CN1528909A - Lactobacillus fixed cell in-situ separating-fermenting lactic-acid production process - Google Patents
Lactobacillus fixed cell in-situ separating-fermenting lactic-acid production process Download PDFInfo
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Abstract
The invention refers to a process for using yeasting method to produce lactic acid. Takes out the mixed liquid of embedding agent and fungus suspending liquid to solidify the cells, the embedding agent is made up of fungus suspending liquid: 2% kala glue liquid: 8-12% polyvinyl alcohol and 2-4 sodium alginate with proportion 1:1:3-5, combines with using pH controller to control the acid rate pH value = 5.0-6.2 automatically, when the pH gauge displays the pH value is 5.0 in the yeasting liquid, the pH control system switches on the power automatically, the circular pump begin to work, when the pH value reaches 6.5, the relay cuts off the power, the circular pump stops, the yeasting is going on, it realizes in situ separation yeast lactic acid generating.
Description
Technical field
The claimed technical scheme of the present invention relates to the preparation method of lactic acid, specifically adopts fermentation process to produce the technology of lactic acid.
Background technology
Having in biological fermentation and the biocatalysis much is product inhibition process, if untimely with product neutralization or separation, the fermentation reaction process can't continue.Lactic fermentation is exactly a typical product inhibition fermenting process.Lactic acid fermented suitable acidity is pH value=5.0～6.2, and fermentation is suppressed when pH value＜5, and lactic acid yield reduces.In the lactic fermentation process because the accumulation of lactic acid causes the pH value of fermented liquid to reduce gradually, severe inhibition growth of milk-acid bacteria and synthesizing of lactic acid.When adopting the traditional zymotic method to produce lactic acid,, add in the lime carbonate in the fermented liquid and the lactic acid that produces in the fermenting process, be converted into calcium lactate for eliminating this restraining effect, subsequently again with calcium lactate and sulfuric acid reaction, and through decolouring, the refining lactic acid that forms of ion-exchange.The traditional zymotic method is produced lactic acid, long, poor, the solid waste disposal difficulty of operational condition of technical process not only, and also because calcium lactate has higher solubleness, the yield of lactic acid generally has only 50～60%.In recent years, the original position isolation technique is applied in lactic fermentation.At " milk-acid bacteria and fermented product production technology a thereof " book, people's such as Tang Fengxia article " application of original position isolation technique in the L-lactic fermentation " (Ningxia agricultural college journal, 2001,22 (2): 70-72) and reported existing several original position isolation technique in some other patents and the non-patent literature, comprising: electrodialysis fermentation method (JP02-286090), absorption exchange process (CN 1332144A), extractive fermentation method (CN 1234387A), film fermentation method (CN 1348992A), tubular fibre fixation method (JP 07-067672), spent ion exchange resin extracts lactic acid (India Aradhana) etc.But these methods still exist following shortcoming: the active cells in the fermenting process in the fermented liquid reduces, transformation efficiency is low, the lactic acid loss is excessive, energy consumption is big, cost is high.US 4,698,303 and US 4,771,001 disclosed substratum fermentation, cell-circulating fermentation, meat soup acidifying fermentation and the isolating continuous fermentation method of lactic acid of producing lactic acid, this method complex process, efficient are not high yet.
Immobilized cell technology is the biotechnology new technology that grows up the beginning of the seventies.This technology can improve cell activity greatly, thus some to immobilized cell produce lactic acid carried out studying (Chang Xiulian. the lactic acid fermented dynamics research of immobilized cell. Shandong Light Ind College journal .1999; It is fixed that the king builds dragon week. and immobilized cell utilizes the research of ion exchange resin extractive fermentation lactic acid. biotechnology .1994; Zheng one boat fourth Xinhua. lactic acid immobilized cell post fermenting process research chemical reaction engineering and technology .1992; Zheng one boat fourth Xinhua. the immobilized cell fermentation of lactic acid and the research of the absorption on polyvinylpyridine resin chemical industry journal .1992 thereof; Liu Xiaolan is sternly multiple. and the immobilized cell fermentation molasses is produced the research China beet sugar industry .1991 of lactic acid; Liu Xiaolan is sternly multiple. the lactic acid fermented research of molasses raw material immobilized cell. and Sichuan sugaring fermentation .1991; Liu Xiaolan is sternly multiple. the research of immobilized cell lactic fermentation kinetics and technology. and the journal .1991 of Qiqihar light industry institute).In these researchs, the immobilized cell method has all adopted entrapping method, mainly is to utilize superpolymer in the process that forms gel microorganism cells to be fixed on its inside.Entrapping method is with respect to other immobilized cell method; have that the influence of simple to operate, pair cell is little, good fixing effect and embedded particles intensity advantages of higher (Lee's broom Rong etc. the method research of carrageenin and gelatin embedding Phanerochaete chrysosporium. Shanghai environmental science and technology .2002,21 (4)).Yet these advantages depend on selected embedding medium and embedding condition.Embedding medium will possess following condition: the source is abundant, and cost is low, has good solidity and toughness, and is nontoxic, can make the substrate of reaction and product be easy to come in and go out carrier and cell is difficult for passing through, and cell breed in carrier soon, grows to stablize, and the life-span is long.Up to the present, the used embedding medium of milk-acid bacteria immobilized cell all is alginate calcium or Lalgine.The physical strength of these embedding mediums is not high, work-ing life short.
