CN104313005B - A kind of high stability lactobacillus micro-capsule and the preparation method and application thereof - Google Patents
A kind of high stability lactobacillus micro-capsule and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of high stability lactobacillus micro-capsules and the preparation method and application thereof, belong to technical field of bioengineering.Lactobacillus cell suspension and sterile casein sodium, the mixed liquor of gelatin and calcium lactate mixes, pass through bacteria-free device for preparing microencapsulated cells, it instills in sterile sodium alginate solution, form lactobacillus suspension core rubber capsule, this capsule containing bacterium is handled with glutamine transaminage, make casein sodium and cross-link gelatin in cyst wall, enhance the mechanical strength of capsule, increase continuous culture and vaccinization process stability, extend the vaccinization time of capsule containing bacterium, improve the service life of the capsule containing bacterium, reduce the unit cost of fermented product, improve the economic benefit and the market competitiveness of enterprise, have a vast market foreground.
Description
Technical field
The present invention is a kind of high stability lactic acid bacteria micro-capsule and the preparation method and application thereof, belongs to biotechnology neck
Domain is that a kind of continuous cultivate of increase improves glue containing bacterium with vaccinization process stability, the extension vaccinization time of capsule containing bacterium
The service life of capsule improves the new technology of the vaccinizations leavening efficiency such as fermented dairy product, fermented fruits and vegetables juice.
Background technology
Traditional liquid leavening refers to sending out the axenic culture of production by mother culture, intermediate leavening, production
Ferment agent etc. is used to produce after expanding proliferation step by step.This kind of leavening is final in use, cell concentration only has 108-109Cfu/ml,
And it is operationally complicated, it is technically not easy to control, often since strain is out of proportion, vigor declines, variable exclusive or pollution and
Lead to unstable product quality.It is external at present not use this kind of leavening substantially, however most of factories at present of China
Family is still produced using traditional liquid starter technique, the main reason is that low production cost.
The throw type leaven of freeze-dried type has obtained very fast promotion and application in recent years.This leavening is the shape in control pH
Inoculation fermentation is carried out under state, centrifugal concentrating is sold with the state for freezing or drying.Viable count can reach 10 in final products10-
1011The advantages of cfu/g, this kind of leavening is：1) strain workshop is saved；2) investment of milk plant is reduced；3) technique mistake is simplified
Journey；4) unstable quality caused by avoiding technical reason, be conducive to fermented dairy product accurately controls and produces standardization.But
Its disadvantage is also apparent from：1) due to the factor of freeze-drier and energy consumption, the production cost of this kind of leavening is higher；2) centrifugation,
Thalline can be damaged to some extent in freezing or drying process and effective thalline quantity is caused to decline；3) thalline is producing
It differs greatly with local environment when preservation and causes spawn activity relatively low, the recovery time is longer, extends fermentation time and (generally wants
6-8 hours).The supply of China's direct putting type freeze dried fermenting preparation is mainly held by external producer at present, such as Denmark's Hansen (Hansen)
Company, French Rhodia (Rhodia) company, Marshed companies of the U.S., Csiro research institutes of Australia etc., these companies
Product occupies the lactic acid bacteria fermenting agent market of China nearly 95%, and price is higher, if delivering directly hair using these companies
The cost of ferment agent, product per ton increases nearly 200 yuan.
Novel, efficient food leavening production technology with independent intellectual property right are developed, above-mentioned two class is overcome
The deficiency of leavening simplifies production technology, and reduces production cost, it appears particularly important.
In recent years, we are tried to explore in the development of microcapsule leaven and application aspect, and pioneering is " biological micro-
The shortcomings that reactor High Density Cultivation technology " avoids conventional suspension culture and limitation utilize suitable coating material, optimization packet
Capsule technique prepares the microcapsules that intensity improves a lot, and culture medium and condition of culture is optimized, by continuously training
It supports, highdensity microencapsulation leavening is made.Microencapsulated cell is detached from culture medium to be not required to by ultrafiltration or refrigerated centrifuge, and
It can be carried out with common centrifugation or microfiltration, therefore greatly reduce production cost, it is often more important that mitigate centrifugation to thalline
Injury.In addition, being also prevented from damage of the oxygen to prebiotic type anaerobic bacteria or facultative anaerobic bacteria after cell encapsulation, bacteriophage is prevented
Infection, plays protective effect of the leavening preparation process to cell.Being packed into specific bioreactor can continuously be connect
Kind, it reuses, greatly reduces the dosage of leavening, reduce the production cost of fermented product.Microencapsulation leavening is in reality at present
Some problems are faced in the application on border, the mechanical strength of capsule is low, and how cracky during use is developed and prepare capsule green wood
Expect and existing capsule material is modified to prepare the microcapsules of high intensity, improves capsule and stablize during vaccinization
Property, it is urgent problem to be solved during microcapsule leaven actual production utilizes.
