CN104946565A - Solid-state continuous fermentation method of Bacillus subtilis - Google Patents
Solid-state continuous fermentation method of Bacillus subtilis Download PDFInfo
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- CN104946565A CN104946565A CN201510378217.0A CN201510378217A CN104946565A CN 104946565 A CN104946565 A CN 104946565A CN 201510378217 A CN201510378217 A CN 201510378217A CN 104946565 A CN104946565 A CN 104946565A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a solid-state continuous fermentation method of Bacillus subtilis, which comprises the following steps: 1) feeding a solid-state fermentation material through a feeding bin, and carrying out high-temperature sterilization in a continuous sterilization device; 2) in a closed purified room, inoculating activated Bacillus subtilis into the sterilized solid-state fermentation material; 3) in the closed purified room, spreading the material obtained in the step 2) on the initial position of a continuous fermentation bed operating at uniform speed, and fermenting for 22-26 hours, wherein the humidity in the purified room is controlled at 80-95%, the purified room is ventilated through a fresh air system, and the fermentation temperature is 37-42 DEG C; and 4) drying the obtained fermentation product in the closed purified room to obtain the dried fermentation product. The method can overcome the defects of low effective viable count and high infectious microbe quantity in the common fermentation process, thereby achieving the goals of enhancing the fermentation viable count, ensuring the product quality and enhancing the use effect.
Description
Technical field
The present invention relates to the zymotechnique of a kind of microorganism---the solid-state continuous fermentation technique of subtilis.
Background technology
Subtilis (Bacillus subtilis), it is a kind of aerobic Gram-positive genus bacillus, it is one of probiotic bacterium of the Ministry of Agriculture of China official approval use, have that anti-microbial property is good, satisfactory stability, microbiotic to be had no drug resistance, to distinguishing features such as animal nontoxicitys, be subject to the favor of numerous investigators, and be applied to the earliest on aquatic products and achieve significant effect.
1872, Cohn definite designation " subtilis " (Bacillus subtilis).It is a kind of aerobic, sporiferous rod-shaped bacterium, the multiple antibiotic such as tens kinds of enzymes such as proteolytic enzyme, amylase, cellulase, dextranase and subtilyne, polymyxin, nystatin can be produced, therefore there is the effect well promoting animal digestion, improve efficiency of feed utilization and suppression harmful intestinal tract bacteria.In addition, subtilis is as typical sporeformer, can acidproof, salt tolerant, high temperature resistant, also certain tolerance is had to common antibiotics, therefore can stomach juice-resistant, bile tolerance, high temperature in resistance to pelletization and extruding, and can, with common antibiotics as conbined usage such as kitasamycin, Zinc-bacitracin, colistin sulfates, be the good selection as fodder additives.
But the result of use difference of subtilis on livestock and poultry is comparatively large, and some product is effective, and some action effect is not obvious.Its major cause may be the difference of fermentation process.Because subtilis is an aerobic bacteria, its reason worked mainly is that the product produced in fermenting process is as enzyme, antibacterial peptide etc., if adopt liquid fermenting mode subtilis, because the protein such as enzyme, antibacterial peptide can be water-soluble, can along with discharge of wastewater during liquid fermenting, both contaminate environment, again reduces the action effect of subtilis.Therefore current many domestic manufacturers adopt common pellet type solid-state fermentation process to produce subtilis, and this method exists following problem:
1. common solid-state fermenting process is simple, and sterilizing is discontinuous, is not suitable for scale operation.
2. in common solid-state fermenting process, quality control is coarse, and cause miscellaneous bacteria content in product higher, intestinal bacteria and Salmonellas easily exceed standard, and are difficult to conform to quality requirements.
3. in common solid-state fermenting process because the conditions such as fermenting process temperature, humidity, pH value can not strictly control, cause leavened prod content low.
4. common solid-state fermenting process sterilization time is long, easily causes product moisture too high, thus affects discharging.
Due to the existence of the problems referred to above, have impact on the application of subtilis in livestock and poultry further.