The king of Harbin Institute of Technology builds people such as dragon and lactic acid original position isolation technique is extracted in immobilized cell method and ion exchange resin extraction is combined; People such as Zheng of Zhejiang University one boat combine immobilized cell method absorption exchange process original position isolation technique.The shortcoming of last method is, from the fermented liquid that flows through, adsorb lactic acid by anionite-exchange resin, thalline, nutrition etc. also flow into resin column simultaneously, be adsorbed or be detained, long-time continuous fermentation like this, a large amount of thalline and suspended substance will block the resin column inlet, and fermented liquid can not be passed through continuously equably.The shortcoming of back one method is, adopting methyl alcohol is that eluent carries out the lactic acid wash-out, not only uneconomical but also not environmental protection.Ion exchange resin also just is used to, obtain in the aftertreatment of fine purification of lactic acid through resin absorption, wash-out from the crude lactic acid clear liquid in suitability for industrialized production as sorbent material at present.Above-mentioned two kinds of methods also all just are seen in experimental study to be reported.
Summary of the invention
Technical problem to be solved by this invention is: provide the method for fixing of the embedding medium cell that a kind of usefulness has excellent mechanical intensity, long service life and the automatic control level milk-acid bacteria immobilized cell original position separate fermentation that the high sepn process that biological respinse and microbial metabolites are removed is fast combined closely to produce lactic acid technology.
The present invention solves this technical problem the technical scheme that is adopted: earlier the preparation weight percent concentration is the mixed solution and 2% carrageenan solutions of the sodium alginate of 8～12% polyvinyl alcohol and 2～4%, with being bacteria suspension by weight: 2% carrageenan solutions: the mixed solution of the mixed solution of 8～12% polyvinyl alcohol and 2～4% sodium alginates=1: 1: 3～5 embedding mediums that are mixed with and bacteria suspension carries out cell fixation to milk-acid bacteria, again in conjunction with controlling lactic acid fermented suitable acidity automatically in pH value=5.0～6.2 with the pH controller, whenever the pH meter that places fermented liquid is shown as the pH value is 5.0 o'clock, the pH Controlling System is connected power supply automatically, recycle pump work, make fermented liquid begin to move to adsorption unit through filter screen, fermented liquid after adsorption unit is removed the lactic acid of fermentation generation returns in the fermentor tank, when the pH meter that places fermented liquid is shown as the pH value when being 6.5, rly. is cut off the electricity supply, recycle pump quits work, fermentation is proceeded, and realizes original position separate fermentation production lactic acid.
The cell fixation step is in the milk-acid bacteria immobilized cell original position separate fermentation production technique of the present invention:
A. the preparation of bacteria suspension: get and be cultured to the exponential growth milk-acid bacteria in latter stage, carried out centrifugal 10 minutes with whizzer, centrifugal force is 8000g～10000g, discards supernatant liquid, the sterile saline of weight such as uses to be dispersed as bacteria suspension;
B. the preparation of the mixed solution of embedding medium and bacteria suspension: earlier the preparation weight percent concentration is the mixed solution and 2% carrageenan solutions of the sodium alginate of 8～12% polyvinyl alcohol and 2～4%, be bacteria suspension more by weight: 2% carrageenan solutions: the mixed solution of 8～12% polyvinyl alcohol and 2～4% sodium alginates=1: 1: 3～5 are mixed with the mixed solution of embedding medium and bacteria suspension, and solvent for use is distilled water;
C. the preparation of linking agent: with weight percent concentration is the distilled water solution of calcium chloride 2～8% and boric acid 1～10%, and regulating the pH value is 6.4～6.7, and it is stand-by to sterilize;
D. the preparation of pearl type immobilized cell particle: get two root holes and directly be the emulsion tube of 3mm, its middle part is contained in the peristaltic pump, at one end load onto the water dropper that internal diameter is 0.5～1.0mm respectively, the water dropper end is separately fixed on the calcium chloride and borate cross-linking agent solution in the c step that magnetic stirring apparatus is housed, no water dropper end immerses respectively in the mixed solution of the embedding medium in b step and bacteria suspension, regulate peristaltic pump speed, can be advisable by Cheng Zhu again as quickly as possible with drip, open peristaltic pump and agitator, two water droppers carry out the preparation of pearl type immobilized cell particle simultaneously, and the fixing lactic acid bacteria pearl that makes is given a baby a bath on the third day after its birth all over standby with sterilized water after hardening 4～12 hours in the c linking agent in step under 4 ℃.