Invention content
The technical problem to be solved by the present invention is to be directed to current microencapsulation leavening during vaccinization, with using
Time increases, and strength of microcapsules weakens, breakage rate increases, and seriously affects inoculation efficiency, reduces production production capacity, increases production cost
The problem of, a kind of high mechanical strength is provided, continuous culture and vaccinization process stability are good, with long service life containing lactic acid bacteria
Microcapsules improve inoculation efficiency, reduce the unit cost of fermented product.
The present invention also technical problems to be solved are to provide the preparation method of above-mentioned high stability lactobacillus micro-capsule.
The last technical problems to be solved of the present invention are to provide the application of above-mentioned high stability lactobacillus micro-capsule.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of preparation method of high stability lactobacillus micro-capsule, it includes the following steps：
(1) by the lactobacillus inoculum of freeze-drying to MRS culture mediums by actication of culture twice；
(2) strain after activating step (1) is inoculated into whey medium or MRS culture mediums, 30-44 DEG C of culture 18-
24 hours；
(3) zymotic fluid that step (2) obtains is discarded through centrifugation except supernatant, remaining thalline is suspended in sterile trehalose
In aqueous solution, the sterile mixed aqueous solution mixing with calcium lactate, casein sodium and gelatin；
(4) the mixing solution for obtaining step (3) sprays into sterile alginic acid by bacteria-free device for preparing microencapsulated cells
In sodium water solution, microcapsules are prepared；
(5) after the microcapsules aseptic water washing that step (4) obtains, it is put into glutamine transaminage (TGase) solution,
It is reacted 5-6 hours under the conditions of 30-40 DEG C；
(6) reaction solution obtained step (5) filters, and discards glutamine transaminage solution, and remaining microcapsules are with sterile
After water rinses, microcapsules are put into calcium lactate aqueous solution and are handled 40-60 minutes, filtered, discard calcium lactate solution, use sterile water
It rinses；
(7) microcapsules that step (6) obtains are put into sterile lactic acid bacteria Multiplying culture liquid, in 30-37 DEG C of constant pH 6-
7, feed supplement is continuously cultivated, and waits for that microcapsules inner cell density reaches 1011Cfu/g or more, stops culture, and filtering removes culture medium, puts
Enter in microcapsules protection liquid, 0-4 DEG C of storage.
In step (1) and (2), the MRS culture medium prescriptions are, per 1L distilled water, 10g peptones, 10g beef to be added
Cream, 5g yeast extracts, 2gK2HPO4, 2g dibasic ammonium citrates, 5g sodium acetates, 20g glucose, 1mL Tween 80s,
0.58gMgSO4.7H2O、0.25gMnSO4.4H2O, mixing, pH6.2-6.4.
In step (2), the whey medium is：Per 1L distilled water, 46g whey powders, 12g soybean separation proteins is added
In vain, 5.3g yeast extracts, 2.6g K2HPO4,1.2g KH2PO4, mixing, pH6.8-7.2.
In step (2), preferably 30-44 DEG C is cultivated 18 hours.
In step (3), in the aqueous trehalose solution, solute trehalose mass percent concentration is that 0.5-0.8% is (excellent
It selects 0.5%)；The mass ratio of thalline and sterile aqueous trehalose solution is 32-38%；The calcium lactate, casein sodium and bright
In the sterile mixed aqueous solution of glue, the mass percentage concentration of solute calcium lactate is the quality percentage of 3-5%, solute casein sodium
A concentration of 5.5-6.5%, solute gelatin mass percentage concentration be 1-1.5%；Lactobacteria-containing aqueous trehalose solution and lactic acid
The mass ratio of the sterile mixed aqueous solution of calcium, casein sodium and gelatin is 1-1.5：6-8 (preferably 1:6).
