CN114410539B - Preparation method of clostridium butyricum solid fermentation material and fermentation material - Google Patents
Preparation method of clostridium butyricum solid fermentation material and fermentation material Download PDFInfo
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- 241000193171 Clostridium butyricum Species 0.000 title claims abstract description 157
- 238000000855 fermentation Methods 0.000 title claims abstract description 136
- 230000004151 fermentation Effects 0.000 title claims abstract description 134
- 239000000463 material Substances 0.000 title claims abstract description 61
- 239000007787 solid Substances 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title abstract description 33
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 48
- 239000002245 particle Substances 0.000 claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 22
- 238000011081 inoculation Methods 0.000 claims abstract description 18
- 238000007789 sealing Methods 0.000 claims abstract description 18
- 230000001580 bacterial effect Effects 0.000 claims abstract description 17
- 238000005507 spraying Methods 0.000 claims abstract description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 26
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 24
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 21
- 241000209094 Oryza Species 0.000 claims description 19
- 235000007164 Oryza sativa Nutrition 0.000 claims description 19
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 13
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 13
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 13
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 13
- 239000011790 ferrous sulphate Substances 0.000 claims description 13
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 13
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 13
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 13
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 13
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 12
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 12
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 12
- 239000008187 granular material Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 235000019764 Soybean Meal Nutrition 0.000 claims description 9
- 239000007789 gas Substances 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
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- 235000015097 nutrients Nutrition 0.000 claims description 9
- 239000004455 soybean meal Substances 0.000 claims description 9
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- 229910052760 oxygen Inorganic materials 0.000 abstract description 20
- 239000002207 metabolite Substances 0.000 abstract description 9
- 230000009604 anaerobic growth Effects 0.000 abstract description 3
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 34
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
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- 239000006041 probiotic Substances 0.000 description 3
- 235000018291 probiotics Nutrition 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
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- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 1
- 101000765308 Aspergillus niger N-(5'-phosphoribosyl)anthranilate isomerase Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000607764 Shigella dysenteriae Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
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- 150000001413 amino acids Chemical class 0.000 description 1
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- 229960001305 cysteine hydrochloride Drugs 0.000 description 1
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- 239000001257 hydrogen Substances 0.000 description 1
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- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
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- 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
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- 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
- C12N1/14—Fungi; Culture media therefor
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention discloses a preparation method of clostridium butyricum solid fermentation material and the fermentation material, wherein the method comprises the following steps: the clostridium butyricum and aspergillus niger are subjected to sectional temperature control culture under a sealed condition to obtain clostridium butyricum solid fermentation material, which comprises the following steps: inoculating clostridium butyricum into a culture medium by utilizing clostridium butyricum spore fermentation liquor; granulating the clostridium butyricum obtained by inoculation to obtain thallus particles; spraying Aspergillus niger fermentation liquor on the thallus particles to inoculate Aspergillus niger to obtain inoculated particles; and sealing the inoculated particles and carrying out sectional temperature control culture to obtain clostridium butyricum solid fermentation material. According to the technical scheme disclosed by the invention, the aspergillus niger consumes oxygen in the clostridium butyricum culture environment to obtain the anaerobic growth environment of clostridium butyricum, so that the bacterial count of clostridium butyricum in a fermentation material can be effectively increased, and the metabolite content of clostridium butyricum can be effectively increased.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a preparation method of clostridium butyricum solid fermentation material and the fermentation material.
Background
Clostridium butyricum (Clostridium butyricum), clostridium butyricum, is a G + The main metabolites of the anaerobic microorganism are butyric acid, acetic acid, hydrogen, carbon dioxide and a small amount of ethanol. Research shows that the probiotics clostridium butyricum can not only be symbiotic with other probiotics in the intestinal tract and promote the growth and proliferation of the probiotics, but also inhibit the growth of intestinal pathogens such as staphylococcus, pathogenic escherichia coli, shigella dysenteriae and the like, reduce the accumulation of harmful substances (amines, indole and the like) secreted by the pathogens in the intestinal tract and regulate the microecological balance of the intestinal tract.
In the prior art, clostridium butyricum preparation is mainly prepared by adopting a liquid fermentation mode, and the preparation cost is higher by adopting the mode, so that the price of the clostridium butyricum preparation is higher. In fungus fermentation technology, a solid fermentation mode is adopted besides a liquid fermentation mode, however, the anaerobic characteristic of clostridium butyricum causes slow development of the solid fermentation technology.
Disclosure of Invention
In order to solve at least one technical problem in the prior art, the embodiment of the invention provides a preparation method of clostridium butyricum solid fermentation material and the fermentation material. The technical proposal is as follows:
in a first aspect, a method for preparing clostridium butyricum solid fermentation material is provided, comprising:
the clostridium butyricum and the aspergillus niger are subjected to sectional temperature control culture under a sealed condition to obtain clostridium butyricum solid fermentation material,
the sectional temperature control culture at least comprises two stages:
the first temperature-controlled culture stage, the temperature is 33-36 ℃,
the second temperature-controlled culture stage is at 36-39 deg.c.