The step of original position separate fermentation production lactic acid is in the milk-acid bacteria immobilized cell original position separate fermentation production technique of the present invention:
A. fermentation mixes saccharification liquid and wheat bran immersion liquid in fermentor tank, and making wheat bran immersion liquid ultimate density is 1.5～2.0%, saccharification liquid ultimate density is 10～12%, regulating the pH value is 6.2～6.4, with pressure 0.1Mpa sterilization 15～30 minutes, under aseptic technique, in fermentor tank, add pearl type fixing lactic acid bacteria pearl of the present invention, connecing pearl amount is 5～6%, and promptly bacterial content is 8～10 times of liquid inoculation amount, and this moment, lactic acid production was 0, open and stir, at 45～50 ℃ of condition bottom fermentations;
The control of B.pH value, the pH value reduces gradually in the fermenting process, pH meter in fermented liquid shows that the pH value is at 5.0 o'clock, and the pH Controlling System is connected power supply automatically, recycle pump work, make fermented liquid begin to move to adsorption unit through filter screen, fermented liquid after adsorption unit is removed the lactic acid of fermentation generation returns in the fermentor tank, and when the pH meter in the fermented liquid showed that the pH value is 6.5, rly. was cut off the electricity supply, recycle pump quits work, and fermentation is proceeded;
C. separation and purification, the lactic acid that fermentation produces adsorbs through adsorption unit, use the pH detection paper, when the pH value of effluent liquid behind first adsorption column absorption lactic acid is 5.0, being this post absorption lactic acid reaches capacity, then fermented liquid is switched and adsorb into second adsorption column, the saturated back incision of second adsorption column absorption the 3rd adsorption column is inhaled post, the rest may be inferred by analogy, adsorption column quantity is decided on industrial scale, and saturated adsorption column is gone out crude lactic acid with 10～40% sulfuric acid wash-outs in the wash-out device, and crude lactic acid enters the decolouring device again, knot screen and concentrating unit, through decolouring, obtain the lactic acid of purity 〉=95% after removal of impurities and concentrated the making with extra care, the adsorption column behind the wash-out is reused after regenerating;
D. circulation, when the pH meter in the fermented liquid is shown as the pH value when being 5.0 once more, the pH Controlling System is connected power supply automatically, recycle pump starts, whole process is with B and C, meanwhile, at any time detect the fermented liquid sugar degree with enzyme membrane bio-sensing analyser, after repeatedly circulation is finished, when the fermented liquid sugar-containing concentration is lower than 2%, replenish dense liquid glucose from the high-order test tank of can sterilizing to fermentor tank, reach till 10～12% until the fermented liquid sugar-containing concentration, whole technology forms an enclosed pH balance fermentation separation system.
The invention has the beneficial effects as follows:, configure the used lactic acid cell fixation embedding medium of technology of the present invention and have following significant advantage: 1. physical strength height through experiment repeatedly.Single alginate calcium or Lalgine embedding medium all almost can not form the pearl body, and the pearl body effect made from the embedding medium of the mixed solution of the sodium alginate of of the present invention 8～12% polyvinyl alcohol and 2～4% and 2% carrageenan solutions is best, and concrete data see Table 1; 2. long service life.Milk-acid bacteria not immobilized cell can only be used once and promptly lost activity, and repeatedly still keeps higher activity and can use repeatedly with milk-acid bacteria immobilized cell of the present invention, and experimental result sees Table 5.Process using immobilized cell of the present invention is controlled lactic acid fermented suitable acidity automatically with using the pH controller, and the meta-bolites of microorganisms producing removed the method that combines fast, compared with prior art, it is also advantageous in that: first, the present invention has avoided ferment a large amount of thalline and suspended substance of long-time continuous to block resin inlet, the problem that fermented liquid can not evenly pass through continuously; The second, milk-acid bacteria immobilized cell of the present invention has good solidity, can make reactant be easy to come in and go out carrier smoothly and cell is difficult for spilling, and that cell is bred in carrier soon, grown is stable, the life-span is long; The 3rd, during the fermentation, do not add any neutralizing agent, avoid owing to producing separation difficulty and the contaminate environment that a large amount of solid wastes cause, removed the technology and the equipment of many precipitations, separation and purification again from, so technology of the present invention and equipment are simple, low consumption, low cost; The 4th, lactic product quality height, the purity of lactic product is not less than 95%; The 6th, the automatic control level height detects the pH value of fermented product at any time, in time separated product, avoid the generation of product inhibition, so the present invention can be widely used in also producing the production of inhibiting organic acid original position separate fermentation because of acidity changes in the reaction process.
Description of drawings
Fig. 1 is a preparation pearl type immobilized cell particle schematic flow sheet.
Fig. 2 is that the present invention produces the lactic acid process flow diagram.
Among the figure, 1. fermentor tank, 2. filtering net, 3. recycle pump, 4. column type adsorption unit, 5. rly., 6.pH controller, 7. the high-order test tank of can sterilizing, 8. wash-out device, 9. decolouring device, 10. knot screen, 11. concentrating uniies, 12. regenerating units, 13. the mixed solution of embedding medium and bacteria suspension, 14. emulsion tubes, 15. peristaltic pumps, 16. water dropper, 17. linking agents, 18. magnetic stirring apparatuss.