In step (4), the bacteria-free device for preparing microencapsulated cells, structural reference Chinese patent
ZL200820035824.2。
In step (4), in the sodium alginate aqueous solution, the mass percentage concentration of solute sodium alginate is 0.4-
1.0%, and the Tween 80 of the also a concentration of 0.1-0.15% containing hundred parts of quality (preferably 0.15%)；The sodium alginate aqueous solution
Dosage is 5-8 times of the mixing solution quality that step (3) obtains.
In step (5), the addition 14-20U/100g microcapsules of glutamine transaminage.
One glutamine transaminage unit of activity (U) is defined as：1 μm of ol L- paddy ammonia of catalysis formation per minute at 37 DEG C
The enzyme amount of the sour mono- hydroximic acids of γ-is an enzyme unit.
In step (6), in the calcium lactate aqueous solution, the mass percentage concentration of solute calcium lactate be 2-4% (preferably
2%).
In step (7), the lactic acid bacteria Multiplying culture formula of liquid is as follows：Per 1L distilled water, addition 20g casoid flours,
30g hydrolyzate of soybean protein, 0.6g yeast powders, 3gK2HPO4, 6g calcium lactates, 10g glucose, 15g lactose, mixing, pH6.8-
7.2；The described microcapsules protection formula of liquid is：Trehalose 0.1wt%, glycerine 8wt%, xylitol 1.2wt%, sucrose 3wt%,
Calcium lactate 0.4wt%, remaining is water.
The high stability lactobacillus micro-capsule that above-mentioned preparation method is prepared is also within the scope of the present invention.
Wherein, the lactic acid bacteria is single lactic acid bacteria or various lactobacillus.
Application of the above-mentioned high stability lactobacillus micro-capsule in milk, fruit juice or vegetable juice vaccinization leavening product
Also within protection scope of the present invention.
Advantageous effect：The present invention utilizes biotechnology, connects made from high stability lactic acid bacteria micro-capsule technology of preparing
Continued access kind lactic acid bacteria fermenting agent, overcomes the problem that existing vaccinization leavening strength of microcapsules is low, breakage rate is high, and raising connects
Kind efficiency, reduces the unit cost of fermented product.
1. micro-capsule inner cell density (> 10 can be effectively improved in incubation11It cfu/g), and can be in vaccinization process
Middle reduction capsule breakage rate.
2. the production cost of leavening is low, it is not required to high speed refrigerated centrifuge (or ultrafiltration), is freeze-dried process, it only need to be through common
Filtering can separating thallus.
3. after lactobacillus cell packing, can prevent thermophilic thalline from infecting, to ensure to ferment successfully.
4. in continuous production process, ratio between each strain can be kept substantially, it is ensured that the wind of continuous production fermented product
Taste is almost the same.
Description of the drawings
Fig. 1 is influence of the vaccinization time to capsule intensity and breakage rate.
Specific implementation mode
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.
Lactic acid bacteria used in following embodiment is all the upper conventional bacterial classification of current production, and the public can directly commercially.
Embodiment 1：
A kind of preparation method of high stability lactobacillus micro-capsule, it includes the following steps：
(1) by the lactobacillus inoculum of freeze-drying to MRS culture mediums by actication of culture twice.
(2) strain after activating step (1) is inoculated into whey medium or MRS culture mediums, and 37 DEG C are cultivated 18 hours.
(3) zymotic fluid that step (2) obtains is discarded through centrifugation (5000rpm, 10min) except supernatant, remaining thalline
It is suspended in sterile aqueous trehalose solution, the sterile mixed aqueous solution mixing with calcium lactate, casein sodium and gelatin；Described
In aqueous trehalose solution, solute trehalose mass percent concentration is 0.5%；The mass ratio of thalline and sterile aqueous trehalose solution
It is 35%；In the sterile mixed aqueous solution of the calcium lactate, casein sodium and gelatin, the quality percentage of solute calcium lactate is dense
Degree is 4%, the mass percentage concentration of solute casein sodium is 6%, the mass percentage concentration of solute gelatin is 1.5%；Containing breast
The mass ratio of the aqueous trehalose solution of sour bacterium and the sterile mixed aqueous solution of calcium lactate, casein sodium and gelatin is 1:6.
(4) the mixing solution for obtaining step (3) (refers to Chinese patent by bacteria-free device for preparing microencapsulated cells
ZL200820035824.2), spray into sterile sodium alginate aqueous solution, microcapsules are prepared；The sodium alginate water
In solution, the mass percentage concentration of solute sodium alginate is 0.6%, and the also Tween 80 containing hundred parts a concentration of 0.15% of quality；
The dosage of the sodium alginate aqueous solution is 6 times of the mixing solution quality that step (3) obtains.
