CN104745566A - Lactic acid bacterium immobilized capsules and preparation method and application thereof - Google Patents
Lactic acid bacterium immobilized capsules and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses lactic acid bacterium immobilized capsules and a preparation method and application thereof. The method comprises the following steps: uniformly mixing centrifugally collected lactic acid bacteria and a sodium alginate aqueous solution with the mass percent concentration of 2% to obtain a mixture; vertically dropping the mixture into a calcium chloride aqueous solution with the mass percent concentration of 1.5% at the speed of 1.5-2.0ml/min; stirring, curing and filtering to obtain gel beads; washing the gel beads; adding the gel beads into a chitosan-acetic acid aqueous solution, of which the volume is ten times of that of the gel beads and the mass percent concentration is 2%; performing a film-forming reaction to obtain a reaction solution; adding a sodium alginate aqueous solution, of which the volume is ten times of that of the reaction solution and the mass percent concentration is 0.15%, into the reaction solution to perform immobilization; filtering to obtain a sediment 1; treating the sediment 1 for 10min with a sodium citrate aqueous solution with the pH value of 5.0 and the concentration of 0.055mol/L; and filtering to obtain a sediment 2, namely the lactic acid bacterium immobilized capsules. The lactic acid bacterium immobilized capsules are used for degrading nitrite in food, and the effects of the lactic acid bacterium immobilized capsules which are reused five times are not reduced.
Description
Technical field
Immobilization capsule that the present invention relates to a kind of milk-acid bacteria and its preparation method and application.
Background technology
Nitrite is the chemical substance be extensively present in physical environment, it is the precursor substance of strong carcinogen nitrosamine, it reacts at the intermediate product secondary amine that stomach can decompose with Protein in Food and forms nitrosamine, and then brings out kinds cancer as cancer of the stomach, liver cancer, large bowel cancer and esophagus cancer etc.Although the addition of country to nitrite in food has strict restriction, because food nitrite is excessive and cause the event of food poisoning to happen occasionally.At present, because a large amount of use of nitrogenous fertilizer and some areas use sanitary sewage and trade effluent to irrigate, cause underground water nitrite pollution serious, grain vegetables nitrite exceeds standard also comparatively general.In addition, also there is nitrite excessive problem in traditional pickling process pickles and various processing meat product always.In addition, find according to an epidemiology survey, cancer district occurred frequently DRINKING WATER nitrite is higher than Di Fa district.
In view of nitrite in food exceeds standard the serious harm caused, people are finding effective control or the method for degrading nitrite hardy, and in biological process degraded food, excessive nitrite is a feasible approach.At present, people have been separated from occurring in nature and have obtained some nitrite degradation bacterium, and wherein, milk-acid bacteria also can degrading nitrite effectively as a kind of probiotic bacterium.
But still there is no the immobilization capsule of milk-acid bacteria for degrading nitrite at present.
Summary of the invention
The object of the present invention is to provide the immobilization capsule of a kind of milk-acid bacteria, the immobilization capsule of this milk-acid bacteria adopts sodium alginate and chitosan as immobilization material.
Two of object of the present invention is the preparation method of the immobilization capsule providing above-mentioned a kind of milk-acid bacteria.
Three of object of the present invention is the nitrite being used for by the immobilization capsule of above-mentioned milk-acid bacteria degrading in food.
Know-why of the present invention
For biological degradation nitrite, immobilized cell is a kind of effective means, and it not only can improve the tolerance of cell Pyrogentisinic Acid, and can reuse.Entrapping method is most widely used process for fixation.Sodium alginate, chitosan due to cheap, widely used embedded material is become to cytotoxic, therefore, its carrier as immobilized cell available.
Adopt Trisodium Citrate by solid for microcapsule core liquefaction, the resistance to mass transfer of the sterically hindered of Growth of Cells and microcapsule can be reduced, contribute to growth and the production of microencapsulated cell.