Further, the step of culturing clostridium butyricum and aspergillus niger together under the sealed condition in a sectional temperature control way comprises the following steps:
inoculating clostridium butyricum into a culture medium by utilizing clostridium butyricum spore fermentation liquor;
granulating the clostridium butyricum obtained by inoculation to obtain thallus particles;
spraying Aspergillus niger fermentation liquor on the thallus particles to inoculate Aspergillus niger to obtain inoculated particles;
and sealing the inoculated particles and carrying out sectional temperature control culture to obtain clostridium butyricum solid fermentation material.
Further, the medium comprises: wheat and/or rice, soybean meal, and inorganic salt nutritional agent.
Further, the culture medium comprises the following components in percentage by mass:
wheat, 50% -60%;
rice, 20% -30%;
6% -26% of bean pulp;
2-4% of inorganic salt nutritional agent.
Further, the inorganic salt nutritional agent comprises a mixture of any one or more of the following: ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, calcium carbonate, ferrous sulfate and sodium bicarbonate.
Further, the inorganic salt nutritional agent comprises the following components in percentage by mass:
ammonium sulfate, 0.6% -1.0%;
anhydrous dipotassium hydrogen phosphate, 0.1% -0.2%;
anhydrous magnesium sulfate, 0.2% -0.5%;
0.5 to 0.8 percent of calcium carbonate;
ferrous sulfate 0.1-0.5%;
sodium bicarbonate 0.5-1.5%.
Further, the mass ratio of ingredients of the culture medium to water is 1 (0.8-0.9).
Further, the sealing the seed particles comprises:
and filling the inoculation particles into a plastic bag of a self-discharging valve, and sealing the bag body to discharge the gas in the bag.
Further, the granulating the inoculated clostridium butyricum to obtain thallus granules, which comprises the following steps:
the inoculated clostridium butyricum is made into cylindrical granules with the diameter of 8mm-12mm and the length of 20mm-30mm, and the temperature of the bacterial granules after granulation is 30-45 ℃.
In a second aspect, there is provided a clostridium butyricum solid fermentation material prepared by the preparation method disclosed in any one of the first aspects, the clostridium butyricum of the fermentation material being (1-2) x 10 in number 8 CFU/g, butyric acid content of 1-5g/Kg and acetic acid content of 0.3-1g/Kg.
The technical scheme provided by the embodiment of the invention has the beneficial effects that at least:
1. according to the technical scheme disclosed by the invention, the aspergillus niger consumes oxygen in the clostridium butyricum culture environment to obtain the anaerobic growth environment of clostridium butyricum, so that the bacterial count of clostridium butyricum in a fermentation material can be effectively improved, and the metabolite content of clostridium butyricum can be effectively improved;
2. according to the technical scheme disclosed by the invention, the clostridium butyricum inoculation culture medium contains nutrients such as a carbon source, a nitrogen source and inorganic salts required by clostridium butyricum, the viscosity of the culture medium is ensured, the pore oxygen content of the culture medium is reduced, the culture medium can further consume oxygen, and an anaerobic environment is provided for the proliferation and metabolism of clostridium butyricum.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for preparing clostridium butyricum solid fermentation material disclosed in the embodiment of the invention.
Detailed Description
Any numerical value or range of numerical values disclosed in the embodiments of the present invention are not limited to the precise range or value, but should be understood to include values close to the numerical value or range of numerical values. In the numerical ranges, one or more new numerical ranges may be combined between the endpoints, between the endpoints and the individual values of the ranges, and between the individual values, and the numerical ranges should be construed as being disclosed in the present invention.
The clostridium butyricum preparation prepared by the existing liquid fermentation mode as described in the background technology only contains a small amount of metabolites or even no metabolites, so that the application value of clostridium butyricum is greatly reduced, however, the clostridium butyricum can only proliferate and metabolize in an anaerobic environment, and the difficulty of adopting a solid fermentation mode is high. Therefore, the invention provides a technical scheme for preparing the clostridium butyricum solid fermentation material, which not only ensures that the prepared clostridium butyricum fermentation material has higher bacterial count and contains higher content of metabolites, but also realizes the preparation of the clostridium butyricum fermentation material by adopting a solid fermentation method in an anaerobic environment.
In a first aspect, the invention discloses a method for preparing clostridium butyricum solid fermentation material, which comprises the following steps:
the clostridium butyricum and aspergillus niger are subjected to sectional temperature control culture under a sealed condition to obtain clostridium butyricum solid fermentation material, wherein the sectional temperature control culture comprises at least two stages: the first temperature-controlled culture stage is at 33-36 deg.c and the second temperature-controlled culture stage is at 36-39 deg.c.