The present invention is further described below in conjunction with Fig. 1, Fig. 2 and embodiment. Although the following example has only been enumerated the technique that the original position separation and fermentation of lactic acid bacteria immobilized cell is produced, but because the present invention can be widely used in producing inhibiting various organic acid production because acidity increases during the fermentation, so claim of the present invention is not limited to the following example.
Embodiment:
Fig. 1 represents that emulsion tube (14) is loaded into peristaltic pump (15), one end of the no water dropper of emulsion tube (14) immerses in the mixed solution (13) of embedding medium and bacteria suspension, and two water droppers (16) on emulsion tube (14) the other end are separately fixed on the cross-linking agent solution (17) that magnetic stirring apparatus (18) are housed; The mixed solution of embedding medium and bacteria suspension (13) by emulsion tube water dropper (16) Cheng Zhu, splashes in the cross-linking agent solution (17) on the magnetic stirring apparatus (18) under the effect of peristaltic pump (15).
Fig. 2 shows that the high-order test tank (7) of can sterilizing is connected with fermentor tank (1), pH electrode in the pH controller (6) is placed in the fermentor tank (1), pH controller (6) links to each other with rly. (5) by electronic circuit, switch with Control Circulation pump (3), filtering net (2) is equipped with in exit, fermentor tank (1) below, use pipeline to be connected with column type adsorption unit (4) herein, between column type adsorption unit (4) and wash-out device (8) and the regenerating unit (12) through recycle pump (3), between wash-out device (8) and the decolouring device (9), between decolouring device (9) and the knot screen (10), all pass through pipe connection between knot screen (10) and the concentrating unit (11).
Embodiment 1
A. cell fixation
(a) preparation of bacteria suspension: milk-acid bacteria is cultured to exponential growth latter stage, and with centrifugal 10 minutes of whizzer, centrifugal force was 9000g, discards supernatant liquid, the sterile saline of weight such as used to be dispersed as bacteria suspension, got this bacteria suspension 22～38.7 grams;
(b) preparation of the mixed solution of embedding medium and bacteria suspension: get 8～12 the gram polyvinyl alcohol and 2～4 the gram sodium alginates be dissolved in 100 ml distilled waters, heating in water bath makes it dissolving, sterilize behind the mixing, after being cooled to 40 ℃, join in the bacteria suspension of (a) with 22～38.7 2% carrageenin distilled water solutions that restrain the bacterium of having gone out, by weight being bacteria suspension: 2% carrageenan solutions: the mixed solution of 8～12% polyvinyl alcohol and 2～4% sodium alginates=1: 1: 3～5 mix;
(c) preparation of linking agent: take by weighing 2～8 gram calcium chloride and 1～10 gram boric acid is dissolved in 100 ml distilled waters, regulating pH value is that 6.4～6.7 sterilizations are stand-by;
(d) preparation of pearl type immobilized cell particle: as shown in Figure 1, get two root holes and directly be the emulsion tube of 3mm, its middle part is contained in the peristaltic pump, at one end loads onto the water dropper of the about 0.5～1.0mm of internal diameter respectively.The water dropper end is separately fixed on the calcium chloride and borate cross-linking agent solution that magnetic stirring apparatus is housed.No water dropper end immerses in the mixed solution of embedding medium and bacteria suspension.Regulate peristaltic pump speed, can be advisable by Cheng Zhu again as quickly as possible with drip.Open peristaltic pump and agitator, two water droppers carry out the preparation of immobilized cell pearl type particulate simultaneously, and the milk-acid bacteria pearl type immobilized cell particle bacterium pearl that makes is given a baby a bath on the third day after its birth all over standby with sterilized water after hardening in linking agent under 4 ℃ 4～12 hours;
B. fermentation
(a) preparation of saccharification liquid: get starchy material 500～1000 weight unit, water 2000～4000 weight unit, 60 ℃ were soaked 1～2 hour, add amylase 5～8U/g starch, here " U " is general enzyme unit alive, under constantly stirring, be warming up to 75～95 ℃, be incubated 0.5～1 hour, detect starch to blue look completely dissolve with the iodine indicator, be pale brown look, filter, supernatant liquid is cooled to 55～60 ℃, add saccharifying enzyme 5～8U/g starch, be incubated 1～2 hour, no longer increase, be saccharification saccharification liquid completely with enzyme membrane bio-sensing analysis-e/or determining pol;
(b) preparation of wheat bran immersion liquid: 30% get wheat bran and in 50 ℃ of water, soaked 24 hours by weight percentage, be filtered into the wheat bran immersion liquid;
(c) preparation of fermention medium: get a certain amount of saccharification liquid and wheat bran immersion liquid and add in the fermentor tank, tap water is settled to 2 liters, making saccharification liquid ultimate density is 10～12%, wheat bran immersion liquid ultimate density is 1.5～2.0%, the pH value of regulating this mixed solution is 6.2～6.4, and pressure is 0.1Mpa sterilization 15～30 minutes;
