CN107955797A - The zonal cooling zymotechnique of clostridium butyricum - Google Patents
The zonal cooling zymotechnique of clostridium butyricum Download PDFInfo
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- CN107955797A CN107955797A CN201711127612.7A CN201711127612A CN107955797A CN 107955797 A CN107955797 A CN 107955797A CN 201711127612 A CN201711127612 A CN 201711127612A CN 107955797 A CN107955797 A CN 107955797A
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Abstract
The invention discloses a kind of zonal cooling zymotechnique of clostridium butyricum, comprise the following steps：（1）Obtain clostridium butyricum bacterium solution；（2）Culture medium is prepared, then puts into fermentation tank, becomes basal fermentation liquid；Backward basal fermentation liquid in add complex enzyme formulation, carry out the fermentation of 12 24h, zymotic fluid A be made；（3）To step 2）In obtained zymotic fluid A carry out low temperature sterilization, and carry out the fermentation of 24 48h, obtain zymotic fluid C.（4）Zymotic fluid C is discharged, while into fermentation tank, supplement adds the zymotic fluid A of equivalent, ferments, is moved in circles with this；(5) by step 3）In after obtained zymotic fluid C carries out vacuum-concentrcted, be spray-dried to obtain finished product by spraying apparatus, the present invention use totally-enclosed circulating fermentation, effectively prevent the possibility of bacterium internal pollution, the raising fermentation viable count of high degree and lifts cell concentration.
Description
Technical field
The invention belongs to technical field of microbial fermentation, more particularly, to a kind of zonal cooling fermentation work of clostridium butyricum
Skill.
Background technology
Clostridium butyricum (Clostridium butyricum) also known as Miyarisan, are a kind of Gram-positive gemma of anaerobism
Bacillus, be 1933 by Chiba, Japan medical university palace enter it is near control doctor and find and report first, therefore be called Clostridium Butyricum.
Nineteen thirty-five, Kingi doctors miyairi isolate clostridium butyricum from the excrement and soil of people, are subsequently found its Anaerobic culturel
Contain less aliphatic acid in filtrate, can inhibit the pathogenic bacteria in enteron aisle, promote beneficial bacterium such as Bifidobacterium and breast in enteron aisle
The growth of bacillus, there are extremely strong whole intestines to act on.
Clostridium butyricum, lactic acid bacteria and Bifidobacterium have a similar function on intestinal environment is adjusted, but Bifidobacterium,
The probioticses such as Bacillus acidi lactici in Clinical practice there are a fatal weakness, that is, easily by the external world during storing and being oral
Various biochemical and physical factor influences and lose activity.Particularly Bifidobacterium is extremely weak to extraneous environmental resistance, and room temperature is protected
Survival bacterium number can be greatly lowered, directly oral also easily to be lessened the curative effect by hydrochloric acid in gastric juice inactivation, and capsulae enterosolubilis need to be made and be protected by
And low-temp storage, in addition antibiotic also has it killing effect, thus should not clinically be taken together with antibiotic.And clostridium butyricum is resistance to
Hot, acidproof, resistance to bile, and it is resistant to common antibiotics, a variety of drawbacks of above-mentioned probiotics can be overcome.Therefore,
It is slow that clostridium butyricum has clinically been widely used in enteric flora disturbance, the urgency caused by a variety of causes such as treatment digestion, children
Property the various intestines problems such as diarrhea, irritable bowel syndrome, Antibiotic-associated Colitis, and on Japan, South Korea and other places as raising
Feed additives are applied to also achieve good effect on livestock and poultry cultivation, are a kind of micro- preferably, with wide development prospect
Ecological agent.
Due to clostridium butyricum strictly anaerobic, exist in yeasting if any oxygen, clostridium butyricum thalline will not be grown
It is even dead；Clostridium butyricum is stringenter for growth Optimal pH requirement, if off-target pH scopes, also will cause thalli growth not
Good, therefore the extremely difficult fermenting and producing in production, according to the report that can consult reference materials, laboratory achievement of most preferably fermenting is that to reach thalline dense at present
800,000,000 CFU/ml are spent, and the optimal fermentation level of industrialized production is 400,000,000 CFU/ml.
The content of the invention
A kind of cell concentration is high, the butyric acid shuttle more than the viable count that ferments in order to overcome the deficiencies of the prior art and provide by the present invention
The zonal cooling zymotechnique of bacterium.