The clostridium butyricum spore fermentation broth can be prepared by anaerobic fermentation of clostridium butyricum strains conventionally used in the field, and preferably is prepared by acid-resistant and oxygen-resistant domestication and has high activity for purifying clostridium butyricum strains. The Aspergillus niger fermentation broth can be fermentation broth formed by culturing Aspergillus niger which is conventionally used in the field, preferably fermentation broth formed by culturing Aspergillus niger strains which are screened by amylase, saccharifying enzyme and protease combined enzyme activity.
In the first temperature control culture stage, the aspergillus niger can grow faster, consume oxygen in the bag and generate hypha, and compound enzymes such as amylase, saccharifying enzyme and protease are generated to soften particles. In the second temperature control culture stage, the growth of Aspergillus niger is not facilitated, but the enzymolysis reaction is facilitated, so that the granular material is converted into better-utilized soluble sugar, peptide and amino acid, and the method is more suitable for the full growth of clostridium butyricum. Thus, the aspergillus niger consumes oxygen in the sealed environment before the clostridium butyricum sufficiently grows, thereby providing an anaerobic environment for the clostridium butyricum. It should be noted that the time of the first temperature-controlled culturing stage is shorter than that of the second temperature-controlled culturing stage, so that clostridium butyricum can have enough time to grow and reproduce.
It should be noted that: the temperature of the first temperature-controlled cultivation stage and the temperature of the second temperature-controlled cultivation stage may both be 36 ℃, but more preferably the temperature of the second temperature-controlled stage is 2 ℃ to 6 ℃ higher than the temperature of the first temperature-controlled stage.
The step of culturing clostridium butyricum and aspergillus niger under sealed conditions in a sectional temperature-controlled manner may be to introduce clostridium butyricum and aspergillus niger into a culture medium in a sectional temperature-controlled manner, but the above method may lead to the aspergillus niger to skim nutrients and growth space of clostridium butyricum in the culture medium, so that, as a preferable case, as shown in fig. 1, the step of culturing clostridium butyricum and aspergillus niger under sealed conditions in a sectional temperature-controlled manner includes:
inoculating clostridium butyricum into a culture medium by utilizing clostridium butyricum spore fermentation liquor;
granulating clostridium butyricum obtained after inoculation to obtain thallus particles;
spraying Aspergillus niger fermentation liquor on the thallus particles to inoculate Aspergillus niger to obtain inoculated particles;
and sealing the inoculated particles and carrying out sectional temperature control culture to obtain the clostridium butyricum solid fermentation material.
As one case, the medium inoculated with clostridium butyricum spore fermentation broth comprises an organic nutrient and an inorganic salt nutrient, specifically comprising wheat and/or rice, and a soybean meal, an inorganic salt nutrient, wherein the inorganic salt nutrient comprises a mixture of any one or more of the following: ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, calcium carbonate, ferrous sulfate and sodium bicarbonate. Further, the culture medium comprises the following components in percentage by mass:
wheat, 50% -60%;
rice, 20% -30%;
6% -26% of bean pulp;
2-4% of inorganic salt nutritional agent.
The above-mentioned wheat and rice are supplied as a carbon source of clostridium butyricum, and the soybean meal is supplied as a nitrogen source of clostridium butyricum, and soybean meal may be used in addition to the soybean meal. Preferably, the rice is crushed rice, preferably, the wheat and the crushed rice are crushed in advance, the grain size of the crushed wheat is not more than 2.5mm, and the grain size of the crushed rice is not more than 1mm. The culture medium according to the proportion ensures the viscosity of the culture medium, reduces the pore oxygen content of the culture medium, and is beneficial to providing an anaerobic environment for the proliferation and metabolism of clostridium butyricum.
As described above, ammonium sulfate may further provide a nitrogen source for Clostridium butyricum. Ferrous sulfate and sodium bicarbonate can consume part of the oxygen and help provide an anaerobic environment for the proliferation and metabolism of clostridium butyricum.
As a specific case, the inorganic salt nutritional agent comprises, by mass:
ammonium sulfate, 0.6% -1.0%;
anhydrous dipotassium hydrogen phosphate, 0.1% -0.2%;
anhydrous magnesium sulfate, 0.2% -0.5%;
0.5 to 0.8 percent of calcium carbonate;
ferrous sulfate 0.1-0.5%;
sodium bicarbonate 0.5-1.5%.
As a further case, the mass ratio of ingredients of the medium to water is 1 (0.8-0.9).
In one aspect, the inoculated clostridium butyricum is granulated to obtain a bacterial granule, comprising:
the inoculated clostridium butyricum is made into cylindrical granules with the diameter of 8mm-12mm and the length of 20mm-30mm, and the temperature of the bacterial granules after granulation is 30-45 ℃.
In one aspect, the method for sealing culture of seed particles specifically comprises:
the inoculation particles are filled into a plastic bag of a self-discharging valve, the bag body is sealed, and the gas in the bag is discharged.
In a second aspect, based on the preparation method of the clostridium butyricum solid fermentation material disclosed in the first aspect, the invention also discloses a clostridium butyricum solid fermentation material prepared by the preparation method, wherein the clostridium butyricum number of the fermentation material is (1-2) multiplied by 10 8 CFU/g, butyric acid content of 1-5g/Kg and acetic acid content of 0.3-1g/Kg.
