 0.28, distilled water 1000ml.Regulate pH to 5.4-6.5 with acetic acid.
(3) get the seed extracting solution 1000ml that step (2) is cultivated, add starch 5-20g, carboxymethyl cellulose 5-20g; Cellulose sodium sulfate 2-5g and calcium chloride 5-20g; Prepare hollow capsules through squeezing and pressing method, the refabrication fermentation medium, the capsule 100ml that gets above-mentioned preparation joins the 1000ml fermentation medium; Logical nitrogen fermentation culture a period of time is at last with getting the lactobacillus reuteri hollow microcapsule after deposited film of capsule and the drying.
Squeezing and pressing method in the described step (3) prepares hollow capsules and is meant above-mentioned 1000ml seed extracting solution, adds starch, carboxymethyl cellulose, cellulose sodium sulfate and the calcium chloride of the bacterium of having gone out, stirs under the aseptic condition, is deployed into appropriate viscosity; This is mixed and can go into a certain amount of sodium alginate soln that has configured the 0.5-1.5% of sterilization by drop, slowly stir, dripped 10 minutes, after reaction a period of time, take out capsule sterilized water washing three times while dripping.
The sodium alginate soln consumption of described 0.5-1.5% is 1000ml.
The described response time is 20-60 minute.
The time of the fermentation culture in the described step (3) is 24-48 hour.
Capsule in the described step (3) applies film and with dry being meant the capsule after the logical nitrogen fermentation culture is washed three times with sterilized water, this capsule is added in the chitosan solution of 0.1%-1%, reacts 1-2 hour, takes out capsule, promptly gets product after the lyophilizing.
The invention has the beneficial effects as follows: the present invention is embedded in Lactobacillus reuteri in the microcapsule of enteric solubility wall material preparation (microcapsule of the enteric solubility wall material preparation that starch, carboxymethyl cellulose, cellulose sodium sulfate, calcium chloride are formed); Can resist the influence of external environment; Guarantee it and can resist gastric acid, arrive intestinal smoothly.In medicine and food industry, the method for a lot of probiotic bacteria microencapsulation has been proposed, but all be after obtaining mycopowder with it through certain means embedding, easy in encapsulation process probiotic bacteria is damaged, thereby influences the stability of probiotic bacteria.Therefore there is certain defective in the method at probiotic microcapsule.And the present invention adopts the wall material of enteric solubility to carry out embedding protection probiotic bacteria outside Lactobacillus reuteri, and a kind of prolongation Lactobacillus reuteri storage period is provided, the method for preparing of a kind of Lactobacillus reuteri hollow capsules preparation of being convenient to store, transport and use.The present invention adopts squeezing and pressing method to prepare benefit and gives birth to hollow microcapsule; Hollow capsules can not produce bulk effect to the growth of microorganism in the capsule, and excellent mass transferring performance is arranged, and has active substance, enzyme and the probiotic bacteria that can protect wherein; Influence with the opposing external environment; Thereby receive killing of gastric acid in the time of prolonging the probiotic bacteria time-to-live at ambient temperature and avoid oral, make its intestinal that arrives safe and sound, the directed characteristics that discharge the performance curative effect.
The specific embodiment
Embodiment 1
The inoculation that present embodiment separates Lactobacillus reuteri, screen behind the purification through isolation medium was cultivated 24 hours down at 37 ℃ to the solid slant culture base.Then the inoculation on the inclined-plane is arrived the 200ml liquid seed culture medium, cultivated 12 hours down for 37 ℃.Then the adding of 200ml seed culture fluid is contained in the 3L fermentation tank of 2L fermentation medium, cultivated 12 hours 37 ℃ of following anaerobism.Get 1000ml seed extracting solution, add starch 20g, carboxymethyl cellulose 12g, cellulose sodium sulfate 2g, the calcium chloride 20g of the bacterium of having gone out, stir under the aseptic condition.This mixed liquor is splashed into sterilized 0.5% sodium alginate soln 1000ml, slowly stir, dripped 10 minutes, react after 30 minutes, take out the capsule sterilized water and wash three times while dripping.Get the above-mentioned capsule for preparing, add and contain in the fermentation tank of 1000ml fermentation medium logical nitrogen cultivation 48 hours.Capsule after the above-mentioned cultivation is taken out,, in the chitosan solution with this capsule adding 0.1%, reacted 1-2 hour with sterilized water washing three times.Take out capsule, get the lactobacillus reuteri microcapsule after the lyophilizing.