To achieve these goals, the present invention uses following technical scheme：A kind of zonal cooling fermentation work of clostridium butyricum
Skill, comprises the following steps：
(1) Tube propagation, triangular flask deep drainpipe, seeding tank are carried out to clostridium butyricum successively and expands culture, obtained with this
Clostridium butyricum bacterium solution；
(2) culture medium is prepared, the pH of culture medium is adjusted to 7.0 30-35min is sterilized under the conditions of 121-128 DEG C afterwards,
Then put into fermentation tank, become basal fermentation liquid；Backward basal fermentation liquid in add complex enzyme formulation, carry out 12-24h's
Fermentation, is made zymotic fluid A；
(3) low temperature sterilization is carried out to obtained zymotic fluid A in step 2), step is added into zymotic fluid A in 1% ratio
1) clostridium butyricum bacterium solution made from, and the fermentation of 24-48h is carried out, obtain zymotic fluid C；
(4) zymotic fluid C made from step 3) is discharged, while supplement adds the zymotic fluid A of equivalent into fermentation tank,
Ferment, moved in circles with this；
(5) by after obtained zymotic fluid C progress vacuum-concentrcted in step 3), it is spray-dried by spraying apparatus
Obtain finished product.
It is of the invention that there is inoculation, fermenting twice process twice, the fermentation of inoculation induced enzyme progress induced enzyme for the first time, second
Secondary inoculation clostridium butyricum bacterium solution carries out the fermentation of clostridium butyricum gemma；Input zymotic fluid for the first time, interval are only needed in fermentation process
Certain time carries out feed supplement, you can zymotic fluid is continuously discharged to outside fermentation tank, thus iterative cycles, avoids intermittent fermentation work
The complicated preparatory process procedure such as the clear tank of skill, feeding, sterilizing, cooling, ensure that and carried out completely in closed pipeline, from
And opportunities for contamination is effectively reduced, reduce production cost.
Further, the culture medium by 2 parts of glucose, 1.0 parts of corn flour, 4.0 parts of soy meal, 0.8 part of dusty yeast,
0.1 part of defoamer plus Drinking Water are formulated.
Preferably, the complex enzyme formulation is protease, lipase, cellulase, zytase, dextranase, sweet dew
One or more in dextranase.
Further, in the step 3), before clostridium butyricum bacterium solution is launched, carbonic acid is added into zymotic fluid A in advance
Hydrogen calcium, calcium chloride, ammonium sulfate, magnesium sulfate, manganese sulfate.
Further, count in parts by weight, the dispensing of the calcium bicarbonate, calcium chloride, ammonium sulfate, magnesium sulfate, manganese sulfate
Measure as 0.05 part of calcium bicarbonate, 0.0015 part of calcium chloride, 0.1 part of ammonium sulfate, 0.015 part of magnesium sulfate, 0.015 part of manganese sulfate.
Further, in the fermenting step of the step 2) temperature of fermentation tank be 37 DEG C, tank pressure be 0.01Mpa.
Further, in the fermenting step of the step 3) temperature of fermentation tank be 37 DEG C, tank pressure be 0.01Mpa, in tank
PH is 6.5.
Further, the fermentation tank include inner tank body, outer tank body, inlet pipe member, between inner tank body and outer tank body
Heating layer, the heat pipes in inner tank body and with the matched seal member of inlet pipe member, the heat pipes with it is described
Heating layer is connected；The seal member includes first seal and the second seal being sheathed in first seal, described
There is hydraulic layer, which passes through the flow liquid opening and heating layer in outer tank body between first seal and second seal
It is connected；By heat pipes in setting and heating layer, water bath with thermostatic control heating, and interior pipe fitting can be carried out to the liquid in fermentation tank
Heated in inside, heating layer makes thermally equivalent inside and outside whole zymotic fluid, and be maintained under 37 DEG C of constant temperature, holding in external heat
Thalline normal growth；Set first seal part to realize preliminary sealing to inlet pipe member, avoid oxygen in the short time from entering to
In fermentation tank, cause thalline dead, set second seal to have realized secondary seal, further by inlet pipe member and big air bound
Open, and the gap formed between first seal and second seal is internally passed through liquid and realizes fluid-tight as hydraulic layer,
Can starvation completely enter in fermentation tank, and seal time length, from the influence of Sealing period, can smoothly ensure thalline
Normal growth；By setting flow liquid opening that heating layer is connected with hydraulic layer, therefore the liquid in heating layer can enter to
In hydraulic layer, to hydraulic layer liquid filling body, and keep the fluid temperature of hydraulic layer identical with fluid temperature in heating layer, avoid outer
Portion's temperature is penetrated to fermentation tank by inlet pipe member, is caused in fermentation tank temperature unstable and is caused thalline not give birth to normally
It is long；At the same time also reduce operational sequence, without in special adding liquid to hydraulic layer to realize fluid-tight, it is easy to operation.
