CN116333943A - Fermentation culture process of strain - Google Patents
Fermentation culture process of strain Download PDFInfo
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- CN116333943A CN116333943A CN202310448408.4A CN202310448408A CN116333943A CN 116333943 A CN116333943 A CN 116333943A CN 202310448408 A CN202310448408 A CN 202310448408A CN 116333943 A CN116333943 A CN 116333943A
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- 230000004151 fermentation Effects 0.000 title claims abstract description 94
- 238000000034 method Methods 0.000 title claims abstract description 38
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- 239000000047 product Substances 0.000 claims abstract description 25
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- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 29
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 29
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- 239000000843 powder Substances 0.000 claims description 25
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 22
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- 239000008103 glucose Substances 0.000 claims description 21
- 239000001888 Peptone Substances 0.000 claims description 20
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- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
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- 229940114124 ferulic acid Drugs 0.000 claims description 9
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- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims description 9
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 9
- 239000001393 triammonium citrate Substances 0.000 claims description 8
- 235000011046 triammonium citrate Nutrition 0.000 claims description 8
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 6
- 229920000053 polysorbate 80 Polymers 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 239000008101 lactose Substances 0.000 claims description 5
- 239000011800 void material Substances 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 10
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- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 description 7
- 229960000511 lactulose Drugs 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 241000287828 Gallus gallus Species 0.000 description 5
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- 241000186000 Bifidobacterium Species 0.000 description 4
- 239000001116 FEMA 4028 Substances 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 4
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 4
- 229960004853 betadex Drugs 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 230000004584 weight gain Effects 0.000 description 4
- 235000019786 weight gain Nutrition 0.000 description 4
- 240000007817 Olea europaea Species 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
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- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
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- 235000013330 chicken meat Nutrition 0.000 description 2
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- 238000004519 manufacturing process Methods 0.000 description 2
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- 230000007269 microbial metabolism Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 238000007605 air drying Methods 0.000 description 1
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- 239000007970 homogeneous dispersion Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 235000001705 insufficient nutrition Nutrition 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
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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
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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
- C12N1/06—Lysis of microorganisms
- C12N1/066—Lysis of microorganisms by physical methods
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention belongs to the field of fermentation, and in particular relates to a fermentation culture process of strains, which comprises the following steps: step 1, inoculating strains into a culture medium for thawing culture to obtain seed liquid; step 2, inoculating the seed solution into a fermentation medium for anaerobic fermentation culture to obtain a fermentation liquor; step 3, adsorbing and filtering the fermentation liquor to obtain filter residues, then placing the filter residues into water for ultrasonic separation, and filtering to obtain crushed prefabricated liquor; and 4, carrying out low-temperature ultrasonic treatment on the crushed prefabricated liquid, centrifuging at a low temperature, and taking supernatant to obtain a fermentation product. The invention solves the defects of the existing strain cultivation process, ensures the resurgence and activity of the strain by thawing cultivation, and greatly improves the strain cultivation effect by matching with secondary fermentation cultivation, and simultaneously provides the effective viable count of the product.
Description
Technical Field
The invention belongs to the field of fermentation, and particularly relates to a fermentation culture process of strains.
Background
The weight of the cultured animals, particularly the cultured aquatic products, is increased by increasing the feed intake, thereby improving the economic benefit. With the development of technology, active products generated by microbial metabolism are more and more emphasized, functional active substances such as small peptide, glucan and the like can be generated in the microbial metabolism process, so that the immune function of animals can be improved, meanwhile, microorganisms can form dominant communities in the digestive tracts of the animals, the micro-ecological balance of the digestive tracts is promoted, and the digestion and absorption capacity of the animals is improved. However, the existing seed liquid cultivation process is simpler, the obtained effective viable count is lower, and the existing market needs cannot be met.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a fermentation culture process of strains, which solves the defects of the conventional strain culture process, ensures the resuscitative and activity of the strains by thawing culture, and greatly improves the strain culture effect by matching with secondary fermentation culture and simultaneously provides the effective viable count of the product.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
a fermentation culture process of strains comprises the following steps:
step 1, inoculating strains into a culture medium for thawing culture to obtain seed liquid;
step 2, inoculating the seed solution into a fermentation medium for anaerobic fermentation culture to obtain a fermentation liquor;
step 3, adsorbing and filtering the fermentation liquor to obtain filter residues, then placing the filter residues into water for ultrasonic separation, and filtering to obtain crushed prefabricated liquor;
and 4, carrying out low-temperature ultrasonic treatment on the crushed prefabricated liquid, centrifuging at a low temperature, and taking supernatant to obtain a fermentation product.