Summary of the invention
The technical problem to be solved in the present invention is to provide the new process for fermenting of a kind of subtilis--the solid-state continuous fermentation method of subtilis, overcome the problem that living bacteria count is not high, miscellaneous bacteria is many occurred in common fermentation method, fermentation viable count is improved to reach, ensure quality product, strengthen the object of result of use.
In order to solve the problems of the technologies described above, the invention provides the solid-state continuous fermentation method of a kind of subtilis, comprising the following steps:
1), by solid fermented material by feeding warehouse charging, in continuous sterilisation equipment, high-temperature sterilization is carried out;
Described solid fermented material is obtained by mixing according to the part by weight of 70 ~ 80%, 15 ~ 25%, 3 ~ 4%, 1% by dregs of beans, wheat bran, fish meal, calcium carbonate;
2), in airtight purification room, in step 1) subtilis after access activation in solid fermented material after the sterilizing of gained, stir;
Inoculum size is: subtilis after the activation of every 100g solid fermented material adapted 8 ~ 10g;
3), in airtight purification room, by step 2) material of gained is sprinkling upon continuously fermenting of at the uniform velocity operating and bed (is specially the initial position of the bed that continuously ferments) carries out fermentation 22 ~ 26 hours (being preferably 24h); The bed that continuously ferments is made up of 5 ~ 6 fermentation beds, and the drop of adjacent fermentation bed is 0.2-0.3 rice, can fall in a rear fermentation bed, thus realize the automatic turning of material after material is transported to the end of previous fermentation bed; The humidity controlled in purification room is 80-95% (being such as 90%); Ventilated to purification room by VMC, leavening temperature controls 37 ~ 42 DEG C (being preferably 37 ~ 40 DEG C);
4), step 3) tunning of gained in airtight purification room in 65 ~ 80 DEG C of dryings 4 ~ 12 hours (being such as 80 DEG C of dryings 10 hours); Obtain drying treatment secondary fermentation product (product as final).
Remarks illustrate: in this step 4) in, step 3) tunning of gained is positioned at the afterbody of the bed that continuously ferments; Tunning directly carries out drying in purification room, both can drying products, can realize again carrying out disinfection sterilization to continuously ferment bed and purification room, thus play at one stroke two energy-conserving actions.
Improvement as the solid-state continuous fermentation method of subtilis of the present invention: described step 3) in, within every 3 ~ 4 hours, ventilate once, ventilated at every turn and changed the air quantity of whole purification room 10 ~ 20% volume.
Solid-state continuous fermentation further improvements in methods as subtilis of the present invention:
Described step 1) high-temperature sterilization be: in 100 ~ 121 DEG C of sterilizings 20 minutes (being preferably 120 ~ 121 DEG C of sterilizings 20 minutes).
In the present invention: step 2) adopt and spray into the inoculation method of formula, thus realize in step 1) bacterial classification after the sterilizing of gained in solid fermented material after access activation; Realize stirring in mixer;
In step 3 of the present invention) in, material moves successively continuously fermenting on bed, can fall in second fermentation bed, until fall into last fermentation bed after material is transported to the end of first fermentation bed.
The present invention has following features:
1, fermentation and drying process are all carried out in airtight purification room, decrease the pollution in fermenting process;
2, fermenting process temperature, humidity have carried out strict control, and fermentation efficiency is high, and fermentation time is short;
3, the high temperature in drying process both can play the effect of dried material, can play the effect of a sterilizing of continuously fermenting again;
4, the bed that continuously ferments adopts tape transport material, material can be made in purification room to enter outlet from initiating terminal, reduce artificial conveyance cost.
5, the fermentation substrate (solid fermented material) that this fermentation adopts is mainly dregs of beans, avoids the density caused owing to adopting wheat bran in a large number in other solid fermentating mode too small, the situation that leavened prod volume is excessive.
Fermentation substrate medium density of the present invention, and protein content is higher, is conducive to subtilis and produces antibacterial peptide and enzyme during the fermentation, in addition, the nutritive ingredient composition of fermentation substrate of the present invention forms similar with animal feedstuff ingredients, can heavy dose ofly in feed add.