Further, closure member matched with the flow liquid opening is equipped with the heating layer and is consolidated with the closure member
Elastic component even, the elastic component are connected with the inner tank body；The second seal, which is equipped with, to be used to promote the closure member
The push rod of activity downwards；Closure member can close flow liquid opening, when the second closure member is not installed, close heating layer, easy to liquid
The full heating layer of body filling, easy to heat fermentation tank in advance, makes temperature in heating tank reach required temperature, is quickly reached easy to zymotic fluid
To required temperature, and whole zymotic fluid is integrally heated evenly, avoid local heating and cause Partial fermentation liquid not ferment
Go out thalline；Elastic component makes closure member be fitted closely with flow liquid opening, realizes and flow liquid opening is sealed；And push rod is set, second
When seal is sheathed on inlet pipe member, push rod is promoted to make closure member movable downwards, opening flow liquid opening makes liquid enter to fluid-tight
In layer, fluid-tight is realized to hydraulic layer, its is easy to operate, easy to use.
Further, the outer tank body inner wall is equipped with stop collar, and the closure member is arranged in the stop collar；The elasticity
A set of duct member is arranged with part, which includes the setting of casing being fixedly arranged in inner tank body and be fixedly arranged under the closure member
Upper casing on end face, the tube portion of trapping are arranged in the upper casing；The pipe outer wall of trapping is convex equipped with one first
Ring, the upper internal surface of sleeve pipe are equipped with and matched second bulge loop of first bulge loop；Stop collar is set, and closure member is located at
In stop collar, closure member is realized spacing, position shifts and causes flow liquid opening complete when avoiding the closure member from closing
Closing, causes liquid in heating layer to flow out；Casing and setting of casing are set on elastic component in setting, and elastic component is realized and is positioned,
Avoid because elastic component double swerve and caused by closure member deviate, it is ensured that closure member successfully closes flow liquid opening, and be arranged to up and down
Casing, does not interfere with elastic component retracted downward, it is ensured that elastic component normal use；In setting the first bulge loop, setting of casing in upper casing
The second bulge loop of interior setting, two bulge loops mutually limit, avoid casing from being separated from from setting of casing, cause device unstable.
In conclusion the present invention has the following advantages：Discharged using zymotic fluid, the mode for being spaced feed supplement carries out circulating fermentation
Operation, workload and the input of worker are reduced without carrying out repeatedly operation, the high degrees such as clear tank, feeding, sterilizing, cooling
Amount, reduces production cost；Simultaneously as circulating fermentation whole process carries out under air-tight state, effectively prevent dirty in thalline
The possibility of dye, the raising fermentation viable count and lifting cell concentration of high degree.
Brief description of the drawings
Fig. 1 is the structure diagram of the present invention.
Fig. 2 is the enlarged drawing at A in Fig. 1.
Fig. 3 is the enlarged drawing at B in Fig. 2.
Fig. 4 is the enlarged drawing at C in Fig. 4.
Embodiment
In order to make those skilled in the art be better understood from the present invention program, below in conjunction with the embodiment of the present invention
Attached drawing, carries out the technical solution in the embodiment of the present invention clear, complete description.
Embodiment 1
A kind of zonal cooling zymotechnique of clostridium butyricum, comprises the following steps：
(1) Tube propagation, triangular flask deep drainpipe, seeding tank are carried out to clostridium butyricum successively and expands culture, obtained with this
Clostridium butyricum bacterium solution；Wherein Tube propagation is：By the clostridium butyricum bacterial strain in cryopreservation tube, the test tube with fluid nutrient medium is placed in
In, cultivate 48h；Test tube is placed in water-bath 10min in 80 DEG C of water afterwards, then accesses to the clostridium butyricum in test tube