The mass ratio of the culture medium in the step 1 is as follows: 5-8% of skimmed milk powder, 0.3-0.5% of cyclodextrin, 1.2-1.4% of corn flour, 0.2-0.4% of glucose, 0.7-1.1% of yeast extract, 0.2-0.3% of peptone, 0.2-0.5% of lactose and 1.1% of dipotassium hydrogen phosphate; the balance being water. The cyclodextrin is beta-cyclodextrin. The culture medium effectively controls a primary fermentation system of a strain in a mode of combining a macromolecular carbon source and a micromolecule, in general, the strain forms slow dissolution from a frozen state, is fragile, can not form rapid growth, can not form rapid absorption of the carbon source, and the defatted milk powder has a good protection effect, can form an excellent protection system in thawing, can form a plurality of hydroxyl groups contained in the defatted milk powder, can form hydrogen bonds with proteins in the strain, plays a role in protecting a primary thawed strain structure, plays a good protection role, and belongs to the macromolecular carbon source, can play a role in protecting the carbon source, provides enough carbonaceous nutrition for the strain, and the defatted milk powder with the macromolecular structure has a relatively slow degradation speed in the strain absorption, can play a role in inhibiting the growth of the strain in the weak strain, and can not bring about rapid absorption of the strain, so that the strain in the thawing in the early stage can not completely absorb the carbon source, and causes a subsequent strain to be inactivated due to insufficient nutrition; the macromolecular protection effect of the skimmed milk powder can reduce the contact of the strain and oxygen, improve the protein stability, namely improve the stability of the strain, obviously improve the initial survival rate of the strain in the addition process of the skimmed milk powder, improve the relative activity of cells by 3-5%, and secondly, the glucose and the isolactose belong to small molecular structures, so that the skimmed milk powder has good absorbability in the use process, and the yeast extract can be quickly degraded and converted into a carbonaceous nutrition material with a small molecular structure, so that the quick absorption of the initial weak strain can be met. Therefore, the carbonaceous material with the large and small molecular structures can ensure the nutrition requirement of the strain in the initial thawing process, and improve the survival rate and the relative activity of the strain. The cyclodextrin itself ring structure can solidify the carbon material and form slow release effect in the whole system, and in the process of strain thawing culture, the slow release structure can effectively control the release of carbon source, thereby achieving the effect of controlling strain to absorb carbon source, and simultaneously, the carbon source can be effectively distributed, thereby achieving the purpose of strain equilibrium absorption. Therefore, the cyclodextrin promotes the control effect of the carbon source through long-time release, thereby achieving the purposes of controlling the thawing process and promoting the survival rate of the strain.