(5) after the microcapsules aseptic water washing that step (4) obtains, glutamine transaminage (TGase) solution, paddy are put into
The addition 18U/100g microcapsules of glutamine transaminase react 5.5 hours under the conditions of 37 DEG C.
(6) reaction solution obtained step (5) filters, and discards glutamine transaminage solution, and remaining microcapsules are with sterile
After water rinses, microcapsules are put into calcium lactate aqueous solution and are handled 50 minutes, in the calcium lactate aqueous solution, solute calcium lactate
Mass percentage concentration be 2%, filtering, discard calcium lactate solution, use aseptic water washing.
(7) microcapsules that step (6) obtains are put into sterile lactic acid bacteria Multiplying culture liquid, in 35 DEG C of constant pH 6-7, are mended
The continuous culture of material, waits for that microcapsules inner cell density reaches 1011Cfu/g or more, stops culture, and filtering removes culture medium, is put into micro-
Capsule is protected in liquid, 0-4 DEG C of storage.
In step (1) and (2), the MRS culture medium prescriptions are, per 1L distilled water, 10g peptones, 10g beef to be added
Cream, 5g yeast extracts, 2gK2HPO4, 2g dibasic ammonium citrates, 5g sodium acetates, 20g glucose, 1mL Tween 80s,
0.58gMgSO4.7H2O、0.25gMnSO4.4H2O, mixing, pH6.2-6.4.
In step (2), the whey medium is：Per 1L distilled water, 46g whey powders, 12g soybean separation proteins is added
In vain, 5.3g yeast extracts, 2.6g K2HPO4,1.2g KH2PO4, mixing, pH6.8-7.2.
In step (7), the lactic acid bacteria Multiplying culture formula of liquid is as follows：Per 1L distilled water, addition 20g casoid flours,
30g hydrolyzate of soybean protein, 0.6g yeast powders, 3gK2HPO4, 6g calcium lactates, 10g glucose, 15g lactose, mixing, pH6.8-
7.2；The described microcapsules protection formula of liquid is：Trehalose 0.1wt%, glycerine 8wt%, xylitol 1.2wt%, sucrose 3wt%,
Calcium lactate 0.4wt%, remaining is water.
Include that micro- glue capsule ﹑ containing a kind of lactic acid bacteria contains various lactobacillus with above-mentioned high stability lactic acid bacteria micro-capsule leavening
Microcapsules, each single lactobacillus micro-capsule mixing lactic acid bacteria micro-capsule leavening, be widely used in milk, soymilk, juice
Vaccinization, mechanical strength and breakage rate the result is shown in Figure 1 during vaccinization.
Breakage rate is less than 5% pair of vaccinization yield and fermented product qualitative effects are not notable, is influenced higher than 10% notable.
The lactic acid bacteria capsule prepared with this method can be significantly higher than 24 days current (wick-containings in bioreactor vaccinization 50-55 days
Micro-capsule leavening vaccinization stability analysis, Food Science, 2013,21,154-157).
Microcapsules mechanical strength detects：It is measured using positive pressing.Capsule is placed on electronic balance, use is appropriately sized
Counterweight to its front gradually pressurization until rupture, the pressure that balance show is changed from small to big, then the mutation to become smaller suddenly, remember
Record the maximum positive pressure i.e. mechanical strength of the microcapsules born at this time.Every batch of microcapsules take 20 at random, use fracture pressure
Average value characterize its mechanical strength.
Capsule breakage rate detects：It is packed into 300 lactobacillus micro-capsules in small-sized biological reactor, carries out vaccinization examination
It tests, takes out capsule within every 3 days, counted up to the quantity of capsule, calculated breakage rate.
Embodiment 2：
A kind of preparation method of high stability lactobacillus micro-capsule, it includes the following steps：
(1) by the lactobacillus inoculum of freeze-drying to MRS culture mediums by actication of culture twice.
(2) strain after activating step (1) is inoculated into whey medium or MRS culture mediums, and 30 DEG C are cultivated 20 hours.
