CN101869139B - Method for preparing lactobacillus cell high-permeability microcapsules and application - Google Patents
Method for preparing lactobacillus cell high-permeability microcapsules and application Download PDFInfo
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- CN101869139B CN101869139B CN2010101857339A CN201010185733A CN101869139B CN 101869139 B CN101869139 B CN 101869139B CN 2010101857339 A CN2010101857339 A CN 2010101857339A CN 201010185733 A CN201010185733 A CN 201010185733A CN 101869139 B CN101869139 B CN 101869139B
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Abstract
The invention provides a method for preparing lactobacillus cell high-permeability microcapsules, and belongs to the technical field of biological engineering. The bacteria-containing microcapsules prepared by using a liquid core cyst lactobacillus preparation method are subjected to high-density culture and biological enzyme treatment to form porous structures on capsule walls so that intra-capsule lactobacillus cells easily penetrate the capsule walls and are released to extra-capsule matrix; therefore, the method improves the efficiency of continuous inoculation, reduces the inoculation cost of unit products (fermented dairy products, fermented juice and the like), and has broad market prospect.
Description
One, technical field
The present invention is a kind of preparation method and application of lactobacillus cell high-permeability microcapsules, belongs to technical field of bioengineering, is a kind of new technology that improves vaccinization starter efficient such as cultured milk prod, fermented fruits and vegetables juice.
Two, background technology
In recent years, along with the raising of China people level of consumption, sour milk output has become the first cultured milk prod of China with the speed increment of annual 25%, is the milk-product of tool profit and development potentiality.
The starter that is used for yoghurt production at present has two types: the subculture formula starter of a quasi-tradition, one type is throw type leaven.Subculture formula starter bacteria containing amount few (10
7-10
8Cfu/ml) can not directly be used for producing, need just can be used for Yoghourt fermentation, the making processes more complicated of starter through the expanding propagation of mother starter, middle starter, productive leavening agent; Aseptic requirement is high; Also exist bacterial classification short duration of service in addition, preserve the difficulty that goes down to posterity, problems such as easy pollution.In order to address these problems, abroad from the end of the nineties, develop direct putting type ferment agent for sour milk, like Hansen Corp. of Denmark, Rhodia of France.DVS fermenting agent used, simplifying the production process, eliminating the bacteria workshop, to avoid lack of technology the manufacturers of microbial strains caused by the problem of poor quality, but the use of DVS leaven Hen cost is high, Currently no DVS fermenting agent production, and in some high-end yogurt used in DVS leaven all imported from abroad.Development has novel, the efficient ferment agent for sour milk production technology of independent intellectual property right, has overcome the deficiency of above-mentioned two types of starters, has promptly simplified production technique, has reduced production cost again, seems particularly important.
Three, summary of the invention
Technical problem the objective of the invention is to have problems such as the cell burst size is low, instability to being used for vaccinization microencapsulation starter production use at present; Research a kind ofly can effectively improve the micro-capsule inner cell density in culturing process, again can be in the vaccinization process the efficient technology that discharges of lactic-acid bacteria cells in the micro-capsule.
Technical scheme is provided by the present invention to provide a kind of lactobacillus cell high-permeability microcapsules preparation method, comprising:
With cryodesiccated lactobacillus inoculum to the MRS substratum with actication of culture twice, then with the activatory bacterial classification inoculation in whey or MRS substratum, cultivated 18 hours for 30-44 ℃; 5000rpm, centrifugal 10min, abandoning supernatant; Thalline being suspended in the aseptic sucrose solution of 2% (w/w), is that the aseptic starch solution of 1%-5% (w/w) (containing 1%-3% calcium chloride) mixes with 10 times of concentration, through microencapsulated cell sterile preparation device (patent No. ZL2008 2 0035824.2); Splash in the aseptic sodium-alginate that contains 0.3%-0.8% (w/w) (containing 0.1% (w/w) tween 80) solution, the gained capsule with aseptic water washing after, put into 1% calcium chloride solution sclerosis 20-40 minute; Filter, discard calcium chloride solution, behind aseptic water washing; Capsule is put into the chitosan starch mixing solutions (chitosan: starch=1: 6), take out behind the 30min, behind aseptic water washing of 1%-3%; Place aseptic improvement whey medium, be cultured to 37 ℃ of constant pH 6.5 that total sugar content is lower than 1% (w/w) in the substratum, change aseptic improvement whey medium; Continuation is cultivated in 37 ℃ of constant pH 6.5, repeats 3 batches, and the microcapsule inner cell density reaches 10
11Cfu/g filters, and removes substratum, promptly processes the high-density lactobacillus micro-capsule.Microcapsule are placed the solution (pH is 5.5-6.5) that contains warm AMS (0.1%-0.3%) and saccharifying enzyme (0.5%-0.8%), and 35-40 ℃ is incubated 2-6 hour, processes lactobacillus cell high-permeability microcapsules.