Further, the preparation method of the culture medium comprises the following steps: a1, adding glucose, peptone and isolactose into water, uniformly stirring, then adding cyclodextrin to form ultrasonic treatment for 10-20min to obtain an initial solution, wherein the stirring speed is 1000-2000r/min, the ultrasonic frequency of ultrasonic treatment is 50-70kHz, and the temperature is 20-30 ℃; the method comprises the steps of uniformly stirring glucose, peptone and isolactose by using water as a solvent to form a homogeneous structure, simultaneously adding cyclodextrin for ultrasonic dispersion, and connecting groups in the cyclodextrin with groups in the glucose, the peptone and the isolactose to achieve an inclusion effect and form a stable saturated system; a2, adding the yeast extract into the primary solution, and performing low-temperature ultrasonic treatment for 10-20min to obtain a prefabricated inclusion liquid; the temperature of the low-temperature ultrasonic treatment is 5-10 ℃, and the ultrasonic frequency is 50-70kHz; when yeast extract is added into an initial solution, most of small molecular materials enter a cyclodextrin pipeline to form an excellent connection inclusion effect, and along with the low-temperature ultrasonic treatment, small molecular carbon sources close to a pipe orifice enter the solution newly and are mixed with the yeast extract and redistributed in the ultrasonic treatment, the mixture is mixed into the cyclodextrin pipeline to form a primary blocking effect, and meanwhile, part of small molecular materials enter a surface solution and are matched with a blocking system after the ultrasonic treatment, and the content of small molecular carbon nutrients continuously rises along with the deep pipeline, so that a good slow-release effect is achieved in the whole system, and the thawing recovery process of strains is met; and a3, adding corn flour, skimmed milk powder and monopotassium phosphate into the prefabricated inclusion liquid, and rapidly stirring to obtain a culture medium, wherein the rapid stirring temperature is 5-10 ℃, and the stirring speed is 2000-3000r/min, and the step is completed by mixing solutes to form a homogeneous state so as to obtain the culture medium with uniform dispersion. The process utilizes cyclodextrin to form inclusion solidification on a micromolecular carbon source, achieves a stable inclusion structure, and forms gradient change by matching with high-frequency vibration of ultrasound to form a stable slow-release control system.
The temperature of the thawing culture in the step 1 is 35-36 ℃ and the time is 10-20h; in the cultivating and thawing process, the strain is slowly thawed by using temperature, and the resuscitating effect is achieved, in the process, the carbon source of the small molecular system is influenced by cyclodextrin to form slow and gradient release, and the content of the small molecular carbon source is gradually increased along with the increase of the resuscitating amount of the strain, so that the requirement of the early-stage carbonaceous absorption of the strain is met, and the weak period is smoothly spent; compared with the prior art, the slow-release structure is matched with the combination of the skimmed milk powder, and can improve the relative activity of strain cells by 5-8%.
The strain adopts bifidobacterium
The inoculation amount of the strain is 5-6% of the mass of the culture medium.
The inoculation amount of the seed liquid in the step 2 is 6-8% of the fermentation medium, and the anaerobic fermentation temperature is 38-39 ℃; the fermentation time is 8-10h, and the pH is 7.1-7.3. The step supplies sufficient carbon to the strain in weak period, so as to greatly improve the activity of the strain and obtain long-term fermentation culture.