Embodiment 2
The inoculation that present embodiment separates Lactobacillus reuteri, screen behind the purification through isolation medium was cultivated 24 hours down at 37 ℃ to the solid slant culture base.Then the inoculation on the inclined-plane is arrived the 200ml liquid seed culture medium, cultivated 12 hours down for 37 ℃.Then the adding of 200ml seed culture fluid is contained in the 3L fermentation tank of 2L fermentation medium, cultivated 12 hours 37 ℃ of following anaerobism.Get 1000ml seed extracting solution, add starch 12g, carboxymethyl cellulose 5g, cellulose sodium sulfate 3g, the calcium chloride 12g of the bacterium of having gone out, stir under the aseptic condition.This mixed liquor is splashed into sterilized 1% sodium alginate soln 1000ml, slowly stir, dripped 10 minutes, react after 20 minutes, take out the capsule sterilized water and wash three times while dripping.Get the above-mentioned capsule for preparing, add and contain in the fermentation tank of 1000ml fermentation medium logical nitrogen cultivation 36 hours.Capsule after the above-mentioned cultivation is taken out,, in the chitosan solution with this capsule adding 0.5%, reacted 1-2 hour with sterilized water washing three times.Take out capsule, get the lactobacillus reuteri microcapsule after the lyophilizing.
Embodiment 3
The inoculation that present embodiment separates Lactobacillus reuteri, screen behind the purification through isolation medium was cultivated 24 hours down at 37 ℃ to the solid slant culture base.Then the inoculation on the inclined-plane is arrived the 200ml liquid seed culture medium, cultivated 12 hours down for 37 ℃.Then the adding of 200ml seed culture fluid is contained in the 3L fermentation tank of 2L fermentation medium, cultivated 12 hours 37 ℃ of following anaerobism.Get 1000ml seed extracting solution, add starch 5g, carboxymethyl cellulose 20g, cellulose sodium sulfate 5g, the calcium chloride 5g of the bacterium of having gone out, stir under the aseptic condition.This mixed liquor is splashed into sterilized 1.5% sodium alginate soln 1000ml, slowly stir, dripped 10 minutes, react after 60 minutes, take out the capsule sterilized water and wash three times while dripping.Get the above-mentioned capsule for preparing, add and contain in the fermentation tank of 1000ml fermentation medium logical nitrogen cultivation 24 hours.Capsule after the above-mentioned cultivation is taken out,, in the chitosan solution with this capsule adding 1%, reacted 1-2 hour with sterilized water washing three times.Take out capsule, get the lactobacillus reuteri microcapsule after the lyophilizing.
Claims (6)
1. the method for preparing of a lactobacillus reuteri hollow microcapsule is characterized in that comprising the steps:
(1) separation of Lactobacillus reuteri, purification: comprise the preparation of strain separating culture medium and the separation screening of strain;
(2) Lactobacillus reuteri seed culture: comprise the preparation of solid slant culture base; The preparation of seed culture medium; The inoculation that will be obtained by step (1) separation screening was then cultivated 24 hours down at 37 ℃ in the solid slant culture base, then the inoculation on the inclined-plane was cultivated 12 hours down for 37 ℃ to liquid seed culture medium; Then the seed culture fluid adding is contained in the 3L fermentation tank of 2L fermentation medium, cultivated 12 hours 37 ℃ of following anaerobism;
(3) get the seed extracting solution 1000ml that step (2) is cultivated, add starch 5-20g, carboxymethyl cellulose 5-20g; Cellulose sodium sulfate 2-5g and calcium chloride 5-20g; Prepare hollow capsules through squeezing and pressing method, the refabrication fermentation medium, the capsule 100ml that gets above-mentioned preparation joins the 1000ml fermentation medium; Logical nitrogen fermentation culture a period of time is at last with getting the lactobacillus reuteri hollow microcapsule after deposited film of capsule and the drying.