In order to more specifically explain the technical scheme disclosed by the invention and embody the technical effects of the preparation method of clostridium butyricum solid fermentation material disclosed by the invention, the technical scheme is described in detail below by combining specific examples and comparative examples.
Although the preparation method of the clostridium butyricum solid fermentation material disclosed by the invention can adopt clostridium butyricum spore fermentation liquor and aspergillus niger fermentation liquor which are conventional in the technical field, in order to fully embody the technical effects of the invention, the clostridium butyricum spore fermentation liquor adopted in the following examples and comparative examples is fermentation liquor of high-activity purified strains subjected to acid-resistant and oxygen-resistant domestication. The Aspergillus niger fermentation liquid is fermentation liquid formed by fermenting amylase, saccharifying enzyme and protease combined enzyme activity screening strains.
The clostridium butyricum bacillus number in clostridium butyricum bacillus fermentation broth is (0.5-1) multiplied by 10 9 CFU/ml, a specific preparation method, comprising: culturing clostridium butyricum primary seed liquid; and (3) performing secondary and tertiary expansion culture on the primary seed liquid in the fermentation tank. The culture medium of the first-stage seed liquid comprises the following components in percentage by mass: glucose 1%, whey powder 0.5%, peptone 1%, yeast extract 0.5%, beef extract 0.5%, K 2 HPO 4 0.2％，MgSO 4 ·7H 2 O 0.1％，NaHCO 3 0.1％，CaCl 2 0.02% of cysteine hydrochloride and 0.1%. The medium pH of the primary seed solution was 7.5 and sterilized at 121℃for 30min. The culture conditions of the first-stage seed liquid are as follows: the culture is carried out for 7-10h at 37 ℃ under the rotation speed of 60 rpm. The medium for secondary and tertiary fermentation expansion culture comprises the following components in percentage by mass: glucose 1.5%, whey powder 2%, peptone 1.5%, yeast extract 0.5%, beef extract 0.5%, K 2 HPO 4 0.2％，MgSO 4 ·7H 2 O 0.1％，CaCO 3 0.1％，MnSO 4 ·H 2 O 0.1％，FeSO 4 ·7H 2 O0.2%. The pH of the medium for the secondary and tertiary fermentation expansion culture is 7.5, and the medium is sterilized for 30min at 121 ℃. And nitrogen is introduced in the cooling process of the secondary culture and the tertiary culture to keep the tank pressure to be 0.05MPa, and the culture conditions are as follows: the temperature is 37+/-1 ℃, the rotating speed is 60-90rpm, the anaerobic fermentation is carried out, and the culture period is 30-40h.
The Aspergillus niger fermentation broth adopts wort (8-10 Baume) as culture medium. The culture conditions of the fermentation tank are as follows: the temperature is 30+/-2 ℃, the rotating speed is 180rpm, the ventilation amount is 1:0.5, the tank pressure is 0.05MPa, and the culture period is 24 hours. And finally forming fine hyphae, wherein the hyphae are sticky.
Example 1
Preparation of clostridium butyricum solid fermentation medium: the preparation is prepared from the following components in percentage by mass: 60% of wheat (pre-crushed, the grain diameter is not more than 2.5 mm), 20% of crushed rice (pre-crushed, the grain diameter is not more than 1 mm), 17% of soybean meal, 1.0% of ammonium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.5% of magnesium sulfate, 0.5% of calcium carbonate, 0.3% of ferrous sulfate, 0.5% of sodium bicarbonate and the feed water ratio of 1:0.9. The sterilization temperature is 115 ℃ and the sterilization time is 20min.
Inoculating clostridium butyricum: and (3) rapidly cooling the sterilized material to 75 ℃ by adopting sterilized air, and inoculating clostridium butyricum spore fermentation liquor accounting for 15% of the mass ratio of the material.
Granulating the inoculated clostridium butyricum: and (3) quickly transferring the clostridium butyricum strain obtained by inoculation into a SET-140 screw extrusion granulator, wherein the extrusion mode is front discharging. The viscous material (about 50 ℃) after inoculation is extruded from the die plate with a hole on a machine head under the action of screw conveying and extrusion, the particle shape is cylindrical, the diameter is 8-12mm, and the length is 20-30 mm.
Inoculating and bagging Aspergillus niger fermentation liquor: the Aspergillus niger fermentation liquid is sprayed on the surface of thallus particles by adopting an external sterile pressure pipeline spray head, then is arranged in a plastic bag (40 x 80 cm) with a self-discharging valve, is arranged at the mouth of the bag as much as possible, and discharges the gas in the bag in an extrusion mode, and the bag is tied up by a self-locking nylon tie.