The MRS substratum:
Peptone 10g Carnis Bovis seu Bubali cream 10g yeast extract 5g
K
2HPO
42g dibasic ammonium citrate 2g sodium acetate 5g
Glucose 20g tween 80 1mL MgSO
4.7H
2O 0.58g
MnSO
4.4H
2O 0.25g zero(ppm) water 1000mL pH 6.2-6.4;
The improvement whey medium:
Whey powder 60g hydrolytic liquid of plant protein 20g yeast powder 0.8g
K
2HPO
44g lime carbonate 8g trehalose 5g
Zero(ppm) water 1000mL pH 6.8-7.2.
With above-mentioned lactobacillus cell high-permeability microcapsules starter; The single bacterium capsule blended lactobacillus starter that comprises single bacteria microcapsule, mixed bacteria microcapsule, different bacterium; Be widely used in milk-product fermentation (as contain bacterium milk, sour milk, cheese, contain probiotic bacterium milk etc.); The soymilk fermentation, the garden spgarden stuff fermentation, acid falls in wine.With the capsule that aforesaid method makes, to pack into through germ-resistant bio-reactor, the vaccinization that is used for cultured milk prod, fermented juice, fermentation vegetable juice gets final product.
Beneficial effect
The present invention utilizes biotechnology; The vaccinization lactobacillus starter that makes with the lactobacillus cell high-permeability microcapsules technology of preparing; Overcome the deficiency of two types of starters in the prior art; Promptly simplified production technique, reduced production cost again, good social benefit and economic benefit must have been arranged.
Major advantage of the present invention and positively effect are following:
1. can effectively improve micro-capsule inner cell density (＞10 in culturing process
11Cfu/g), again can be in the vaccinization process in the micro-capsule lactic-acid bacteria cells efficiently discharge;
2. the production cost of starter is low, does not need high speed frozen centrifugation (or ultrafiltration), and the lyophilize operation only needs through common filtration with regard to separable thalline.
3. behind the lactic-acid bacteria cells packing, can prevent to have a liking for thalline and infect, thereby guarantee to ferment successfully;
4. in continuous flow procedure, can keep the ratio of coccus and bacillus basically, guarantee the local flavor basically identical of continuous production sour milk.
Four, embodiment
Method 1: with cryodesiccated lactobacillus inoculum to the MRS substratum with actication of culture twice, then with the activatory bacterial classification inoculation in whey or MRS substratum, cultivated 18 hours for 30-44 ℃; 5000rpm, centrifugal 10min, abandoning supernatant; Thalline being suspended in the aseptic sucrose solution of 2% (w/w), is that the aseptic starch solution of 1%-5% (w/w) (containing 1%-3% calcium chloride) mixes with 10 times of concentration, through microencapsulated cell sterile preparation device (patent No. ZL2008 2 0035824.2); Splash in the aseptic sodium-alginate that contains 0.3%-0.8% (w/w) (containing 0.1% (w/w) tween 80) solution, the gained capsule with aseptic water washing after, put into 1% calcium chloride solution sclerosis 20-40 minute; Filter, discard calcium chloride solution, behind aseptic water washing; Capsule is put into the chitosan starch mixing solutions (chitosan: starch=1: 6), take out behind the 30min, behind aseptic water washing of 1%-3%; Place aseptic improvement whey medium, be cultured to 37 ℃ of constant pH 6.5 that total sugar content is lower than 1% (w/w) in the substratum, change aseptic improvement whey medium; Continuation is cultivated in 37 ℃ of constant pH 6.5, repeats 3 batches, and the microcapsule inner cell density reaches 10
11Cfu/g filters, and removes substratum, promptly processes the high-density lactobacillus micro-capsule.Microcapsule are placed the solution (pH is 5.5-6.5) that contains warm AMS (0.1%-0.3%) and saccharifying enzyme (0.5%-0.8%), and 35-40 ℃ is incubated 2-6 hour, processes lactobacillus cell high-permeability microcapsules.
With the capsule that aforesaid method makes, pack into through germ-resistant bio-reactor, be used for the vaccinization of cultured milk prod, fermented juice, fermentation vegetable juice.
With the lactobacillus starter that above-mentioned lactobacillus cell high-permeability microcapsules prepares and utilisation technology makes; The single bacterium capsule blended lactobacillus starter that comprises single bacteria microcapsule, mixed bacteria microcapsule, different bacterium; Be widely used in milk-product fermentation (as contain bacterium milk, sour milk, cheese, contain probiotic bacterium milk etc.); The soymilk fermentation, the garden spgarden stuff fermentation, acid falls in wine.