(d) under the aseptic technique, add A and go on foot prepared milk-acid bacteria pearl type immobilized cell particle bacterium pearl in fermentor tank, connecing the pearl amount is 5～6%, be that bacterial content is 8～10 times of liquid inoculation amount, this moment, lactic acid production was 0, opened and stirred, at 45～50 ℃, the condition bottom fermentation;
The control of C.pH value, the pH value reduces gradually in the fermenting process, pH meter in fermented liquid shows that the pH value is at 5.0 o'clock, and the pH Controlling System is connected power supply automatically, recycle pump work, make fermented liquid begin to move to adsorption unit through filter screen, fermented liquid after adsorption unit is removed the lactic acid of fermentation generation returns in the fermentor tank, and when the pH meter in the fermented liquid showed that the pH value is 6.5, rly. was cut off the electricity supply, recycle pump quits work, and fermentation is proceeded;
D. separation and purification, the lactic acid that fermentation produces adsorbs through adsorption unit, use the pH detection paper, when the pH value of effluent liquid behind first adsorption column absorption lactic acid is 5.0, being this post absorption lactic acid reaches capacity, then fermented liquid is switched and adsorb into second adsorption column, the saturated back incision of second adsorption column absorption the 3rd adsorption column is inhaled post, saturated adsorption column is gone out crude lactic acid with 10～40% sulfuric acid wash-outs in the wash-out device, crude lactic acid enters the decolouring device again, knot screen and concentrating unit, through decolouring, obtain the lactic acid of purity 〉=95% after removal of impurities and concentrated the making with extra care, the adsorption column behind the wash-out is reused after regenerating;
E. circulation, when the pH meter in the fermented liquid is shown as the pH value when being 5.0 once more, the pH Controlling System is connected power supply automatically, and recycle pump starts, and whole process is with C and D, meanwhile, at any time detect the fermented liquid sugar degree with enzyme membrane bio-sensing analyser, after repeatedly circulation is finished, when the fermented liquid sugar-containing concentration is lower than 2%, to fermentor tank, replenish dense liquid glucose from the high-order test tank of can sterilizing, reach till 10～12% until the fermented liquid sugar-containing concentration.
Embodiment 2
Select polyvinyl alcohol, sodium alginate, carrageenin to be mixed with the embedding medium of four kinds of different ratioss according to the percentage concentration in the table 1, be used for making pearl type immobilized cell particle bacterium pearl respectively, the intensity of burying the bacterium pearl in order to following method test pack: between two slide glasss, place the embedded particles that particle diameter equates, it is crushed until particle to put counterweight on slide glass, intensity with the weight characterizing particles of counterweight, survey its every index, the results are shown in Table 1.
The quality test of the embedding medium of table 1 heterogeneity concentration preparation
The poly-second marine alga carrageenin bacterial content transformation efficiency machinery of preface particle phenomenon
Number sour sodium of enol (%) (%) is (%) intensity (g) (%)
(％)
1612 0.1 90.20＜50 adhesions
2822 0.1 90.63＞50 is good
3 10 32 0.1 90.79＞50 is good
4 12 42 0.1 90.22＞50 is better
Experiment showed, with single alginate calcium or Lalgine to be that embedding medium almost can not form the pearl body.When list prepared the pearl body with polyvinyl alcohol or sodium alginate as embedding medium, embedding medium then easily sticked together when crosslinked and forms a glob of condensation product.And when adopting carrageenin and sodium alginate to prepare the pearl body as embedding medium, intensity can not meet the demands.Through experiment repeatedly, the present invention just finds out best embedding medium composition proportion: 2% carrageenan solutions: the mixed solution of 8～12% polyvinyl alcohol and 2～4% sodium alginates=1: 3～5, the every index of making thus of pearl body is all better, physical strength meets the requirements, and has overcome the particle adhesion phenomenon again.
Embodiment 3
Press the explained hereafter lactic acid of embodiment 1, and fixing lactic acid bacteria pearl of the present invention is used three times repeatedly.Under identical condition, re-using not, the fixing lactic acid bacteria pearl produces.The production concentration result contrasts as shown in table 2.
Table 2 fixing lactic acid bacteria pearl of the present invention and immobilized bacterium liquid contrast experiment not
| Reaction system | Immobilization is 1 time | Immobilization is 2 times | Immobilization is 3 times | Immobilization 1 time | Immobilization 2 times | Immobilization 3 times |
| Production concentration (g/L) | 93.5 | 0 | 0 | 86.0 | 83.6 | 81.1 |
Find out that from test-results fixing lactic acid bacteria pearl of the present invention is though production concentration can be reused and can keep reactive behavior a little less than immobilized bacterium liquid not when using for the first time.