Technical scheme of the present invention
An immobilization capsule for milk-acid bacteria, is prepared by a method comprising the following steps and forms:
(1), the preparation of wet milk-acid bacteria thalline
The milk-acid bacteria that picking one ring inclined-plane is preserved is in the MRS liquid nutrient medium of 5 ml, and 37 DEG C of static gas wave refrigerator 20h, obtain first-generation bacterial classification;
By first-generation bacterial classification, by volume percentage calculation, the inoculum size with 10% is inoculated into MR liquid S substratum, and 37 DEG C of static gas wave refrigerator 20h, obtain s-generation bacterial classification;
By s-generation bacterial classification, by volume percentage calculation, the inoculum size with 10% is inoculated in MRS liquid nutrient medium, and 37 DEG C of static gas wave refrigerator 12h, obtain nutrient solution 1;
The concentration being incorporated as nutrient solution 1 volume 0.01 times in the nutrient solution 1 of gained is that the 0.8-1mol/L nitrite aqueous solution carries out cultivation 8h, obtains nutrient solution 2;
By the nutrient solution 2 of gained in 4 DEG C, the centrifugal 10min of 4000rpm, collect thalline, with the centrifugal 10min of 4000rpm after sterilized water vibration washing, repeat sterilized water vibration washing, centrifugation step once, the precipitation obtained is wet milk-acid bacteria thalline;
(2), being 2% by wet milk-acid bacteria thalline and mass percent concentration, sodium alginate aqueous solution mixes, and after static 15-20min, obtains mixed solution;
Above-mentioned wet milk-acid bacteria thalline and mass percent concentration are the consumption of the sodium alginate aqueous solution of 2%, in wet milk-acid bacteria thalline: mass percent concentration be 2% sodium alginate aqueous solution be that the ratio of 1g:10ml calculates;
(3), the mixed solution of step (2) gained is drawn in asepsis injector, be in the calcium chloride water of 1.5% with the mass percent concentration that is vertically added dropwise to of the speed of 1.5-2ml/min, stir with 150r/min rotating speed, solidification 20-30min, remove calcium chloride water by filter method, obtain gel beads;
(4), the gel beads sterile distilled water of step (3) gained is washed three times, then the mass percent concentration adding gel beads volume 10 times be 2% chitosan aqueous acetic acid carry out film formation reaction 15min, the mass percent concentration being incorporated as its volume 10 times in the reaction solution of gained be 0.15% sodium alginate aqueous solution be fixed 10min, then filter with ordinary filter paper, gained precipitation 1 pH5.0, concentration is the sodium citrate aqueous solution effect 10min of 0.055mol/L, then 100 object screen filtrations are used, gained precipitation 2 is the immobilization capsule of milk-acid bacteria, the diameter of the immobilization capsule of milk-acid bacteria is 1.0-2.5mm.
The application of immobilization capsule in degrading nitrite of the milk-acid bacteria of above-mentioned gained, immobilization capsule by described milk-acid bacteria joins in the nitrite aqueous solution, static placement 72h, namely the degradation treatment of nitrite is completed, nitrite degradation rate reaches 96-98%, and after the immobilization capsule of milk-acid bacteria reuses five times, effect does not reduce, nitrite degradation rate is still all at 96-98%;
The add-on of the immobilization capsule of described milk-acid bacteria, the immobilization capsule in milk-acid bacteria: the nitrite in the nitrite aqueous solution is that the ratio of 1g:10-15 μm of ol calculates;
The nitrite aqueous solution of the described nitrite aqueous solution to be mass percent concentration be 0.2g/L, being preferably mass percent concentration is the sodium nitrite in aqueous solution of 0.2g/L.
Beneficial effect of the present invention
The immobilization capsule of milk-acid bacteria of the present invention, owing to adopting sodium alginate and chitosan as immobilization material embedded lactobacillus, it can be used for excessive nitrite salt in degraded food.
Further, the immobilization capsule of milk-acid bacteria of the present invention, owing to adopting Trisodium Citrate by solid for microcapsule core liquefaction in preparation process, thus the resistance to mass transfer of the sterically hindered of Growth of Cells and microcapsule can be reduced, therefore the milk-acid bacteria immobilization capsule of gained has good biocompatibility, is easy to the features such as small molecules diffusion, acid and alkali-resistance, therefore this milk-acid bacteria immobilization capsule is repeatedly applied to the process of water nitrite, and degradation rate is higher, and stability is better.The immobilization capsule of milk-acid bacteria reuses five times, in 72h to mass percent concentration be the degradation rate of the nitrite of 0.2g/L at 96-98%, therefore the immobilization capsule of milk-acid bacteria is used for excessive nitrite salt in Biochemical method food and has high efficiency.