Sealing and sectional temperature control culture: placing the plastic bags filled with the sprayed thalli particles in a fermentation chamber, orderly arranging the fermentation bags, and culturing by adopting a sectional temperature control mode. The room temperature is controlled at 33 ℃ in the initial stage (the first day) of culture, so that the aspergillus niger is accelerated to grow, oxygen in the bag is consumed, hypha is generated, compound enzymes such as amylase, saccharifying enzyme and protease are generated, the particles are softened, and then the room temperature is adjusted to 39 ℃ for continuous culture for 3 days, and the temperature condition is unfavorable for the growth of the aspergillus niger but favorable for enzymolysis reaction, so that the method is more suitable for the full growth of clostridium butyricum.
After the fermentation is completed, the obtained clostridium butyricum solid fermentation material has obvious butyric acid taste, and the bacterial number is 2 multiplied by 10 8 CFU/g, butyric acid content of 5g/Kg and acetic acid content of 1g/Kg.
Example 2
Preparation of clostridium butyricum solid fermentation medium: the preparation is prepared from the following components in percentage by mass: 50% of wheat (pre-crushed, the grain diameter is not more than 2.5 mm), 20% of crushed rice (pre-crushed, the grain diameter is not more than 1 mm), 26% of soybean meal, 0.8% of ammonium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.5% of magnesium sulfate, 0.5% of calcium carbonate, 0.5% of ferrous sulfate, 1.5% of sodium bicarbonate and the feed water ratio of 1:0.8. The sterilization temperature is 115 ℃ and the sterilization time is 20min.
Inoculating clostridium butyricum: and (3) rapidly cooling the sterilized material to 70 ℃ by adopting sterilized air, and inoculating clostridium butyricum spore fermentation liquor accounting for 15% of the mass ratio of the material.
Granulating the inoculated clostridium butyricum: and (3) quickly transferring the clostridium butyricum strain obtained by inoculation into a SET-140 screw extrusion granulator, wherein the extrusion mode is front discharging. The viscous material (about 50 ℃) after inoculation is extruded from the die plate of the die head under the action of screw conveying and extrusion, the particle shape is cylindrical, the diameter is 8mm-12mm, and the length is 20mm-30mm.
Inoculating and bagging Aspergillus niger fermentation liquor: the Aspergillus niger fermentation liquid is sprayed on the surface of thallus particles by adopting an external sterile pressure pipeline spray head, then is arranged in a plastic bag (40 x 80 cm) with a self-discharging valve, is arranged at the mouth of the bag as much as possible, and discharges the gas in the bag in an extrusion mode, and the bag is tied up by a self-locking nylon tie.
Sealing and sectional temperature control culture: placing the plastic bags filled with the sprayed thalli particles in a fermentation chamber, orderly arranging the fermentation bags, and culturing by adopting a sectional temperature control mode. The room temperature is controlled at 35 ℃ in the initial stage (the first day) of culture, so that the growth of the aspergillus niger is accelerated, oxygen in the bag is consumed, hypha is generated, compound enzymes such as amylase, saccharifying enzyme and protease are generated, the particles are softened, and then the room temperature is adjusted to 39 ℃ for continuous culture for 3 days, and the temperature condition is unfavorable for the growth of the aspergillus niger but favorable for enzymolysis reaction, so that the method is more suitable for the full growth of clostridium butyricum.
After the fermentation is completed, the obtained clostridium butyricum solid fermentation material has obvious butyric acid taste, and the bacterial number is 1.3X10 8 CFU/g, butyric acid content of 3g/Kg and acetic acid content of 0.4g/Kg.
Example 3
Preparation of clostridium butyricum solid fermentation medium: the preparation is prepared from the following components in percentage by mass: 55% of wheat (pre-crushed, the grain diameter is not more than 2.5 mm), 25% of crushed rice (pre-crushed, the grain diameter is not more than 1 mm), 18% of bean pulp, 0.6% of ammonium sulfate, 0.1% of dipotassium hydrogen phosphate, 0.2% of magnesium sulfate, 0.5% of calcium carbonate, 0.1% of ferrous sulfate, 0.5% of sodium bicarbonate and the feed water ratio of 1:0.9. The sterilization temperature is 115 ℃ and the sterilization time is 20min.
Inoculating clostridium butyricum: the sterilization material is quickly cooled to 70 ℃ by adopting sterilization air, and clostridium butyricum spore fermentation liquor is inoculated according to the mass ratio of 10 percent of the material.
Granulating the inoculated clostridium butyricum: and (3) quickly transferring the clostridium butyricum strain obtained by inoculation into a SET-140 screw extrusion granulator, wherein the extrusion mode is front discharging. The viscous material (about 50 ℃) after inoculation is extruded from the die plate of the die head under the action of screw conveying and extrusion, the particle shape is cylindrical, the diameter is 8mm-12mm, and the length is 20mm-30mm.
Inoculating and bagging Aspergillus niger fermentation liquor: the Aspergillus niger fermentation liquid is sprayed on the surface of thallus particles by adopting an external sterile pressure pipeline spray head, then is arranged in a plastic bag (40 x 80 cm) with a self-discharging valve, is arranged at the mouth of the bag as much as possible, and discharges the gas in the bag in an extrusion mode, and the bag is tied up by a self-locking nylon tie.