Embodiment 4
Get Semen Maydis powder 1000g, water 4000mL, 60 ℃ were soaked 1～2 hour, add amylase 5～8U/g starch, under constantly stirring, be warming up to 75～95 ℃, be incubated 0.5～1 hour, detect starch to blue completely dissolve with the iodine indicator, liquid is pale brown look, filters, and supernatant liquid is cooled to 55～60 ℃, add saccharifying enzyme 5～8U/g starch, be incubated 1～2 hour, no longer increase, be saccharification saccharification liquid completely with enzyme membrane bio-sensing analysis-e/or determining pol; Get wheat bran by 30% and in 50 ℃ of water, soaked 24 hours, be filtered into the wheat bran immersion liquid; Saccharification liquid and wheat bran immersion liquid are added in the 3L fermentor tank, use the tap water constant volume, make the two final concentration be respectively 10～12% and 1.5～2.0%, cumulative volume 2000mL, regulating the pH value is 6.2～6.4, sterilization is 15 minutes under 0.1Mp pressure, when treating that temperature is reduced to 45～50 ℃, the milk-acid bacteria pearl type immobilized cell particle bacterium pearl that adds 110g embodiment 1 under aseptic technique keeps leavening temperature at 45 ℃, and the pH meter in the fermentor tank is shown as 6.2.The total reducing sugar amount of the fermented liquid in the fermentor tank is 233g at this moment, and lactic acid production is 0; Open and stir fermentation, when pH meter in the fermented liquid shows that for the first time the pH value is 5.0, pH controller automatic control relay is connected power supply, recycle pump is started working, make fermented liquid flow to the column type adsorption unit that is filled with weak base anion-exchange resin through filter screen, the fermented liquid after the column type adsorption unit is removed the lactic acid of fermentation generation turns back to again in the fermentor tank, when pH meter in the fermented liquid shows that the pH value is 6.5, rly. is cut off the electricity supply, and fermentation is proceeded; When pH meter in the fermented liquid showed that once more the pH value is 5.0, pH controller automatic control relay was connected power supply, and recycle pump is started working, and whole process is with last; At any time detect the fermented liquid sugar degree with enzyme membrane bio-sensing analyser, after nine circulations, the fermented liquid sugar-containing concentration is less than 2%.The product acid amount of each process sees Table 3.
Table 3 is mended preceding nine round-robin fermentation of sugar result for the first time
| Time (accumulative total h) | Total reducing sugar amount (g/L) | Produce acid amount (accumulative total g/L) |
| ????0 | ????116.5 | ????0 |
| ????6 | ????109.5 | ????6.3 |
| ????12 | ????98.5 | ????16.2 |
| ????24 | ????79.2 | ????33.76 |
| ????30 | ????58.6 | ????52.3 |
| ????36 | ????44.4 | ????65.83 |
| ????48 | ????36.5 | ????72.62 |
| ????54 | ????31.5 | ????77.12 |
| ????60 | ????28.0 | ????80.24 |
| ????72 | ????25.1 | ????82.79 |
Embodiment 5
After nine circulations of embodiment 4, add through the saccharification liquid after concentrating from the high-order test tank stream to fermentor tank of can sterilizing, promptly dense liquid glucose till the fermented liquid sugar-containing concentration is 10～12%, carries out nine circulations again and produces.The test result that these nine times circulations produce sees Table 4.By table 4 result as can be seen, continuous fermentation process of the present invention is stable.For improving unit efficiency, after fermentation 48～72 hours, begin to add dense liquid glucose, till the fermented liquid sugar-containing concentration is 10～12%.By that analogy, circulating fermentation.
Embodiment 6
After fermentation 48～72 hours, begin the circulating fermentation that repetition embodiment 5 adds dense liquid glucose and embodiment 4, by that analogy, carry out semicontinuous fermentation.Along with continuing of fermentation time, bacterium pearl fermentation capacity weakens to some extent, when bacterium pearl fermentation capacity is that the bacterium pearl produces the acid amount when being lower than initial products acid amount 60%, then fermentation termination.Present embodiment is mended sugar 16 times altogether, and each average sugar amount of mending is equivalent to clean sugared 142.9g, lasts more than 30 day, and the fermentation and acid total amount is 2049g.The test result milk-acid bacteria pearl of the present invention as can be seen type immobilized cell particle bacterium pearl of listing by table 5 use repeatedly bacterium pearl activity after 16 times still when initial 60%, visible milk-acid bacteria pearl type immobilized cell particle bacterium pearl of the present invention can be used the long period repeatedly.