6, effectively prevent the pollution of miscellaneous bacteria.
7, adopt zymotechnique of the present invention, effectively can realize the uniform fermentation of fermentation substrate, shorten fermentation time.
The fermentation of bacillus subtilis amount of 8, being fermented by this processing method can reach 400 × 10
8cFU/g, fermentation efficiency is far above ordinary solid leavened prod.
In sum, the invention provides one and to continuously ferment a solid-state fermentation technology, effectively reduce living contaminants chance, and improve fermentation viable count, thus ensure that product addition is few, result of use good.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is process flow sheet of the present invention.
Embodiment
The solid-state continuous fermentation method of embodiment 1, a kind of subtilis, carry out following steps successively:
1), by solid fermented material by feeding warehouse charging, high-temperature sterilization (in 120-121 DEG C of sterilizing 20 minutes) is carried out at continuous sterilisation equipment (being such as continuous steaming pot for cooking rice);
Solid fermented material is obtained by mixing according to the part by weight of 80%:15%:4%:1% by dregs of beans, wheat bran, fish meal, calcium carbonate.
2), in airtight purification room, in step 1) subtilis after access activation in solid fermented material after the sterilizing of gained, stir;
Inoculum size is: subtilis after the activation of every 100g solid fermented material adapted 8g;
Subtilis selects the subtilis being numbered CGMCC9383 at China Microbiological kind culture presevation management committee's common micro-organisms center;
Remarks illustrate: have in invention that the subtilis of the above-mentioned CGMCC9383 of being numbered is 2014106622732 at application number " composite fungus agent and the continuous fermentation method of complex microorganism adopted " and clearly inform.
Activation is: by above-mentioned subtilis in beef-protein medium 37 DEG C carry out activation treatment, must activate after subtilis.
3), in airtight purification room, by step 2) material of gained is sprinkling upon on the bed that continuously ferments that at the uniform velocity operates and carries out fermentation 24h; Fermentation bed of continuously fermenting has 6, and have certain drop (drop of adjacent fermentation bed is 0.2-0.3 rice), material can be fallen in second fermentation bed after being transported to the end of first fermentation, realize the automatic turning of material, move successively, until to last fermentation bed, the humidity controlled in purification room is 90%; Ventilated by VMC, within every 4 hours, ventilate once, ventilated at every turn and changed the air quantity of whole purification room 20% volume, keep leavening temperature 37-40 DEG C (that is, controlling purification room temperature is 37-40 DEG C).
4), by step 3) tunning of gained in airtight purification room in 80 DEG C of dryings 10 hours; Obtain drying treatment secondary fermentation product (product as final).
The survey report of the bacillus subtilis formulation that this technique obtains is as described in Table 1:
Table 1: the subtilis quality measurements that different process is produced
The subtilis that comparative example 1, regular fermentation process are produced, its processing step is specific as follows:
1), by solid fermented material high-temperature sterilization (in 120 ~ 121 DEG C of sterilizings 20 minutes);
The component of this solid state fermentation thing and content are: wheat bran 64%, analysis for soybean powder 18%, Semen Maydis powder 15%, kH
2pO
42%, CaCl
21%.
2), in step 1) subtilis after access activation in solid fermented material after the sterilizing of gained, stir; Inoculum size is: subtilis after the activation of every 100g solid fermented material adapted 10g;
After activation, subtilis is with embodiment 1.
3), by step 2) material is laid on pallet, and ferment, leavening temperature is at 37-40 DEG C, and fermentation time is 48 hours.Upset or the stirring of material is manually carried out in fermenting process.
4), step 3) tunning of gained in fluidized-bed in 80 DEG C of dryings 10 hours; Obtain product.
Comparative example 2-1, the solid state fermentation thing made into by the solid fermented material in embodiment 1 in comparative example 1, that is, the component of this solid state fermentation thing and content are: wheat bran 64%, analysis for soybean powder 18%, Semen Maydis powder 15%, kH
2pO
42%, CaCl
21%.
All the other are equal to embodiment 1.