It it is 37 DEG C in temperature in the seed culture medium of 500ml (loading amount 200ml), in culture 8h under anaerobic condition；Triangular flask deep layer
Cultivate and be：Clostridium butyricum after seed culture medium culture is accessed into the 5L triangles that liquid amount is 2L by 5% inoculum concentration
Continue to cultivate 8h in bottle；Seeding tank expands culture：Clostridium butyricum after triangular flask culture is put into by 5% inoculum concentration
Culture is enlarged in 50L seeding tanks；
(2) by glucose 2kg, corn flour 1.0kg, soy meal 4.0kg, dusty yeast 0.8kg, defoamer 0.1kg, adds life
Culture medium is made in drinking water, backward culture medium in put into NaHCO3, NaHCO3Input amount be until by the pH of culture medium
Adjust untill 7.0；Culture is based on afterwards to sterilize 30min under the conditions of 121 DEG C, is then put into the fermentation tank of 1000L, into
For basic zymotic fluid；Backward basal fermentation liquid in add protease, lipase, cellulase, zytase, dextranase,
Mannase, specific input amount are decided according to the actual requirements；Under conditions of 37 DEG C, 0.01Mpa tank pressures to fermentation tank
Interior material carries out the fermentation of 12h, and zymotic fluid A is made；
(3) low temperature sterilization is carried out to obtained zymotic fluid A in step 2)；Then with calcium bicarbonate 0.05kg, calcium chloride
The ratio of 0.0015kg, ammonium sulfate 0.1kg, magnesium sulfate 0.015kg, manganese sulfate 0.015kg add above-mentioned thing into zymotic fluid A
Material；Clostridium butyricum bacterium solution made from step 1) is added into zymotic fluid A in 1% ratio again afterwards；Backward culture medium in throw
Enter NaHCO3, NaHCO3Input amount to be adjusted until by the pH of culture medium untill 6.5；After 37 DEG C, 0.01Mpa tank pressures
Under conditions of in fermentation tank material carry out 24h fermentation, obtain zymotic fluid C；
Above-mentioned data are the optimal cases by being obtained after many experiments, and experiment is proved when pH is less than 6.5 or is higher than
When 6.5, the inactivation phenomenon of high degree, in the zymotic fluid finally obtained, the work of clostridium butyricum occurs in clostridium butyricum bacterium solution
Bacterium number is too low, and obtained feed and normal diet difference are very few；And when pH is in 6.5, and temperature is less than 37 DEG C, clostridium butyricum
Still it is difficult to maintain in the optimal state of activation；And when temperature is higher than 37 DEG C, substantial amounts of clostridium butyricum will appear from inactivating, system
The zymotic fluid viable count obtained, which even will also be less than, uses zymotic fluid made from common preparation method；So as to by the contrast to this
After experiment, determine, only when pH value is equal to 6.5, and fermentation temperature is equal to 37 DEG C, can cause the butyric acid in zymotic fluid
The viable count of clostridium reaches highest, reaches 20-25 × 108The viable count of CFU/ml, so as to become optimal case.
(4) zymotic fluid C is discharged, while into fermentation tank, supplement adds the zymotic fluid A of equivalent, ferments, with
This moves in circles；After the zymotic fluid C given off is carried out vacuum-concentrcted using vacuum decompressioning and concentrating tank afterwards, pass through spraying
Equipment is spray-dried to obtain finished product.
Further, as shown in Figs 1-4, the fermentation tank includes inner tank body 1, outer tank body 2, heating layer 12, heating tube
Part 4 and seal member 5, the inner tank body 1 are located in outer tank body 2, and have gap between two tank bodies, which is described
Heating layer 12；The heat pipes 4 are spiral to be located in inner tank body 1, and 4 one end of heat pipes is connected with the heating layer 12
Logical, the other end is connected with the temperature bath outside fermentation tank, and thermostatted water is pumped into heating by the temperature bath by the pump housing
In pipe fitting 4, while the water in heat pipes 4 is entered in heating layer 12, and heating layer 12 is filled completely, so as to fulfill to fermentation tank
Overall heating, the bulk temperature in fermentation tank is reached 37 DEG C；Further, the inlet pipe member 3 sets outer tank body 2 again
On, and be connected through two tank bodies with the chamber in inner tank body 1, which is with female metal tube.