Table 4 continues nine circulating fermentation results after replenishing for the first time and supplying with dense liquid glucose
| Time (accumulative total h) | Total reducing sugar amount (g/L) | Produce acid amount (accumulative total g/L) |
| ????0 | ????108.8 | ????0 |
| ????6 | ????101.5 | ????6.6 |
| ????12 | ????91.5 | ????15.6 |
| ????24 | ????73.5 | ????32.2 |
| ????30 | ????55.5 | ????49.5 |
| ????36 | ????41.2 | ????62.3 |
| ????48 | ????33.2 | ????69.4 |
| ????54 | ????27.2 | ????74.8 |
| ????60 | ????22.7 | ????78.9 |
| ????72 | ????20.4 | ????81.0 |
Table 5 milk-acid bacteria pearl of the present invention type immobilized cell particle bacterium pearl is used the active testing result repeatedly
| Add sugared number of times | ????1 | ????2 | ????3 | ????4 |
| Bacterium pearl activity (lactic acid g/L) | ????80.96 | ????78.53 | ????76.10 | ????72.86 |
| Add sugared number of times | ????5 | ????6 | ????7 | ????8 |
| Bacterium pearl activity (lactic acid g/L) | ????71.24 | ????68.82 | ????66.39 | ????64.78 |
| Add sugared number of times | ????9 | ????10 | ????11 | ????12 |
| Bacterium pearl activity (lactic acid g/L) | ????62.34 | ????60.72 | ????58.29 | ????56.67 |
| Add sugared number of times | ????13 | ????14 | ????15 | ????16 |
| Bacterium pearl activity (lactic acid g/L) | ????55.05 | ????53.43 | ????51.00 | ????47.32 |
Embodiment 7
The NK-D301 resin of handling well is housed in adsorption column.PH meter was shown as 5.0 o'clock in fermented liquid, and the pH Controlling System is connected power supply automatically, and recycle pump work makes fermented liquid begin to move to adsorption unit through filter screen, and fermented liquid begins to pass through adsorption column; When the fermented liquid pH meter was shown as 6.5, rly. was cut off the electricity supply, and recycle pump quits work, and fermented liquid temporarily stops to pass through adsorption column.At any time measure lactic acid concn and pH value that adsorption column is imported and exported,, when adsorption column effluent liquid pH value reaches import fermented liquid pH value, be adsorption column and adsorbed saturated through circulation repeatedly.Import and export the fermented liquid lactic acid concn by detecting adsorption column, this adsorption column reaches more than 85% the adsorption efficiency of lactic acid as calculated.
Switch second adsorption column after first adsorption column absorption lactic acid is saturated, the 3rd adsorption column is switched in the saturated back of second adsorption column, and the rest may be inferred by analogy.Adsorption column that will be saturated is collected crude lactic acid with 25% sulfuric acid wash-out simultaneously.At any time detect with bariumchloride, find that in collecting liquid sulfuric acid stops when occurring collecting.Then collect lactic acid and vitriolic mixed solution, till not having lactic acid, this mixed solution is as the elutriant of next adsorption column.
By the decolouring device of the gac of handling well is housed, by the knot screen and the concentrating unit of the NK-001x7 strong acidic ion resin post of handling well are housed, warp concentrates the lactic acid that obtains purity 〉=95% again with the crude lactic acid liquid behind the wash-out.This flow process extract yield is about 80%.
The enzyme membrane bio-sensing analyser of used mensuration fermented liquid total reducing sugar amount and lactic acid content is the analysis of SBA-40C enzyme membrane bio-sensing in the foregoing description, and lactic acid purity uses high pressure liquid chromatograph WATERS 600E to measure.
Claims (3)
1. milk-acid bacteria immobilized cell original position separate fermentation is produced lactic acid technology, it is characterized in that: earlier the preparation weight percent concentration is the mixed solution and 2% carrageenan solutions of the sodium alginate of 8～12% polyvinyl alcohol and 2～4%, with being bacteria suspension by weight: 2% carrageenan solutions: the mixed solution of the mixed solution of 8～12% polyvinyl alcohol and 2～4% sodium alginates=1: 1: 3～5 embedding mediums that are mixed with and bacteria suspension carries out cell fixation to milk-acid bacteria, again in conjunction with controlling lactic acid fermented suitable acidity automatically in pH value=5.0～6.2 with the pH controller, whenever the pH meter that places fermented liquid is shown as the pH value is 5.0 o'clock, the pH Controlling System is connected power supply automatically, recycle pump work, make fermented liquid begin to move to adsorption unit through filter screen, fermented liquid after adsorption unit is removed the lactic acid of fermentation generation returns in the fermentor tank, when the pH meter that places fermented liquid is shown as the pH value when being 6.5, rly. is cut off the electricity supply, recycle pump quits work, fermentation is proceeded, and realizes original position separate fermentation production lactic acid.
2. milk-acid bacteria immobilized cell original position separate fermentation according to claim 1 is produced lactic acid technology, and it is characterized in that: the step of cell fixation is in the milk-acid bacteria immobilized cell original position separate fermentation production technique:
A. the preparation of bacteria suspension: get and be cultured to the exponential growth milk-acid bacteria in latter stage, carried out centrifugal 10 minutes with whizzer, centrifugal force is 8000g～10000g, discards supernatant liquid, the sterile saline of weight such as uses to be dispersed as bacteria suspension;
B. the preparation of the mixed solution of embedding medium and bacteria suspension: earlier the preparation weight percent concentration is the mixed solution and 2% carrageenan solutions of the sodium alginate of 8～12% polyvinyl alcohol and 2～4%, be bacteria suspension more by weight: 2% carrageenan solutions: the mixed solution of 8～12% polyvinyl alcohol and 2～4% sodium alginates=1: 1: 3～5 are mixed with the mixed solution of embedding medium and bacteria suspension, and solvent for use is distilled water;
C. the preparation of linking agent: with weight percent concentration is the distilled water solution of calcium chloride 2～8% and boric acid 1～10%, and regulating the pH value is 6.4～6.7, and it is stand-by to sterilize;
D. the preparation of pearl type immobilized cell particle: get two root holes and directly be the emulsion tube of 3mm, its middle part is contained in the peristaltic pump, at one end load onto the water dropper that internal diameter is 0.5～1.0mm respectively, the water dropper end is separately fixed on the calcium chloride and borate cross-linking agent solution in the c step that magnetic stirring apparatus is housed, no water dropper end immerses respectively in the mixed solution of the embedding medium in b step and bacteria suspension, regulate peristaltic pump speed, can be advisable by Cheng Zhu again as quickly as possible with drip, open peristaltic pump and agitator, two water droppers carry out the preparation of pearl type immobilized cell particle simultaneously, and the fixing lactic acid bacteria pearl that makes is given a baby a bath on the third day after its birth all over standby with sterilized water after hardening 4～12 hours in the c linking agent in step under 4 ℃.