Sealing and sectional temperature control culture: placing the plastic bags filled with the sprayed thalli particles in a fermentation chamber, orderly arranging the fermentation bags, and culturing by adopting a sectional temperature control mode. The room temperature is controlled at 33 ℃ in the initial stage (the first day) of culture, so that the aspergillus niger is accelerated to grow, oxygen in the bag is consumed, hypha is generated, compound enzymes such as amylase, saccharifying enzyme and protease are generated, the particles are softened, and then the room temperature is adjusted to 36 ℃ for continuous culture for 3 days, and the temperature condition is unfavorable for the growth of the aspergillus niger but favorable for enzymolysis reaction, so that the method is more suitable for the full growth of clostridium butyricum.
After the fermentation is completed, the obtained clostridium butyricum solid fermentation material has obvious butyric acid taste, and the bacterial number is 1 multiplied by 10 8 CFU/g, butyric acid content of 1g/Kg and acetic acid content of 0.4g/Kg.
Example 4
Preparation of clostridium butyricum solid fermentation medium: the preparation is prepared from the following components in percentage by mass: 60% of wheat (pre-crushed, the grain diameter is not more than 2.5 mm), 30% of crushed rice (pre-crushed, the grain diameter is not more than 1 mm), 6% of bean pulp, 0.8% of ammonium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.5% of magnesium sulfate, 0.5% of calcium carbonate, 0.5% of ferrous sulfate, 1.5% of sodium bicarbonate and the feed water ratio of 1:0.8. The sterilization temperature is 115 ℃ and the sterilization time is 20min.
Inoculating clostridium butyricum: and (3) rapidly cooling the sterilized material to 75 ℃ by adopting sterilized air, and inoculating clostridium butyricum spore fermentation liquor accounting for 15% of the mass ratio of the material.
Granulating the inoculated clostridium butyricum: and (3) quickly transferring the clostridium butyricum strain obtained by inoculation into a SET-140 screw extrusion granulator, wherein the extrusion mode is front discharging. The viscous material (about 50 ℃) after inoculation is extruded from the die plate of the die head under the action of screw conveying and extrusion, the particle shape is cylindrical, the diameter is 8mm-12mm, and the length is 20mm-30mm.
Inoculating and bagging Aspergillus niger fermentation liquor: the Aspergillus niger fermentation liquid is sprayed on the surface of thallus particles by adopting an external sterile pressure pipeline spray head, then is arranged in a plastic bag (40 x 80 cm) with a self-discharging valve, is arranged at the mouth of the bag as much as possible, and discharges the gas in the bag in an extrusion mode, and the bag is tied up by a self-locking nylon tie.
Sealing and sectional temperature control culture: placing the plastic bags filled with the sprayed thalli particles in a fermentation chamber, orderly arranging the fermentation bags, and culturing by adopting a sectional temperature control mode. The room temperature is controlled at 35 ℃ in the initial stage (the first day) of culture, so that the growth of the aspergillus niger is accelerated, oxygen in the bag is consumed, hypha is generated, compound enzymes such as amylase, saccharifying enzyme and protease are generated, the particles are softened, and then the room temperature is adjusted to 37 ℃ for continuous culture for 3 days, and the temperature condition is unfavorable for the growth of the aspergillus niger but favorable for enzymolysis reaction, so that the method is more suitable for the full growth of clostridium butyricum.
After the fermentation is completed, the obtained clostridium butyricum solid fermentation material has obvious butyric acid taste, and the bacterial number is 1.7X10 8 CFU/g, butyric acid content of 4.5g/Kg and acetic acid content of 0.8g/Kg.
Comparative example 1
In the comparative example, saccharomycetes are used as oxygen consumption strains in clostridium butyricum fermentation culture environment.
Preparation of clostridium butyricum solid fermentation medium: the preparation is prepared from the following components in percentage by mass: 60% of wheat (pre-crushed, the grain diameter is not more than 2.5 mm), 20% of crushed rice (pre-crushed, the grain diameter is not more than 1 mm), 17% of soybean meal, 1.0% of ammonium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.5% of magnesium sulfate, 0.5% of calcium carbonate, 0.3% of ferrous sulfate, 0.5% of sodium bicarbonate and the feed water ratio of 1:0.9. The sterilization temperature is 115 ℃ and the sterilization time is 20min. Inoculating clostridium butyricum: the sterilized materials are quickly cooled to 70 ℃ by adopting sterilized air, and clostridium butyricum spore fermentation liquor accounting for 15% of the mass ratio of the materials is inoculated into a culture container.
Adding saccharomycetes: yeast is added into the culture container.
Sealing and temperature controlling culture: sealing the culture container, and fermenting at 36 deg.c. Yeast can grow and reproduce in both aerobic and anaerobic environments. At the initial stage of fermentation, saccharomycetes grow preferentially and consume oxygen in the culture container. After the oxygen in the culture container is depleted, the culture container is provided with anaerobic growth conditions of clostridium butyricum, and clostridium butyricum grows and grows.