Sealed to ensure to realize inlet pipe member, a seal member 5, the sealing are provided with the inlet pipe member
Component 5 includes first seal 51 and second seal 52, and the first seal 51 carries externally threaded truncated cone-shaped for one
Metal closures, therefore first seal 51 can be screwed into inlet pipe member 3, and 51 upper end of first seal is equipped with a sealing
Cap 511, which is equipped with a concave ring 512, and is equipped with a sealing ring 513 in concave ring 512, which is O shape rubbers
Glue sealing ring；The inner wall of 3 upper end of inlet pipe member be equipped with the sealing ring 513 it is matched sealing concave ring 313, should
The convex ribs 314 to raise up are equipped with sealing concave ring 313, it is described close after the first seal 51 is screwed into inlet pipe member 3
513 side wall of seal and the side wall of the sealing concave ring 313 fit closely, and the convex ribs 314 make sealing ring 513 that local change occur
Shape, thus sealing ring 513 elastic force effect under, fitted closely with convex ribs 314, gap will not be left, prevent completely liquid from
The junction of first seal 51 and inlet pipe member 3 is entered in inner tank body 1；Further, 52 sets of the second seal
It is located at outside inlet pipe member 3, and hydraulic layer 53 is equipped between second seal 52 and first seal 51, which is first
Gap between seal 51 and second seal 52, and two flow liquid openings 21, two flow liquids of person are provided with outer tank body 2
Opening 21 makes hydraulic layer 53 be connected with heating layer 12, so that heating layer 12 to be symmetrical arranged by the two flow liquid openings 21
Interior water can enter to hydraulic layer 53 by a flow liquid opening 21, then return to heating layer by another flow liquid opening 21
53。
Further, opened and closed to be realized to flow liquid opening 21, two closings are equipped with the heating layer 12
Part 61, two closure members 61 are respectively allocated at two flow liquid openings 21, i.e., each flow liquid opening 21 is realized by a seal 61
Sealing, the seal 61 is a metallic plate；In the second seal be equipped be used for promote the seal 61 to
The lower push rod 521 by activity, which is metallic rod；Specifically, activity is equipped with the side wall of the second seal 52
Chamber 529, the push rod 521 are located in movable chamber 529；The movable chamber bottom is set for opening, therefore push rod 521 can portion
Divide and stretch out movable chamber, so as to push closure member 61, and rubber piston 528 is installed with push rod 521, the rubber piston 528 and institute
The inner wall for stating movable chamber 529 fits closely, and the inner wall of movable chamber 529 is smooth setting, therefore liquid can not be from movable chamber 529
Interior outflow, avoids that leakage situation occurs；In order to strengthen leakproofness of the seal 61 to flow liquid opening 21, in the flow liquid opening 21
Periphery be equipped with two concave concave rings 219 from inside to outside, be equipped with the upper surface of the seal 61 two with it is described recessed
The matched leakage-proof ring 619 of ring 219, the leakage circle are O-shaped rubber ring, and closure member 61 closes flow liquid opening 21, and leakage-proof ring 619 is caught in
To concave ring 219, and leakage-proof ring 619 is fitted closely with 219 inner wall of concave ring；In order to keep closure member 61 to close flow liquid opening 21,
Two elastic components 62 are fixed with the bottom surface of the closure member 61, which is spring, and two elastic components 62 are installed in
In the inner tank body 1, so that closure member 61 keeps closing flow liquid opening 21 under the effect of 62 elastic force of elastic component.
Further, in order to ensure accuracy that closure member 61 closes flow liquid opening 21, in the inner wall of the outer tank body 2
A stop collar 22 is equipped with, which includes the first body 221 and the second body 222, first body 221
Cross-sectional diameter is identical with the diameter of closure member, i.e., the side wall of closure member 61 fits with 221 inner wall of the first body, and second
The cross-sectional diameter of body 222 is more than the cross-sectional diameter of the first body 221, and two bodies are seamlessly connected, therefore seal
61 by external force when pushing down on, and seal 61 is pushed into out of first body 221 in second body 222, seal 61 and the
The side wall of two bodies 222 has gap, therefore water is flowed into or from from the gap；Further in order to avoid elastic component 62 is sent out
Raw double swerve, is arranged with a setting of casing 71, upper end is arranged with casing 72 on one, the upper casing from 62 lower end of elastic component
72 are connected with the lower face of closure member 61, and setting of casing 71 and the outer wall of the inner tank body 1 are connected, and 71 part of setting of casing is arranged in
In upper casing 72, and one second bulge loop 721 is installed with the inner wall of the upper casing 72, the upper end of 71 outer wall of setting of casing
The first bulge loop 711 is installed with, second bulge loop 721 and the first bulge loop 711 mutually limit, so that upper casing 72 can not be under
It is separated from casing 71, it is ensured that the stability of device.
In order to enable the liquid circulation flowing in heat pipes 4, heating layer 12 and hydraulic layer 53, sets on the shell body
There is a water outlet pipe fitting 81 being connected with heating layer 12, the height higher than the second seal 52 of the water outlet pipe fitting 81, therefore
The liquid in hydraulic layer 53 can be kept to be full of；The discharge pipe being connected with the inner tank body is equipped with the bottom of fermentation tank
82；The thermometer 83 and PH meters 84 stretched in inner tank body is equipped with the side wall of the fermentation tank.