3. milk-acid bacteria immobilized cell original position separate fermentation according to claim 1 is produced lactic acid technology, it is characterized in that: the step of original position separate fermentation production lactic acid is in the milk-acid bacteria immobilized cell original position separate fermentation production technique:
A. fermentation mixes saccharification liquid and wheat bran immersion liquid in fermentor tank, and making wheat bran immersion liquid ultimate density is 1.5～2.0%, saccharification liquid ultimate density is 10～12%, regulating the pH value is 6.2～6.4, with pressure 0.1Mpa sterilization 15～30 minutes, under aseptic technique, in fermentor tank, add pearl type fixing lactic acid bacteria pearl of the present invention, connecing pearl amount is 5～6%, and promptly bacterial content is 8～10 times of liquid inoculation amount, and this moment, lactic acid production was 0, open and stir, at 45～50 ℃ of condition bottom fermentations;
The control of B.pH value, the pH value reduces gradually in the fermenting process, pH meter in fermented liquid shows that the pH value is at 5.0 o'clock, and the pH Controlling System is connected power supply automatically, recycle pump work, make fermented liquid begin to move to adsorption unit through filter screen, fermented liquid after adsorption unit is removed the lactic acid of fermentation generation returns in the fermentor tank, and when the pH meter in the fermented liquid showed that the pH value is 6.5, rly. was cut off the electricity supply, recycle pump quits work, and fermentation is proceeded;
C. separation and purification, the lactic acid that fermentation produces adsorbs through adsorption unit, use the pH detection paper, when the pH value of effluent liquid behind first adsorption column absorption lactic acid is 5.0, being this post absorption lactic acid reaches capacity, then fermented liquid is switched and adsorb into second adsorption column, the saturated back incision of second adsorption column absorption the 3rd adsorption column is inhaled post, the rest may be inferred by analogy, adsorption column quantity is decided on industrial scale, and saturated adsorption column is gone out crude lactic acid with 10～40% sulfuric acid wash-outs in the wash-out device, and crude lactic acid enters the decolouring device again, knot screen and concentrating unit, through decolouring, obtain the lactic acid of purity 〉=95% after removal of impurities and concentrated the making with extra care, the adsorption column behind the wash-out is reused after regenerating;
D. circulation, when the pH meter in the fermented liquid is shown as the pH value when being 5.0 once more, the pH Controlling System is connected power supply automatically, recycle pump starts, whole process is with B and C, meanwhile, at any time detect the fermented liquid sugar degree with enzyme membrane bio-sensing analyser, after repeatedly circulation is finished, when the fermented liquid sugar-containing concentration is lower than 2%, replenish dense liquid glucose from the high-order test tank of can sterilizing to fermentor tank, reach till 10～12% until the fermented liquid sugar-containing concentration, whole technology forms an enclosed pH balance fermentation separation system.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101397538B (en) * | 2008-10-27 | 2012-07-18 | 东莞市英芝堂生物工程有限公司 | Novel circulation-type packed bed reactor |
| CN104031857A (en) * | 2014-04-17 | 2014-09-10 | 江南大学 | High-density lactobacillus culture method based on ion exchange |
| CN109486645A (en) * | 2018-12-20 | 2019-03-19 | 山西紫林醋业股份有限公司 | The method for targeting mostly micro- strain brewage blending mature vinegar using immobilization |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101497880B (en) * | 2009-03-11 | 2011-11-09 | 清华大学 | Novel method for improving PVA immobilized microorganism |
| CN102086463A (en) * | 2010-12-06 | 2011-06-08 | 天津科技大学 | Biological constant temperature separating tank and separating method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101397538B (en) * | 2008-10-27 | 2012-07-18 | 东莞市英芝堂生物工程有限公司 | Novel circulation-type packed bed reactor |
| CN104031857A (en) * | 2014-04-17 | 2014-09-10 | 江南大学 | High-density lactobacillus culture method based on ion exchange |
| CN104031857B (en) * | 2014-04-17 | 2016-08-17 | 江南大学 | A kind of high-intensity culture method for lactobacilli based on ion exchange |
| CN109486645A (en) * | 2018-12-20 | 2019-03-19 | 山西紫林醋业股份有限公司 | The method for targeting mostly micro- strain brewage blending mature vinegar using immobilization |
| CN109486645B (en) * | 2018-12-20 | 2021-11-19 | 山西紫林醋业股份有限公司 | Method for brewing and blending fragrant mature vinegar by applying immobilized targeted multi-microbe strain |
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