Embodiment
Below by specific embodiment, the present invention is set forth further, but do not limit the present invention.
The measuring method of nitrite content adopts the hydrochloric acid-naphthalene-ethylenediamine method in GB 5009.33-2010.
MRS liquid nutrient medium used in embodiments of the invention, by often liter of calculating, containing 5.0 g glucose, 10.0 g extractum carniss, 5.0 g yeast extract pastes, 10.0 g soy peptones, 5.0 g anhydrous sodium acetates, 2.0 g Triammonium citrates, 1.0 g dipotassium hydrogen phosphates, 0.2 g magnesium sulfate, 0.05 g manganous sulfate, surplus is deionized water, pH value 7.0.
Milk-acid bacteria used in embodiments of the invention is plant lactobacillus h2, by Shanghai Institute Of Technology's biotechnology room isolation identification and preservation, can externally business provide.
embodiment 1
An immobilization capsule for milk-acid bacteria, is prepared by a method comprising the following steps and forms:
(1), the preparation of wet milk-acid bacteria thalline
The milk-acid bacteria that picking one ring inclined-plane is preserved is in the MRS liquid nutrient medium of 5ml, and 37 DEG C of static gas wave refrigerator 20h, obtain first-generation bacterial classification;
By first-generation bacterial classification, by volume percentage calculation, the inoculum size with 10% is inoculated into MRS liquid nutrient medium, and 37 DEG C of static gas wave refrigerator 20h, obtain s-generation bacterial classification;
By s-generation bacterial classification, by volume percentage calculation, the inoculum size with 10% is inoculated in MRS liquid nutrient medium, and 37 DEG C of static gas wave refrigerator 12h, obtain nutrient solution 1;
The concentration being incorporated as nutrient solution 1 volume 0.01 times in the nutrient solution 1 of gained is that the 0.8-1mol/L nitrite aqueous solution carries out cultivation 8h, obtains nutrient solution 2;
By the nutrient solution 2 of gained in 4 DEG C, the centrifugal 10min of 4000rpm, collect thalline, with the centrifugal 10min of 4000rpm after sterilized water vibration washing, repeat sterilized water vibration washing, centrifugation step once, the precipitation obtained is wet milk-acid bacteria thalline;
(2) sodium alginate aqueous solution that milk-acid bacteria thalline and 20mL mass percent concentration be 2% that, by 2g wets mixes, and after static 10-15min, obtains mixed solution;
(3), the mixed solution of step (2) gained is drawn in the asepsis injector of 5mL, be in the calcium chloride water of 1.5% with the mass percent concentration that is vertically added dropwise to of the speed of 1.5-2.0ml/min, stir with 150r/min rotating speed, solidification 20-30min, remove calcium chloride water by filter method, obtain gel beads;
(4), the gel beads sterile distilled water of step (3) gained is washed three times, then the mass percent concentration adding gel beads volume 10 times be 2% chitosan aqueous acetic acid carry out film formation reaction 15min, the mass percent concentration being incorporated as its volume 10 times in the reaction solution of gained be 0.15% sodium alginate aqueous solution be fixed 10min, then filter with ordinary filter paper, gained precipitation 1 pH5.0, concentration is the sodium citrate aqueous solution effect 10min of 0.055mol/L, then 100 object screen filtrations are used, gained precipitation 2 is the immobilization capsule of milk-acid bacteria, the diameter of the immobilization capsule of milk-acid bacteria is 1.0-2.5mm after testing.