(3) zymotic fluid that step (2) obtains is discarded through centrifugation (5000rpm, 10min) except supernatant, remaining thalline
It is suspended in sterile aqueous trehalose solution, the sterile mixed aqueous solution mixing with calcium lactate, casein sodium and gelatin；Described
In aqueous trehalose solution, solute trehalose mass percent concentration is 0.6% (preferably 0.5%)；Thalline and sterile seaweed syrup
The mass ratio of solution is 32%；In the sterile mixed aqueous solution of the calcium lactate, casein sodium and gelatin, solute calcium lactate
Mass percentage concentration be 3%, the mass percentage concentration of solute casein sodium is 5.5%, the quality percentage of solute gelatin is dense
Degree is 1%；The quality of lactobacteria-containing aqueous trehalose solution and the sterile mixed aqueous solution of calcium lactate, casein sodium and gelatin
Than being 1：8.
(4) the mixing solution for obtaining step (3) (refers to Chinese patent by bacteria-free device for preparing microencapsulated cells
ZL200820035824.2), spray into sterile sodium alginate aqueous solution, microcapsules are prepared；The sodium alginate water
In solution, the mass percentage concentration of solute sodium alginate is 0.4%, and the also Tween 80 containing hundred parts a concentration of 0.1% of quality；Institute
The dosage for stating sodium alginate aqueous solution is 5 times of the mixing solution quality that step (3) obtains.
(5) after the microcapsules aseptic water washing that step (4) obtains, glutamine transaminage (TGase) solution, paddy are put into
The addition 14U/100g microcapsules of glutamine transaminase react 5 hours under the conditions of 30 DEG C.
(6) reaction solution obtained step (5) filters, and discards glutamine transaminage solution, and remaining microcapsules are with sterile
After water rinses, microcapsules are put into calcium lactate aqueous solution and are handled 40 minutes, in the calcium lactate aqueous solution, solute calcium lactate
Mass percentage concentration be 4%, filtering, discard calcium lactate solution, use aseptic water washing.
(7) microcapsules that step (6) obtains are put into sterile lactic acid bacteria Multiplying culture liquid, in 30 DEG C of constant pH 6-7, are mended
The continuous culture of material, waits for that microcapsules inner cell density reaches 1011Cfu/g or more, stops culture, and filtering removes culture medium, is put into micro-
Capsule is protected in liquid, 0-4 DEG C of storage.
The MRS culture mediums, whey medium, lactic acid bacteria Multiplying culture liquid, microcapsules protect the same embodiment of formula of liquid
1。
Embodiment 3：
A kind of preparation method of high stability lactobacillus micro-capsule, it includes the following steps：
(1) by the lactobacillus inoculum of freeze-drying to MRS culture mediums by actication of culture twice.
(2) strain after activating step (1) is inoculated into whey medium or MRS culture mediums, and 44 DEG C are cultivated 24 hours.
(3) zymotic fluid that step (2) obtains is discarded through centrifugation (5000rpm, 10min) except supernatant, remaining thalline
It is suspended in sterile aqueous trehalose solution, the sterile mixed aqueous solution mixing with calcium lactate, casein sodium and gelatin；Described
In aqueous trehalose solution, solute trehalose mass percent concentration is 0.8%；The mass ratio of thalline and sterile aqueous trehalose solution
It is 38%；In the sterile mixed aqueous solution of the calcium lactate, casein sodium and gelatin, the quality percentage of solute calcium lactate is dense
Degree is 5%, the mass percentage concentration of solute casein sodium is 6.5%, the mass percentage concentration of solute gelatin is 1.5%；Contain
The mass ratio of the aqueous trehalose solution of lactic acid bacteria and the sterile mixed aqueous solution of calcium lactate, casein sodium and gelatin is 1.5：8.
(4) the mixing solution for obtaining step (3) (refers to Chinese patent by bacteria-free device for preparing microencapsulated cells
ZL200820035824.2), spray into sterile sodium alginate aqueous solution, microcapsules are prepared；The sodium alginate water
In solution, the mass percentage concentration of solute sodium alginate is 1.0%, and the also Tween 80 containing hundred parts a concentration of 0.15% of quality；
The dosage of the sodium alginate aqueous solution is 8 times of the mixing solution quality that step (3) obtains.
(5) after the microcapsules aseptic water washing that step (4) obtains, glutamine transaminage (TGase) solution, paddy are put into
The addition 20U/100g microcapsules of glutamine transaminase react 6 hours under the conditions of 40 DEG C.