Embodiment 2
A kind of zonal cooling zymotechnique of clostridium butyricum, comprises the following steps：
(5) Tube propagation, triangular flask deep drainpipe, seeding tank are carried out to clostridium butyricum successively and expands culture, obtained with this
Clostridium butyricum bacterium solution；Wherein Tube propagation is：By the clostridium butyricum bacterial strain in cryopreservation tube, the test tube with fluid nutrient medium is placed in
In, cultivate 48h；Test tube is placed in water-bath 10min in 80 DEG C of water afterwards, then accesses to the clostridium butyricum in test tube
It it is 37 DEG C in temperature in the seed culture medium of 500ml (loading amount 200ml), in culture 8h under anaerobic condition；Triangular flask deep layer
Cultivate and be：Clostridium butyricum after seed culture medium culture is accessed into the 5L triangles that liquid amount is 2L by 5% inoculum concentration
Continue to cultivate 8h in bottle；Seeding tank expands culture：Clostridium butyricum after triangular flask culture is put into by 5% inoculum concentration
Culture is enlarged in 50L seeding tanks；
(6) by glucose 2kg, corn flour 1.0kg, soy meal 4.0kg, dusty yeast 0.8kg, defoamer 0.1kg, adds life
Culture medium is made in drinking water, backward culture medium in put into NaHCO3, NaHCO3Input amount be until by the pH of culture medium
Adjust untill 7.0；Culture is based on afterwards to sterilize 35min under the conditions of 128 DEG C, is then put into the fermentation tank of 1000L, into
For basic zymotic fluid；Backward basal fermentation liquid in add protease, lipase, cellulase, zytase, dextranase,
Mannase, specific input amount are decided according to the actual requirements；Under conditions of 37 DEG C, 0.01Mpa tank pressures to fermentation tank
Interior material carries out the fermentation of 24h, and zymotic fluid A is made；
(7) low temperature sterilization is carried out to obtained zymotic fluid A in step 2)；Then with calcium bicarbonate 0.05kg, calcium chloride
The ratio of 0.0015kg, ammonium sulfate 0.1kg, magnesium sulfate 0.015kg, manganese sulfate 0.015kg add above-mentioned thing into zymotic fluid A
Material；Clostridium butyricum bacterium solution made from step 1) is added into zymotic fluid A in 1% ratio again afterwards；Backward culture medium in throw
Enter NaHCO3, NaHCO3Input amount to be adjusted until by the pH of culture medium untill 6.5；After 37 DEG C, 0.01Mpa tank pressures
Under conditions of in fermentation tank material carry out 48h fermentation, obtain zymotic fluid C；
Above-mentioned data are the optimal cases by being obtained after many experiments, and experiment is proved when pH is less than 6.5 or is higher than
When 6.5, the inactivation phenomenon of high degree, in the zymotic fluid finally obtained, the work of clostridium butyricum occurs in clostridium butyricum bacterium solution
Bacterium number is too low, and obtained feed and normal diet difference are very few；And when pH is in 6.5, and temperature is less than 37 DEG C, clostridium butyricum
Still it is difficult to maintain in the optimal state of activation；And when temperature is higher than 37 DEG C, substantial amounts of clostridium butyricum will appear from inactivating, system
The zymotic fluid viable count obtained, which even will also be less than, uses zymotic fluid made from common preparation method；So as to by the contrast to this
After experiment, determine, only when pH value is equal to 6.5, and fermentation temperature is equal to 37 DEG C, can cause the butyric acid in zymotic fluid
The viable count of clostridium reaches highest, reaches 20-25 × 108The viable count of CFU/ml, so as to become optimal case.
(8) zymotic fluid C is discharged, while into fermentation tank, supplement adds the zymotic fluid A of equivalent, ferments, with
This moves in circles；After the zymotic fluid C given off is carried out vacuum-concentrcted using vacuum decompressioning and concentrating tank afterwards, pass through spraying
Equipment is spray-dried to obtain finished product.
Fermentation tank in the present embodiment is identical with the structure in embodiment 1, and so it will not be repeated.