comparative examples 1
An immobilization capsule for milk-acid bacteria, is prepared by a method comprising the following steps and forms:
(1), the preparation of wet milk-acid bacteria thalline
The milk-acid bacteria that picking one ring inclined-plane is preserved is in the MRS liquid nutrient medium of 5ml, and 37 DEG C of static gas wave refrigerator 20h, obtain first-generation bacterial classification;
By first-generation bacterial classification, by volume percentage calculation, the inoculum size with 10% is inoculated into MRS liquid nutrient medium, and 37 DEG C of static gas wave refrigerator 20h, obtain s-generation bacterial classification;
By s-generation bacterial classification, by volume percentage calculation, inoculum size with 10% is inoculated in MRS liquid nutrient medium, 37 DEG C of static gas wave refrigerator 12h, the concentration being incorporated as nutrient solution 1 volume 0.01 times in the nutrient solution 1 of gained is that the 0.8-1mol/L nitrite aqueous solution carries out cultivation 8h, by the nutrient solution 2 of gained in 4 DEG C, the centrifugal 10min of 4000rpm, collect thalline, with the centrifugal 10min of 4000rpm after sterilized water vibration washing, repeat sterilized water vibration washing, centrifugation step once, the precipitation obtained is wet milk-acid bacteria thalline;
(2) sodium alginate aqueous solution that milk-acid bacteria thalline and 20mL mass percent concentration be 2% that, by 2g wets mixes, and after static 15-20min, obtains mixed solution;
(3), the mixed solution of step (2) gained is drawn in the asepsis injector of 5mL, be in the calcium chloride water of 1.5% with the mass percent concentration that is vertically added dropwise to of the speed of 1.5-2.0ml/min, stir with 150r/min rotating speed, solidification 20-30min, remove calcium chloride water by filter method, obtain gel beads;
(4), the gel beads sterile distilled water of step (3) gained is washed three times, then the mass percent concentration adding gel beads volume 10 times be 2% chitosan aqueous acetic acid carry out film formation reaction 15min, the mass percent concentration being incorporated as its volume 10 times in the reaction solution of gained be 0.15% sodium alginate aqueous solution be fixed 10min, then filtered process removing chitosan solution, obtains the immobilization capsule of milk-acid bacteria.
application Example 1
By the application of immobilization capsule in nitrite degradation of the milk-acid bacteria of gained in embodiment 1 and comparative examples 1, step is as follows:
Be in the aqueous solution of the Sodium Nitrite of 2% by embodiment 1 and comparative examples 1, the immobilization capsule of the milk-acid bacteria of gained joins mass percent concentration respectively; control temperature is 37 DEG C, 100rpm shaking culture 72h respectively; measure content of sodium nitrite respectively, result shows that the degradation rate of Sodium Nitrite is respectively 98%, 86%;
The add-on of the immobilization capsule of described milk-acid bacteria, the milk-acid bacteria in the immobilization capsule of milk-acid bacteria: the ratio that the Sodium Nitrite in the nitrite aqueous solution is 1g:10-15 μm of ol calculates.
The immobilization capsule of the milk-acid bacteria in the nutrient solution after above-mentioned shaking culture 72h is taken out, three times are washed with sterilized water, and then the immobilization capsule of milk-acid bacteria after washing is joined mass percent concentration is in the aqueous solution of the Sodium Nitrite of 2%, 37 DEG C, 100rpm shaking culture 72h;
Repeat above-mentioned steps four times, adopt hydrochloric acid-naphthalene-ethylenediamine method to record the degradation curve of nitrite.
The immobilization capsule of the milk-acid bacteria of above-described embodiment 1 gained uses 1-5 time, and it is respectively 98 %, 98 %, 97%, 97%, 96% to nitrite degradation rate;
The immobilization capsule of the milk-acid bacteria of above-mentioned comparative examples 1 gained uses 1-5 time, and it is respectively 86%, 81%, 72%, 64%, 50% to nitrite degradation rate.
The above results indicates that the immobilization capsule of the milk-acid bacteria of the embodiment of the present invention 1 gained is higher than the degradation rate of immobilization capsule to nitrite of the milk-acid bacteria of comparative examples 1 gained and stability better, and after recycling 5 times, its degradation rate still can reach 96%.
In sum, the immobilization capsule of a kind of milk-acid bacteria provided by the invention, owing to adopting Trisodium Citrate by solid for microcapsule core liquefaction in preparation process, thus the resistance to mass transfer of the sterically hindered of Growth of Cells and microcapsule can be reduced, therefore the milk-acid bacteria immobilization capsule of gained has good biocompatibility, is easy to the features such as small molecules diffusion, acid and alkali-resistance, therefore the immobilization capsule of this milk-acid bacteria is repeatedly applied to the process of water nitrite, and degradation rate is higher, and stability is better.
The above is only the citing of embodiments of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvement, these improvement also should be considered as protection scope of the present invention.