(6) reaction solution obtained step (5) filters, and discards glutamine transaminage solution, and remaining microcapsules are with sterile
After water rinses, microcapsules are put into calcium lactate aqueous solution and are handled 60 minutes, in the calcium lactate aqueous solution, solute calcium lactate
Mass percentage concentration be 4%, filtering, discard calcium lactate solution, use aseptic water washing.
(7) microcapsules that step (6) obtains are put into sterile lactic acid bacteria Multiplying culture liquid, in 37 DEG C of constant pH 6-7, are mended
The continuous culture of material, waits for that microcapsules inner cell density reaches 1011Cfu/g or more, stops culture, and filtering removes culture medium, is put into micro-
Capsule is protected in liquid, 0-4 DEG C of storage.
The MRS culture mediums, whey medium, lactic acid bacteria Multiplying culture liquid, microcapsules protect the same embodiment of formula of liquid
1。
Embodiment 4：Fermented soybean milk produces.
Streptococcus thermophilus (S.thermophilus) obtained, De Shi lactobacillus bulgaricus as described in Example 1
(L.dellrueckii subsp.bulgraricus), lactobacillus acidophilus (:Lactobacillus acidophilus), plant
Lactobacillus (L.plantarum), lactobacillus fermenti (L.fermentum), brevibacterium casei (Brevibacteriumcasei) are single
Kind or mixed microcapsule leaven are packed into the bioreactor through sterilization, by the feed inlet of the bioreactor and pasteurize
The outlet of device (with cooling) is connected, and reactor outlet is connected with fermentation tank, is continuously pumped into pasteurize soymilk, while from biology
Soymilk containing bacterium is flowed out in reactor, strain density is 10 in control milk containing bacterium6-107cfu/ml.Soymilk containing bacterium flows into next stage fermentation
Tank can produce various fermented soybean milks in 30-37 DEG C of ferment at constant temperature.The leavening once put into can be used continuously 50-55 days, at
This reduces 60-70% than straight formula leavening thoroughly, while avoiding the defect using subculture formula leavening unstable quality.
Embodiment 5：Fermented dairy product produces.
Streptococcus thermophilus (S.thermophilus), De Shi bulgarian milks obtained as described in Example 1
Bacillus (L.dellrueckii subsp.bulgraricus), Lactobacillus casei (L.casei), bifidobacterium longum
((B.longum), lactobacillus fermenti (L.fermentum) single or mixed microcapsule leaven are packed into the biology through sterilization instead
Device is answered, the feed inlet of the bioreactor is connected with the outlet of pasteurizer (with cooling), reactor outlet and fermentation
Tank is connected, and is continuously pumped into pasteurize milk, while milk containing bacterium is flowed out from bioreactor, controls strain density in milk containing bacterium and exists
106-107cfu/ml.Milk containing bacterium flows into next stage fermentation tank, and in 37-42 DEG C of ferment at constant temperature, the Yoghourt of production, physics and chemistry and sense organ refer to
Mark is all better, meets standard GB/T 2746-1999.The leavening once put into can be used continuously 50-55 days, cost
60-70% is reduced than straight formula leavening thoroughly, while avoiding the defect using subculture formula leavening unstable quality.
Embodiment 6：Fermented fruits and vegetables juice produces.
It is obtained by lactobacillus plantarum (L.plantarum), nutrition lactobacillus as described in Example 1
(L.alimentarius), Lactococcus lactis (L.lactis), Luo Wang acetobacters (Acetobacterlovaniensis), Han Shi
Acetobacter (Gluconacetobacterhansenii) is prepared into single or mixed microcapsule leaven, is packed into through sterilization
The feed inlet of the bioreactor is connected by bioreactor with the outlet of pasteurizer (with cooling), reactor outlet
It is connected with fermentation tank, is continuously pumped into pasteurize juice, while flowing out juice containing bacterium from bioreactor, control contains bacterium
Strain density is 10 in juice5-106cfu/ml.Juice containing bacterium flows into next stage fermentation tank, 25-35 DEG C of ferment at constant temperature, production
Various fermented fruits and vegetables juice beverages.The leavening once put into can be used continuously 50-55 days, and cost is reduced than straight formula leavening thoroughly
60-70%, while avoiding the defect using subculture formula leavening unstable quality.