Embodiment 3
A kind of zonal cooling zymotechnique of clostridium butyricum, comprises the following steps：
(9) Tube propagation, triangular flask deep drainpipe, seeding tank are carried out to clostridium butyricum successively and expands culture, obtained with this
Clostridium butyricum bacterium solution；Wherein Tube propagation is：By the clostridium butyricum bacterial strain in cryopreservation tube, the test tube with fluid nutrient medium is placed in
In, cultivate 48h；Test tube is placed in water-bath 10min in 80 DEG C of water afterwards, then accesses to the clostridium butyricum in test tube
It it is 37 DEG C in temperature in the seed culture medium of 500ml (loading amount 200ml), in culture 8h under anaerobic condition；Triangular flask deep layer
Cultivate and be：Clostridium butyricum after seed culture medium culture is accessed into the 5L triangles that liquid amount is 2L by 5% inoculum concentration
Continue to cultivate 8h in bottle；Seeding tank expands culture：Clostridium butyricum after triangular flask culture is put into by 5% inoculum concentration
Culture is enlarged in 50L seeding tanks；
(10) by glucose 2kg, corn flour 1.0kg, soy meal 4.0kg, 0.8 kg of dusty yeast, defoamer 0.1kg, adds life
Culture medium is made in drinking water living, backward culture medium in put into NaHCO3, NaHCO3Input amount be until by culture medium
PH is adjusted untill 7.0；Culture is based on afterwards to sterilize 35min under the conditions of 125 DEG C, is then put into the fermentation tank of 1000L, into
For basic zymotic fluid；Backward basal fermentation liquid in add protease, lipase, cellulase, zytase, dextranase,
Mannase, specific input amount are decided according to the actual requirements；Under conditions of 37 DEG C, 0.01Mpa tank pressures to fermentation tank
Interior material carries out the fermentation of 18h, and zymotic fluid A is made；
(11) low temperature sterilization is carried out to obtained zymotic fluid A in step 2)；Then with calcium bicarbonate 0.05kg, calcium chloride
0.0015kg, ammonium sulfate 0.1kg, the ratio of magnesium sulfate 0.015 kg, manganese sulfate 0.015kg add above-mentioned thing into zymotic fluid A
Material；Clostridium butyricum bacterium solution made from step 1) is added into zymotic fluid A in 1% ratio again afterwards；Backward culture medium in throw
Enter NaHCO3, NaHCO3Input amount to be adjusted until by the pH of culture medium untill 6.5；After 37 DEG C, 0.01Mpa tank pressures
Under conditions of in fermentation tank material carry out 30h fermentation, obtain zymotic fluid C；
Above-mentioned data are the optimal cases by being obtained after many experiments, and experiment is proved when pH is less than 6.5 or is higher than
When 6.5, the inactivation phenomenon of high degree, in the zymotic fluid finally obtained, the work of clostridium butyricum occurs in clostridium butyricum bacterium solution
Bacterium number is too low, and obtained feed and normal diet difference are very few；And when pH is in 6.5, and temperature is less than 37 DEG C, clostridium butyricum
Still it is difficult to maintain in the optimal state of activation；And when temperature is higher than 37 DEG C, substantial amounts of clostridium butyricum will appear from inactivating, system
The zymotic fluid viable count obtained, which even will also be less than, uses zymotic fluid made from common preparation method；So as to by the contrast to this
After experiment, determine, only when pH value is equal to 6.5, and fermentation temperature is equal to 37 DEG C, can cause the butyric acid in zymotic fluid
The viable count of clostridium reaches highest, reaches 20-25 × 108The viable count of CFU/ml, so as to become optimal case.
(12) zymotic fluid C is discharged, while into fermentation tank, supplement adds the zymotic fluid A of equivalent, ferments, with
This moves in circles；After the zymotic fluid C given off is carried out vacuum-concentrcted using vacuum decompressioning and concentrating tank afterwards, pass through spray
Mist equipment is spray-dried to obtain finished product.
Fermentation tank in the present embodiment is identical with the fermentation jar structure in embodiment 1, and so it will not be repeated.
Table 1：The clostridium butyricum product bacterium number and the bacterium of the product obtained by normal fermentation that this preparation process is fermented
Number comparison sheet
| Zymotechnique | Regular fermentation process | Zymotechnique of the present invention |
| Experiment one | 2×108CFU/ml | 22×108CFU/ml |
| Experiment two | 1.8×108CFU/ml | 21×108CFU/ml |
| Experiment three | 2.5×108CFU/ml | 25×108CFU/ml |
| Experiment four | 3.0×108CFU/ml | 23×108CFU/ml |
| Experiment five | 2.6×108CFU/ml | 21×108CFU/ml |
Result of the test shows：This preparation process can effectively improve the viable count of clostridium butyricum product after fermentation.
Test report：
Experiment purpose：Effect of this product to the growth promotion and adjustment intestinal flora of weanling pig
Experimental period：1 day~November 11 October in 2015
Experimental method：The piglet 192 of 21 ages in days wean is chosen, is divided into 4 treatment groups, each handles 4 repetitions, each
12 are repeated, blank control group feeding basal diet, experiment 2-4 groups add 100,200 and 300mg/ in basal diet respectively
(i.e. clostridium butyricum content is respectively 1.0 × 10 to the clostridium butyricum preparation of kg8、2.0×108、3.0×108Cfu/kg feeds), examination
Test the phase 21 days.During the experiment observation each group swinery feeding and drinking-water situation, record feed consumption rate, the weight of 21 days after measure wean,
Calculate average daily gain, average day feeding and feed-weight ratio；Collect excrement sample during off-test, measure wherein Escherichia coli, lactic acid
Bacillus and the quantity of Bifidobacterium.Experimental result：Such as following table.
Table 2：Influence of the clostridium butyricum to Growth Performance of Weaning Piglets
Note：The different letters of colleague's mark represent significant difference.