After the fermentation is completed, the obtained clostridium butyricum solid fermentation material has no obvious butyric acid taste, and the bacterial count is 0.4x10 8 CFU/g, butyric acid content of 1.2g/Kg and acetic acid content of 0.2g/Kg.
Comparative example 2
The comparative example adopts the preparation method of clostridium butyricum solid fermentation material disclosed in the embodiment of the invention, but saccharomycete is used as an oxygen consumption strain in clostridium butyricum fermentation culture environment.
Preparation of clostridium butyricum solid fermentation medium: the preparation is prepared from the following components in percentage by mass: 60% of wheat (pre-crushed, the grain diameter is not more than 2.5 mm), 30% of crushed rice (pre-crushed, the grain diameter is not more than 1 mm), 6% of bean pulp, 0.8% of ammonium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.5% of magnesium sulfate, 0.5% of calcium carbonate, 0.5% of ferrous sulfate, 1.5% of sodium bicarbonate and the feed water ratio of 1:0.8. The sterilization temperature is 115 ℃ and the sterilization time is 20min.
Inoculating clostridium butyricum: the sterilized materials are quickly cooled to 70 ℃ by adopting sterilized air, and clostridium butyricum spore fermentation liquor accounting for 15% of the mass ratio of the materials is inoculated into a culture container.
Granulating the inoculated clostridium butyricum: rapidly transferring into a SET-140 screw extrusion granulator, wherein the extrusion mode is front discharging. The viscous material (about 50 ℃) after inoculation is extruded from the die plate of the die head under the action of screw conveying and extrusion, the particle shape is cylindrical, the diameter is 8mm-12mm, and the length is 20mm-30mm.
Inoculating and bagging yeast fermentation liquor: the saccharomycete fermentation liquor is sprayed onto the surface of thallus grains by using an external sterile pressure pipeline nozzle, and then is filled into a plastic bag (40 x 80 cm) with a self-discharging valve, the bag is filled into a bag mouth as much as possible, and the gas in the bag is discharged in an extrusion mode, so that the bag is tied up by a self-locking nylon tie.
Sealing and temperature controlling culture: placing the plastic bags filled with the sprayed thalli particles in a fermentation chamber, orderly arranging the fermentation bags, and culturing by adopting a sectional temperature control mode. The room temperature is controlled at 33 ℃ in the initial stage (the first day) of the culture, so that the growth of the saccharomycetes is accelerated, oxygen in the bag is consumed, then the room temperature is adjusted to 39 ℃ for continuous culture for 3 days, and the temperature is more suitable for the growth of clostridium butyricum. Although the yeast consumes oxygen in the bag, complex enzymes such as amylase, saccharifying enzyme and protease are not produced, which is unfavorable for softening particles and slowing the growth of clostridium butyricum.
After the fermentation is completed, the obtained clostridium butyricum solid fermentation material has no obvious butyric acid taste, and the bacterial number is 0.2x10 8 CFU/g, butyric acid content of 0.5g/Kg and acetic acid content of 0.2g/Kg.
Comparative example 3
The present comparative example uses a medium commonly used in the art as a fermentation medium for clostridium butyricum.
Preparation of clostridium butyricum solid fermentation medium: the preparation is prepared from the following components in percentage by mass: 50% of bran and 50% of bean flour, and the ratio of feed to water is 1:0.9. The sterilization temperature is 115 ℃ and the sterilization time is 20min.
Inoculating clostridium butyricum: the sterilized materials are quickly cooled to 70 ℃ by adopting sterilized air, and clostridium butyricum spore fermentation liquor accounting for 15% of the mass ratio of the materials is inoculated into a culture container.
Granulating the inoculated clostridium butyricum: rapidly transferring into a SET-140 screw extrusion granulator, wherein the extrusion mode is front discharging. The viscous material (about 50 ℃) after inoculation is extruded from the die plate with a hole on a machine head under the action of screw conveying and extrusion, the particle shape is nearly cylindrical, the diameter is about 10mm, and the length is 20mm-30mm. The thallus particles prepared by the solid culture medium are loose and have slightly poor viscosity.
Inoculating and bagging Aspergillus niger fermentation liquor: the Aspergillus niger fermentation liquid is sprayed on the surface of thallus particles by adopting an external sterile pressure pipeline spray head, then is arranged in a plastic bag (40 x 80 cm) with a self-discharging valve, is arranged at the mouth of the bag as much as possible, and discharges the gas in the bag in an extrusion mode, and the bag is tied up by a self-locking nylon tie.