Comparative example 2-2, the solid fermented material in embodiment 1 is made into as follows:
Solid fermented material is obtained by mixing according to the part by weight of 15%, 80%, 4%, 1% by dregs of beans, wheat bran, fish meal, calcium carbonate.
All the other are equal to embodiment 1.
Comparative example 2-3, the solid fermented material in embodiment 1 is made into as follows:
Solid fermented material is obtained by mixing according to the part by weight of 16:83:1 by dregs of beans, wheat bran, calcium carbonate.
All the other are equal to embodiment 1.
Comparative example 3-1, by the step 3 of embodiment 1) make the step 3 of same comparative example 1 into), all the other are equal to embodiment 1.
Comparative example 3-2, by the step 2 of embodiment 1) ~ 4) make the step 2 of same comparative example 1 into) ~ 4), step 1) with embodiment 1.
The quality measurements of the subtilis of above-mentioned comparative example 2-1 ~ comparative example 3-2 gained is as shown in table 2.
The subtilis object bacteria that table 2 different process is produced and miscellaneous bacteria number measurement result
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (4)
1. the solid-state continuous fermentation method of subtilis, is characterized in that comprising the following steps:
1), by solid fermented material by feeding warehouse charging, in continuous sterilisation equipment, high-temperature sterilization is carried out;
Described solid fermented material is obtained by mixing according to the part by weight of 70 ~ 80%, 15 ~ 25%, 3 ~ 4%, 1% by dregs of beans, wheat bran, fish meal, calcium carbonate;
2), in airtight purification room, in step 1) subtilis after access activation in solid fermented material after the sterilizing of gained, stir;
Inoculum size is: subtilis after the activation of every 100g solid fermented material adapted 8 ~ 10g;
3), in airtight purification room, by step 2) material of gained is sprinkling upon on the bed that continuously ferments that at the uniform velocity operates and carries out fermentation 22 ~ 26 hours; The bed that continuously ferments is made up of 5 ~ 6 fermentation beds, and the drop of adjacent fermentation bed is 0.2-0.3 rice, can fall in a rear fermentation bed, thus realize the automatic turning of material after material is transported to the end of previous fermentation bed; The humidity controlled in purification room is 80-95%; Ventilated to purification room by VMC, leavening temperature controls at 37 ~ 42 DEG C;
4), step 3) tunning of gained in airtight purification room in 65 ~ 80 DEG C of dryings 4 ~ 12 hours; Obtain drying treatment secondary fermentation product.
2. the solid-state continuous fermentation method of subtilis according to claim 1, is characterized in that: described step 3) in, within every 3 ~ 4 hours, ventilate once, ventilated at every turn and changed the air quantity of whole purification room 10 ~ 20% volume.
3. the solid-state continuous fermentation method of subtilis according to claim 2, is characterized in that:
Described step 1) high-temperature sterilization be: in 100 ~ 121 DEG C of sterilizings 20 minutes.
4., according to the solid-state continuous fermentation method of the arbitrary described subtilis of claims 1 to 3, it is characterized in that:
Solid fermented material is obtained by mixing according to the part by weight of 80%, 15%, 4%, 1% by dregs of beans, wheat bran, fish meal, calcium carbonate.
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Cited By (3)
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| CN108220208A (en) * | 2018-03-16 | 2018-06-29 | 青岛农业大学 | A kind of zinc-rich bacterial strain and its application |
| CN111733117A (en) * | 2020-08-17 | 2020-10-02 | 中国科学院烟台海岸带研究所 | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof |
| CN115287212A (en) * | 2022-04-14 | 2022-11-04 | 国家林业和草原局竹子研究开发中心 | Salt-tolerant growth-promoting bacterium ACP81 and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111733117A (en) * | 2020-08-17 | 2020-10-02 | 中国科学院烟台海岸带研究所 | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof |
| CN111733117B (en) * | 2020-08-17 | 2020-12-08 | 中国科学院烟台海岸带研究所 | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof |
| CN115287212A (en) * | 2022-04-14 | 2022-11-04 | 国家林业和草原局竹子研究开发中心 | Salt-tolerant growth-promoting bacterium ACP81 and application thereof |
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