Claims (5)
1. an immobilization capsule for milk-acid bacteria, is characterized in that the immobilization capsule of described milk-acid bacteria is prepared by a method comprising the following steps and forms:
(1), being 2% by wet milk-acid bacteria thalline and the mass percent concentration of collected by centrifugation, sodium alginate aqueous solution mixes, and after static 10-25min, obtains mixed solution;
Above-mentioned wet milk-acid bacteria thalline and mass percent concentration are the consumption of the sodium alginate aqueous solution of 2%, in wet milk-acid bacteria thalline: mass percent concentration be 2% sodium alginate aqueous solution be that the ratio of 1g:10ml calculates;
(2), the mixed solution of step (1) gained is drawn in asepsis injector, be in the calcium chloride water of 1.5% with the mass percent concentration that is vertically added dropwise to of the speed of 1.5-2.0ml/min, stir with 150r/min rotating speed, solidification 20-30min, by the method removing calcium chloride water filtered, obtain gel beads;
(3), the gel beads sterile distilled water of step (2) gained is washed three times, then the mass percent concentration adding gel beads volume 10 times be 2% chitosan aqueous acetic acid carry out film formation reaction 15min, the mass percent concentration being incorporated as its volume 10 times in the reaction solution of gained be 0.15% sodium alginate aqueous solution be fixed 10min, then filter with ordinary filter paper, gained precipitation 1 pH5.0, concentration is the sodium citrate aqueous solution effect 10min of 0.055mol/L, then 100 object screen filtrations are used, the precipitation 2 of gained is the immobilization capsule of milk-acid bacteria.
2. the preparation method of the immobilization capsule of a kind of milk-acid bacteria as claimed in claim 1, is characterized in that specifically comprising the steps:
(1), the preparation of wet milk-acid bacteria thalline
The milk-acid bacteria that picking one ring inclined-plane is preserved is in the MRS liquid nutrient medium of 5.0ml, and 37 DEG C of static gas wave refrigerator 20h, obtain first-generation bacterial classification;
By first-generation bacterial classification, by volume percentage calculation, the inoculum size with 10% is inoculated into MRS liquid nutrient medium, and 37 DEG C of static gas wave refrigerator 20h, obtain s-generation bacterial classification;
By s-generation bacterial classification, by volume percentage calculation, the inoculum size with 10% is inoculated in MRS liquid nutrient medium, and 37 DEG C of static gas wave refrigerator 12h, obtain nutrient solution 1;
The concentration being incorporated as nutrient solution 1 volume 0.01 times in the nutrient solution 1 of gained is that the 0.8-1mol/L nitrite aqueous solution carries out cultivation 8h, obtains nutrient solution 2;
By the nutrient solution 2 of gained in 4 DEG C, the centrifugal 10min of 4000rpm, collect thalline, with the centrifugal 10min of 4000rpm after sterilized water vibration washing, repeat sterilized water vibration washing, centrifugation step once, the precipitation obtained is wet milk-acid bacteria thalline;
(2), being 2% by wet milk-acid bacteria thalline and mass percent concentration, sodium alginate aqueous solution mixes, and after static 10-25min, obtains mixed solution;
Above-mentioned wet milk-acid bacteria thalline and mass percent concentration are the consumption of the sodium alginate aqueous solution of 2%, in wet milk-acid bacteria thalline: mass percent concentration be 2% sodium alginate aqueous solution be that the ratio of 1g:10ml calculates;
(3), the mixed solution of step (2) gained is drawn in asepsis injector, be in the calcium chloride water of 1.5% with the mass percent concentration that is vertically added dropwise to of the speed of 1.5-2.0ml/min, stir with 150r/min rotating speed, solidification 20-30min, remove calcium chloride water by filter method, obtain gel beads;
(4), the gel beads sterile distilled water of step (3) gained is washed three times, then the mass percent concentration being incorporated as gel beads volume 10 times be 2% chitosan aqueous acetic acid carry out film formation reaction 15min, the mass percent concentration being incorporated as its volume 10 times in the reaction solution of gained be 0.15% sodium alginate aqueous solution be fixed 10min, then filter with ordinary filter paper, gained precipitation 1 pH5.0, concentration is the sodium citrate aqueous solution effect 10min of 0.055mol/L, then 100 object screen filtrations are used, gained precipitation 2 is the immobilization capsule of milk-acid bacteria.