Sealing and sectional temperature control culture: placing the plastic bags filled with the sprayed thalli particles in a fermentation chamber, orderly arranging the fermentation bags, and culturing by adopting a sectional temperature control mode. At the initial stage (the first day) of culture, the room temperature is controlled at 33 ℃, so that the aspergillus niger accelerates the growth, consumes oxygen in the bag, generates hypha, generates amylase, saccharifying enzyme, protease and other complex enzymes, and then adjusts the room temperature to 39 ℃ for continuous culture for 3 days, and the clostridium butyricum grows slowly due to loose thallus particles and low thallus quantity and metabolite.
After the fermentation is completed, the obtained clostridium butyricum solid fermentation material has obvious butyric acid taste, and the bacterial number is 0.5X10 8 CFU/g, butyric acid content of 0.2g/Kg and acetic acid content of 0.1g/Kg.
As can be seen from the comparison of the above examples 1 to 4 with comparative example 1, the preparation method of clostridium butyricum solid fermentation material disclosed in the present invention adopts Aspergillus niger as the oxygen consuming strain in the clostridium butyricum growth and propagation process, and can obtain clostridium butyricum with higher bacterial count than that obtained by adopting saccharomycetes. As the saccharomycete can grow and reproduce in both aerobic and anaerobic environments, nutrients and growth space of clostridium butyricum are greatly occupied in fermentation, and the bacterial count of clostridium butyricum is not high.
As can be seen from the comparison of examples 1 to 4 with comparative example 2, the clostridium butyricum solid fermentation material obtained by the method for preparing clostridium butyricum solid fermentation material disclosed in the example of the present invention has a higher clostridium butyricum count and a higher metabolite content than the yeast-sprayed solid fermentation material even though the same granulation and other strain fermentation solutions were used to inoculate the granule preparation method.
As can be seen from the comparison of examples 1 to 4 described above with comparative example 3, the clostridium butyricum culture medium disclosed in the examples of the present invention can significantly increase the bacterial count of clostridium butyricum solid fermentation material and increase the clostridium butyricum metabolite content in clostridium butyricum solid fermentation.
The foregoing has described in detail the technical solutions provided herein, and specific examples have been used herein to illustrate the principles and embodiments of the present application, the above examples being provided only to assist in understanding the methods of the present application and their core ideas; also, as will occur to those of ordinary skill in the art, many modifications are possible in view of the teachings of the present application, both in the detailed description and the scope of its applications. In view of the foregoing, this description should not be construed as limiting the application.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (6)
1. A method for preparing clostridium butyricum solid fermentation material, which is characterized by comprising the following steps:
the clostridium butyricum and the aspergillus niger are subjected to sectional temperature control culture under a sealing condition, and a solid fermentation method is adopted to prepare and obtain clostridium butyricum solid fermentation material under an anaerobic environment,
the sectional temperature control culture at least comprises two stages:
the first temperature-controlled culture stage, the temperature is 33-36 ℃,
the second temperature-controlled culture stage, the temperature is 36-39 ℃,
the clostridium butyricum and aspergillus niger are subjected to sectional temperature control culture under a sealed condition, and the method comprises the following steps:
inoculating clostridium butyricum into a culture medium by utilizing clostridium butyricum spore fermentation liquor;
granulating the clostridium butyricum obtained by inoculation to obtain thallus particles;
spraying Aspergillus niger fermentation liquor on the thallus particles to inoculate Aspergillus niger to obtain inoculated particles;
sealing the inoculated particles and carrying out sectional temperature control culture to obtain clostridium butyricum solid fermentation material;
the ingredients of the culture medium comprise the following components in percentage by mass:
50% -60% of wheat,
20% -30% of rice,
6% -26% of soybean meal,
2% -4% of inorganic salt nutritional agent,
the wheat is pre-crushed wheat, and the grain size of the crushed wheat is not more than 2.5mm;
the rice is crushed rice, and the grain diameter of the crushed rice is not more than 1mm;
the sum of the mass percentages of the components in the ingredients of the culture medium is 100 percent.
2. The method of claim 1, wherein the inorganic salt nutrient comprises a mixture of any one or more of: ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, calcium carbonate, ferrous sulfate and sodium bicarbonate.
3. The method of claim 2, wherein the inorganic salt nutrient comprises, in mass percent:
ammonium sulfate, 0.6% -1.0%;
anhydrous dipotassium hydrogen phosphate, 0.1% -0.2%;
anhydrous magnesium sulfate, 0.2% -0.5%;
0.5 to 0.8 percent of calcium carbonate;
ferrous sulfate 0.1-0.5%;
sodium bicarbonate 0.5-1.5%.
4. The method of claim 1, wherein the mass ratio of ingredients of the medium to water is 1 (0.8-0.9).
5. The method of claim 1, wherein said sealing said seed particles comprises:
and filling the inoculation particles into a plastic bag of a self-discharging valve, and sealing the bag body to discharge the gas in the bag.
6. The method of claim 1, wherein granulating the inoculated clostridium butyricum to obtain a granule comprises:
the inoculated clostridium butyricum is made into cylindrical granules with the diameter of 8mm-12mm and the length of 20mm-30mm, and the temperature of the bacterial granules after granulation is 30-45 ℃.
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