Claims (4)
1. a kind of preparation method of high stability lactobacillus micro-capsule, which is characterized in that it includes the following steps：
(1) by the lactobacillus inoculum of freeze-drying to MRS culture mediums by actication of culture twice；
(2) strain after activating step (1) is inoculated into whey medium or MRS culture mediums, and 30-44 DEG C of culture 18-24 is small
When；
(3) zymotic fluid that step (2) obtains is discarded through centrifugation except supernatant, it is water-soluble that remaining thalline is suspended in sterile trehalose
In liquid, the sterile mixed aqueous solution mixing with calcium lactate, casein sodium and gelatin；
(4) the mixing solution for obtaining step (3) sprays into sterile sodium alginate water by bacteria-free device for preparing microencapsulated cells
In solution, microcapsules are prepared；
(5) after the microcapsules aseptic water washing that step (4) obtains, it is put into glutamine transaminage solution, in 30-40 DEG C of condition
Lower reaction 5-6 hours；
(6) reaction solution for obtaining step (5) filters, and discards glutamine transaminage solution, remaining microcapsules are rushed with sterile water
After washing, microcapsules are put into calcium lactate aqueous solution and are handled 40-60 minutes, filtered, discard calcium lactate solution, rushed with sterile water
It washes；
(7) microcapsules that step (6) obtains are put into sterile lactic acid bacteria Multiplying culture liquid, in 30-37 DEG C of constant pH 6-7, are mended
The continuous culture of material, waits for that microcapsules inner cell density reaches 1011Cfu/g or more, stops culture, and filtering removes culture medium, is put into micro-
Capsule is protected in liquid, 0-4 DEG C of storage；
In step (1) and (2), the MRS culture medium prescriptions are, per 1L distilled water, be added 10g peptones, 10g beef extracts,
5g yeast extracts, 2gK2HPO4, 2g dibasic ammonium citrates, 5g sodium acetates, 20g glucose, 1mL Tween 80s,
0.58gMgSO4.7H2O、0.25gMnSO4.4H2O, mixing, pH6.2-6.4；In step (2), the whey medium is：Often
46g whey powders, 12g soybean protein isolates, 5.3g yeast extracts, 2.6g K is added in 1L distilled water2HPO4、1.2g KH2PO4, mix
It is even, pH 6.8-7.2；
In step (3), in the aqueous trehalose solution, solute trehalose mass percent concentration is 0.5-0.8%；Thalline with
The mass ratio of sterile aqueous trehalose solution is 32-38%；The sterile mixing of the calcium lactate, casein sodium and gelatin is water-soluble
In liquid, the mass percentage concentration of solute calcium lactate is 3-5%, the mass percentage concentration of solute casein sodium be 5.5-6.5%,
The mass percentage concentration of solute gelatin is 1-1.5%；Lactobacteria-containing aqueous trehalose solution and calcium lactate, casein sodium and bright
The mass ratio of the sterile mixed aqueous solution of glue is 1-1.5: 6-8；
In step (4), in the sodium alginate aqueous solution, the mass percentage concentration of solute sodium alginate is 0.4-1.0%, and
The also Tween 80 of hundred parts of a concentration of 0.1-0.15% containing quality；The dosage of the sodium alginate aqueous solution is what step (3) obtained
5-8 times of mixing solution quality；
In step (5), the addition 14-20U/100g microcapsules of glutamine transaminage；
In step (6), in the calcium lactate aqueous solution, the mass percentage concentration of solute calcium lactate is 2-4%；
In step (7), the lactic acid bacteria Multiplying culture formula of liquid is as follows：Per 1L distilled water, it is big that 20g casoid flours, 30g is added
Legumin hydrolyzate, 0.6g yeast powders, 3gK2HPO4, 6g calcium lactates, 10g glucose, 15g lactose, mixing, pH6.8-7.2；Institute
The microcapsules protection liquid stated is that formula is：Trehalose 0.1wt%, glycerine 8wt%, xylitol 1.2wt%, sucrose 3wt%, lactic acid
Calcium 0.4wt%, remaining is water.
2. the high stability lactobacillus micro-capsule that the preparation method described in claim 1 is prepared.
3. high stability lactobacillus micro-capsule according to claim 2, which is characterized in that the lactic acid bacteria is single breast
Sour bacterium or various lactobacillus.
4. the high stability lactobacillus micro-capsule described in claim 2 is in milk, fruit juice or vegetable juice vaccinization leavening
The application of product.
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