The above-mentioned culture medium prescription that is used for the milk-acid bacteria cultivation:
The MRS substratum:
Peptone 10g Carnis Bovis seu Bubali cream 10g yeast extract 5g
K
2HPO
42g dibasic ammonium citrate 2g sodium acetate 5g
Glucose 20g tween 80 1mL MgSO
4.7H
2O 0.58g
MnSO
4.4H
2O 0.25g zero(ppm) water 1000mL pH 6.2-6.4
The improvement whey medium:
Whey powder 60g hydrolytic liquid of plant protein 20g yeast powder 0.8g
K
2HPO
44g lime carbonate 8g trehalose 5g
Zero(ppm) water 1000mL pH 6.8-7.2
The lactobacillus starter application implementation example that above-mentioned lactobacillus cell high-permeability microcapsules preparation and utilisation technology make (annotate: the used milk-acid bacteria of following examples all is to produce at present to go up conventional bacterial classification, and the public can obtain):
Embodiment 1. fermented fruits and vegetables juice productions
As stated above plant lactobacillus (L.plantarum), nutrition probiotic lactobacillus (L.alimentarius), lactococcus lactis ssp (L.lactis), Luo Wang acetobacter (Acetobacter lovaniensis), Han Shi acetobacter (Gluconacetobacter hansenii) are prepared into single the kind or the blended microcapsule leaven; Pack into through germ-resistant bio-reactor; The opening for feed of this bio-reactor is connected with the outlet of pasteurizing unit (band cooling); Reactor outlet links to each other with fermentor tank; Be continuously pumped into the pasteurize garden spgarden stuff, from bio-reactor, flow out simultaneously and contain the bacterium garden spgarden stuff, control contains in the bacterium garden spgarden stuff nectar degree 10
5-10
6Cfu/ml.Contain the bacterium garden spgarden stuff and flow into the next stage fermentor tank, 20-30 ℃ of ferment at constant temperature produced various fermented fruits and vegetables juice beverages.The general starter that once drops into can use 15-30 days continuously, and cost reduces 50-60% than straight penetrate type starter, has avoided the unsettled defective of use subculture formula starter quality simultaneously.
Embodiment 2. cultured milk prod productions
(single kind of (B.longum), lactobacillus fermentum (L.fermentum) or blended microcapsule leaven are packed into through germ-resistant bio-reactor for the thermophilus streptococcus that makes as stated above (S.thermophilus), De Shi lactobicillus bulgaricus (L.dellrueckii subsp.bulgraricus), lactobacterium casei (L.casei), bifidus longum bb; The opening for feed of this bio-reactor is connected with the outlet of pasteurizing unit (band cooling); Reactor outlet links to each other with fermentor tank; Be continuously pumped into pasteurize milk; From bio-reactor, flow out simultaneously and contain bacterium milk, control contains in the bacterium milk nectar degree 10
6-10
7Cfu/ml.Contain bacterium milk and flow into the next stage fermentor tank,,, can produce various cultured milk prods if at 30-42 ℃ of ferment at constant temperature if directly can be produced probiotic bacterium fresh milk.The general starter that once drops into can use 15-30 days continuously, and cost reduces 50-60% than straight penetrate type starter, has avoided the unsettled defective of use subculture formula starter quality simultaneously.
Embodiment 3. fermented soybean milk productions
The thermophilus streptococcus that makes as stated above (S.thermophilus), De Shi lactobicillus bulgaricus (L.dellrueckii subsp.bulgraricus), Lactobacterium acidophilum, plant lactobacillus (L.plantarum), lactobacillus fermentum (L.fermentum), the single kind of brevibacterium casei (Brevibacterium casei) or blended microcapsule leaven are packed into through germ-resistant bio-reactor; The opening for feed of this bio-reactor is connected with the outlet of pasteurizing unit (band cooling); Reactor outlet links to each other with fermentor tank; Be continuously pumped into pasteurize soymilk; From bio-reactor, flow out simultaneously and contain bacterium soymilk, control contains in the bacterium milk nectar degree 10
6-10
7Cfu/ml.Contain bacterium soymilk and flow into the next stage fermentor tank,, can produce various fermenting bean milks at 30-42 ℃ of ferment at constant temperature.The general starter that once drops into can use 15-30 days continuously, and cost reduces 50-60% than straight penetrate type starter, has avoided the unsettled defective of use subculture formula starter quality simultaneously.