2. the method for preparing of lactobacillus reuteri hollow microcapsule as claimed in claim 1; It is characterized in that: the squeezing and pressing method in the described step (3) prepares hollow capsules and is meant above-mentioned 1000ml seed extracting solution; The starch, carboxymethyl cellulose, cellulose sodium sulfate and the calcium chloride that add the bacterium of having gone out; Stir under the aseptic condition, be deployed into appropriate viscosity; This mixed liquor is splashed into a certain amount of sodium alginate soln that has configured the 0.5-1.5% of sterilization, slowly stir, dripped 10 minutes, after reaction a period of time, take out capsule sterilized water washing three times while dripping.
3. the method for preparing of lactobacillus reuteri hollow microcapsule as claimed in claim 2 is characterized in that: the sodium alginate soln consumption of described 0.5-1.5% is 1000ml.
4. the method for preparing of lactobacillus reuteri hollow microcapsule as claimed in claim 2 is characterized in that: the described response time is 20-60 minute.
5. the method for preparing of lactobacillus reuteri hollow microcapsule as claimed in claim 1 is characterized in that: the time of the fermentation culture in the described step (3) is 24-48 hour.
6. the method for preparing of lactobacillus reuteri hollow microcapsule as claimed in claim 1; It is characterized in that: capsule applies film and dry being meant the capsule after the logical nitrogen fermentation culture with sterilized water washing three times in the described step (3); This capsule is added in the chitosan solution of 0.1%-1%; Reacted 1-2 hour, and took out capsule, promptly get product after the lyophilizing.
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| CN101946850B (en) * | 2009-12-11 | 2013-03-13 | 中国农业科学院饲料研究所 | Lactobacillus reuteri fermented liquid feed, preparation method and application thereof |
| CA2946464A1 (en) * | 2014-05-05 | 2015-11-12 | Giovanni Mogna | Adjunctive therapy to chemotherapy treatments for tumors, acquired immunodeficiency syndrome and leukemias |
| CN104970368B (en) * | 2015-06-30 | 2017-11-17 | 安徽大学 | Preparation method of gastric acid-resistant bacillus licheniformis microcapsules with high survival rate |
| CN107929219B (en) * | 2017-11-21 | 2020-05-08 | 江南大学 | Method for improving stability of lactic acid bacteria in toothpaste |
| CN109673848A (en) * | 2018-12-29 | 2019-04-26 | 广州智特奇生物科技股份有限公司 | A kind of enteric capsulating material, intestinal sustained releasing tannic acid and preparation method thereof |
| CN111621443B (en) * | 2020-06-17 | 2022-07-22 | 江南大学 | Freeze-drying protective agent and application thereof in freeze-drying lactobacillus reuteri |
| CN113080470A (en) * | 2021-04-13 | 2021-07-09 | 苏州赛迈尔生物科技有限公司 | Composition beneficial to intestinal regulation and preparation method thereof |
| CN114717220B (en) * | 2021-06-09 | 2023-06-27 | 青岛蔚蓝生物股份有限公司 | Lactobacillus reuteri microcapsule and preparation method thereof |
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Patent Citations (4)
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| CN1251388A (en) * | 1999-03-22 | 2000-04-26 | 山东农业大学 | Micro-capsule peridium growth regulator |
| CN1569043A (en) * | 2003-07-17 | 2005-01-26 | 中国农业大学 | Coated micro capsule of lactic acid bacteria and its preparation |
| CN1487078A (en) * | 2003-08-14 | 2004-04-07 | 南京农业大学 | Lactobacillus cell microcyst culturing process and produced leaven |
| CN1969889A (en) * | 2006-12-04 | 2007-05-30 | 济南赛拜斯生物工程有限公司 | Enteric-coated multilayer encapsulated probiotic microcapsule and preparation method thereof |
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