3. the immobilization capsule of milk-acid bacteria as claimed in claim 1 is used for the degraded of nitrite in food.
4. the application of immobilization capsule in nitrite in food degraded of milk-acid bacteria as claimed in claim 3, the immobilization capsule of described milk-acid bacteria is it is characterized in that to join in the nitrite aqueous solution, static placement 72h, namely completes the degradation treatment of nitrite;
The add-on of the immobilization capsule of described milk-acid bacteria, the immobilization capsule in milk-acid bacteria: the nitrite in the nitrite aqueous solution is that the ratio of 1g:10-15 μm of ol calculates;
The nitrite aqueous solution of the described nitrite aqueous solution to be mass percent concentration be 0.2g/L.
5. the application of immobilization capsule in nitrite in food degraded of milk-acid bacteria as claimed in claim 4, is characterized in that described mass percent concentration be the nitrite aqueous solution of 0.2g/L to be mass percent concentration is the sodium nitrite in aqueous solution of 0.2g/L.
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| CN106035739A (en) * | 2016-05-23 | 2016-10-26 | 中国农业大学 | Layer-by-layer self-assembled solidified linseed oil and method for layer-by-layer self-assembled solidification of compound fat |
| WO2018103118A1 (en) * | 2016-12-07 | 2018-06-14 | 深圳先进技术研究院 | Fermented soybean whey beverage and preparation method thereof |
| CN108669565A (en) * | 2018-04-25 | 2018-10-19 | 广东石油化工学院 | A kind of preparation method of microcapsules |
| CN108740244A (en) * | 2018-05-11 | 2018-11-06 | 中玺（天津）枣业技术工程中心 | Cartoon jujube sugar and processing method |
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| CN114698769A (en) * | 2022-03-15 | 2022-07-05 | 大连工业大学 | Method for immobilizing black pigment of agaric and application of agaric in preparation of jelly |
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| CN105200032A (en) * | 2015-10-27 | 2015-12-30 | 长春力太生物技术有限公司 | Preparation method and application of immobilized cells |
| CN105200032B (en) * | 2015-10-27 | 2019-02-19 | 长春力太生物技术有限公司 | A kind of preparation method and applications of phase solidification cell |
| CN106035739A (en) * | 2016-05-23 | 2016-10-26 | 中国农业大学 | Layer-by-layer self-assembled solidified linseed oil and method for layer-by-layer self-assembled solidification of compound fat |
| CN106035739B (en) * | 2016-05-23 | 2019-05-17 | 中国农业大学 | A kind of method that LBL self-assembly solidifies linseed oil and its compounds grease |
| WO2018103118A1 (en) * | 2016-12-07 | 2018-06-14 | 深圳先进技术研究院 | Fermented soybean whey beverage and preparation method thereof |
| CN108669565A (en) * | 2018-04-25 | 2018-10-19 | 广东石油化工学院 | A kind of preparation method of microcapsules |
| CN108740248A (en) * | 2018-05-11 | 2018-11-06 | 中玺（天津）枣业技术工程中心 | Jujube haw sheet and production method |
| CN108740244A (en) * | 2018-05-11 | 2018-11-06 | 中玺（天津）枣业技术工程中心 | Cartoon jujube sugar and processing method |
| CN108740971A (en) * | 2018-05-14 | 2018-11-06 | 中玺（天津）枣业技术工程中心 | Green mustard jujube western-style food garnishes and production method |
| CN112239758A (en) * | 2019-07-19 | 2021-01-19 | 高雄科技大学 | Probiotic composition, method for the production thereof and use thereof |
| CN111996184A (en) * | 2020-08-19 | 2020-11-27 | 云南省农业科学院农产品加工研究所 | Immobilization method of lactic acid bacteria and application of lactic acid bacteria in preparation of fruit and vegetable beverage |
| CN111996184B (en) * | 2020-08-19 | 2023-10-10 | 云南省农业科学院农产品加工研究所 | Immobilization method of lactic acid bacteria and application of lactic acid bacteria in preparation of fruit and vegetable beverage |
| CN114698769A (en) * | 2022-03-15 | 2022-07-05 | 大连工业大学 | Method for immobilizing black pigment of agaric and application of agaric in preparation of jelly |
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