Claims (4)
1. the preparation method of a lactobacillus cell high-permeability microcapsules comprises:
With cryodesiccated lactobacillus inoculum to the MRS substratum with actication of culture twice, then with the activatory bacterial classification inoculation in whey or MRS substratum, cultivated 5000rpm 18 hours for 30-44 ℃; Centrifugal 10min, abandoning supernatant is suspended in thalline in the aseptic sucrose solution of mass ratio 2%; With 10 times to contain mass ratio 1%-3% calcium chloride and concentration be that the aseptic starch solution of 1%-5% (w/w) mixes, through microencapsulated cell sterile preparation device, splash into and aseptic contain 0.1% (w/w) tween 80 and concentration is in the sodium alginate soln of 0.3%-0.8% (w/w); The gained capsule with aseptic water washing after, put into 1% calcium chloride solution sclerosis 20-40 minute, filter; Discard calcium chloride solution, behind aseptic water washing, capsule is put into the chitosan starch mixing solutions of 1%-3%; Chitosan wherein: starch=1: 6, take out behind the 30min, behind aseptic water washing; Place aseptic improvement whey medium, be cultured to 37 ℃ of constant pH 6.5 that total sugar content is lower than 1% (w/w) in the substratum, change aseptic improvement whey medium; Continuation is cultivated in 37 ℃ of constant pH 6.5, repeats 3 batches, and the microcapsule inner cell density reaches 10
11Cfu/g filters, and removes substratum; Promptly process the high-density lactobacillus micro-capsule, microcapsule are placed the solution that contains 0.1%-0.3% temperature AMS and 0.5%-0.8% saccharifying enzyme, pH is 5.5-6.5; 35-40 ℃ is incubated 2-6 hour, processes lactobacillus cell high-permeability microcapsules, wherein
The MRS substratum:
Peptone 10g Carnis Bovis seu Bubali cream 10g yeast extract 5g
K
2HPO
42g dibasic ammonium citrate 2g sodium acetate 5g
Glucose 20g tween 80 1mL MgSO
47H
2O 0.58g
MnSO
44H
2O 0.25g zero(ppm) water 1000mL pH 6.2-6.4;
The improvement whey medium:
Whey powder 60g hydrolytic liquid of plant protein 20g yeast powder 0.8g
K
2HPO
44g lime carbonate 8g trehalose 5g
Zero(ppm) water 1000mL pH 6.8-7.2.
2. the lactobacillus cell high-permeability microcapsules starter product of the said method of claim 1 preparation.
3. according to the said lactobacillus cell high-permeability microcapsules starter of claim 2 product, comprise single bacterium capsule blended lactobacillus starter of single bacteria microcapsule, mixed bacteria microcapsule, different bacterium.
4. the application of claim 2 or 3 said lactobacillus cell high-permeability microcapsules starter products; It is characterized in that; Claim 2 or 3 said lactobacillus cell high-permeability microcapsules starter products are packed into through germ-resistant bio-reactor, be used for the vaccinization of cultured milk prod, fermented juice, fermentation vegetable juice.
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| CN102334590B (en) * | 2011-08-10 | 2013-04-03 | 广东工业大学 | Method for recycling waste yogurt |
| CN104000025B (en) * | 2014-06-11 | 2016-03-23 | 北京昕地美饲料科技有限公司 | A kind of preparation method of stabilisation lactic acid bacteria product |
| CN104313005B (en) * | 2014-10-22 | 2018-10-30 | 江苏省农业科学院 | A kind of high stability lactobacillus micro-capsule and the preparation method and application thereof |
| CN107287073A (en) * | 2017-07-06 | 2017-10-24 | 泸州向林老窖股份有限公司 | A kind of compound Koji and its production technology |
| CN108641886A (en) * | 2018-05-22 | 2018-10-12 | 中玺（天津）枣业技术工程中心 | Jujube vinegar beverage and production method |
| CN112535181B (en) * | 2020-12-04 | 2021-09-24 | 江苏省农业科学院 | Chrysanthemum essential oil microcapsule and preparation method and application thereof |
| CN114657039B (en) * | 2022-04-19 | 2024-03-22 | 海天醋业(广东)有限公司 | Yellow wine and preparation method thereof |
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| CN1487078A (en) * | 2003-08-14 | 2004-04-07 | 南京农业大学 | Lactobacillus cell microcyst culturing process and produced leaven |
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| CN1487078A (en) * | 2003-08-14 | 2004-04-07 | 南京农业大学 | Lactobacillus cell microcyst culturing process and produced leaven |
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| 王英 等.液芯胶囊的强度及生物相容性研究.《江苏农业科学》.2010,(第2期),270-271. * |
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