Result of the test shows：Clostridium butyricum produced by the invention can significantly improve being averaged for weanling pig and increase day by day and be averaged
Daily ingestion amount, reduces feed-weight ratio, reduces the quantity of Escherichia coli in excrement, on the quantity of lactic acid bacteria without influence is influenced, significantly carries
The breeding of high Bifidobacterium.
Obviously, described embodiment is only the part of the embodiment of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without making creative work
Embodiment, should all belong to the scope of protection of the invention.
Claims (10)
1. a kind of zonal cooling zymotechnique of clostridium butyricum, comprises the following steps：
(1) Tube propagation, triangular flask deep drainpipe, seeding tank are carried out to clostridium butyricum successively and expands culture, butyric acid is obtained with this
Clostridium bacterium solution；
(2) culture medium is prepared, the pH of culture medium is adjusted to 7.0 afterwards, 30-35min is sterilized under the conditions of 121-128 DEG C, so
Put into afterwards in fermentation tank, become basal fermentation liquid；Backward basal fermentation liquid in add complex enzyme formulation, carry out the hair of 12-24h
Ferment, is made zymotic fluid A；
(3) low temperature sterilization is carried out to obtained zymotic fluid A in step 2), adds step 1) system into zymotic fluid A in 1% ratio
The clostridium butyricum bacterium solution obtained, and the fermentation of 24-48h is carried out, obtain zymotic fluid C；
(4) zymotic fluid C is discharged, while into fermentation tank, supplement adds the zymotic fluid A of equivalent, ferments, is followed with this
Ring is reciprocal；
(5) by after obtained zymotic fluid C progress vacuum-concentrcted in step 3), it is spray-dried to obtain by spraying apparatus
Finished product.
2. the zonal cooling zymotechnique of clostridium butyricum according to claim 1, it is characterised in that：The culture medium is by Portugal
2 parts of grape sugar, 1.0 parts of corn flour, 4.0 parts of soy meal, 0.8 part of dusty yeast, 0.1 part of defoamer plus Drinking Water are formulated.
3. the zonal cooling zymotechnique of clostridium butyricum according to claim 1, it is characterised in that：The complex enzyme formulation
For the one or more in protease, lipase, cellulase, zytase, dextranase, mannase.
4. the zonal cooling zymotechnique of clostridium butyricum according to claim 1, it is characterised in that：In the step 3),
Before clostridium butyricum bacterium solution is launched, calcium bicarbonate, calcium chloride, ammonium sulfate, magnesium sulfate, sulfuric acid are added into zymotic fluid A in advance
Manganese.
5. the zonal cooling zymotechnique of clostridium butyricum according to claim 4, it is characterised in that：Count in parts by weight,
The calcium bicarbonate, calcium chloride, ammonium sulfate, magnesium sulfate, the injected volume of manganese sulfate are 0.05 part of calcium bicarbonate, calcium chloride 0.0015
Part, 0.1 part of ammonium sulfate, 0.015 part of magnesium sulfate, 0.015 part of manganese sulfate.
6. the zonal cooling zymotechnique of clostridium butyricum according to claim 1, it is characterised in that：The hair of the step 2)
In ferment link the temperature of fermentation tank be 37 DEG C, tank pressure be 0.01Mpa.
7. the zonal cooling zymotechnique of clostridium butyricum according to claim 1, it is characterised in that：The hair of the step 3)
In ferment link the temperature of fermentation tank be 37 DEG C, tank pressure be 0.01Mpa, PH is 6.5 in tank.
A kind of 8. zonal cooling zymotechnique of clostridium butyricum according to claim 1, it is characterised in that：The fermentation tank
Including inner tank body (1), outer tank body (2), inlet pipe member (3), the heating layer (12) between inner tank body (1) and outer tank body (2), set
In the heat pipes (4) in inner tank body (1) and with inlet pipe member (3) matched seal member (5).
A kind of 9. zonal cooling zymotechnique of clostridium butyricum according to claim 8, it is characterised in that：The heating tube
Part (4) is connected with the heating layer (12)；The seal member (5) includes first seal (51) and is sheathed on the first sealing
Second seal (52) on part (51), has hydraulic layer (53) between the first seal (51) and second seal (52),
The hydraulic layer (53) is connected by the flow liquid opening (21) in outer tank body (2) with heating layer (12).
A kind of 10. zonal cooling zymotechnique of clostridium butyricum according to claim 9, it is characterised in that：The heating
The elasticity being connected with the matched closure member of the flow liquid opening (21) (61) and with the closure member (61) is equipped with layer (12)
Part (62), the elastic component (62) are connected with the inner tank body (1)；The second seal (52) is equipped with described for promoting
The push rod (521) of closure member (61) activity downwards.
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