The mass ratio of the fermentation medium in the step 2 is as follows: 2-4% of glucose, 1.5-2% of corn extract, 1.5-3% of yeast powder, 1.0-2% of beef peptone, 1.5-3% of ferulic acid, 0.5-1% of sodium acetate, 0.2-0.3% of triammonium citrate, 0.1-0.5% of ammonium bicarbonate, 0.1% of tween-80, 0.05% of MgSO4, 0.02% of MnSO40, 0.05% of monopotassium phosphate and the balance of water; the method comprises the steps of adding micro-nano ammonium bicarbonate into olive oil, rapidly stirring, filtering and then carrying out high-level airing to obtain an olive oil coated ammonium bicarbonate material, wherein the mass ratio of the ammonium bicarbonate to the olive oil is 1:3-4, the rapid stirring speed is 1000-2000r/min, and the high-level airing temperature is 10-20 ℃; the air drying is based on the fluidity of olive oil to achieve stable separation, to form a structural system with olive oil wrapped in ammonium bicarbonate, and the olive oil is wrapped in carbon in a thin layerAmmonium acid hydroxide surface; in the use process, the olive oil belongs to hydrophobicity, and can isolate ammonium bicarbonate from water molecules to form structural stability; in addition, in the fermentation process, ammonium bicarbonate can be promoted to decompose to form internal decomposition, so that the internal pressure is increased, an oil film is damaged, generated carbon dioxide, water and ammonia gas are released into the water, and the effect of supplementing nitrogen elements is achieved. Further, the preparation method of the fermentation medium comprises the following steps: b1, glucose, corn extract, yeast powder, beef peptone, ferulic acid, sodium acetate, tri-ammonium citrate, tween-80 and MgSO 4 、MnSO 4 Adding potassium dihydrogen phosphate into water, stirring, and sterilizing to obtain a prefabricated culture medium, wherein the stirring speed of the stirring is 1000-2000r/min, and sterilizing is performed in an autoclave at 121deg.C; the pressure is 103.4kPa, and the time is 40-60min; the step utilizes an autoclave sterilization mode to form a homogeneous culture medium prefabricated liquid; and b2, cooling the prefabricated culture medium to 30-31 ℃, then adding olive oil to wrap ammonium bicarbonate, and homogenizing and stirring to obtain the fermentation culture medium, wherein the temperature of the homogenizing and stirring is 30 ℃, and the stirring speed is 1000-2000 r/min. The culture preparation process can sterilize a carbon source and then stir the carbon source, so that the stability of ammonium bicarbonate and the homogeneous dispersion in a culture medium are realized.
The adsorption filtration in the step 3 adopts active carbon, and the void structure of the active carbon is gradually reduced from top to bottom; the activated carbon with the reduced pore size gradient can achieve a good adsorption effect, and bacterial sludge and sediment are collected in the activated carbon; in order to reduce the release of materials in the earlier stage of filtration, the filtrate is repeatedly filtered for 2-3 times to achieve the effect of intercepting components, and the filter residues are a mixture of sediment, bacterial sludge and active carbon.
The mass ratio of filter residues to distilled water in the step 3 is 1:4-7, the ultrasonic separation temperature is 20-30 ℃, the ultrasonic frequency is 80-90kHz, and the step utilizes an ultrasonic mode to crush bacterial mud and thalli in the precipitate, so that the thalli are crushed, and meanwhile, soluble matters in the thalli are rapidly dissolved; realizing the crushing of the prefabricated liquid. Further, the secondary filter residue after the filtration of the crushed prefabricated liquid is put into distilled water for secondary low-temperature ultrasonic treatment, the filter residue is repeatedly subjected to ultrasonic treatment for 2-4 times, and the filtered filtrate is combined into the crushed prefabricated liquid, wherein the temperature of the low-temperature ultrasonic treatment is 5-10 ℃ and the ultrasonic frequency is 80-90kHz; the repeated treatment can effectively improve the treatment effect of the sediment and the bacterial mud, fully dissolve the soluble matters in the bacterial body, reduce the adsorptivity of the active carbon to the material and reduce the loss of the soluble matters.
The temperature of the low-temperature ultrasonic treatment in the step 4 is 1-5 ℃, the ultrasonic frequency is 50-70kHz, the time is 0.5-1h, and the temperature of the low-temperature centrifugation is 5-10 ℃; the step utilizes a low-temperature ultrasonic mode to realize further dispersion of the residual thalli, thereby achieving a high-quality thallus crushing effect. The volume of the fermentation product is 8-10 times of that of the fermentation liquid.
The ratio of the fermentation product to water in the use process is 1:10000.
From the above description, it can be seen that the present invention has the following advantages:
1. the invention solves the defects of the existing strain cultivation process, ensures the resurgence and activity of the strain by thawing cultivation, and greatly improves the strain cultivation effect by matching with secondary fermentation cultivation, and simultaneously provides the effective viable count of the product.
2. The invention utilizes cyclodextrin to form stable slow release effect, thereby achieving the supply of micromolecular carbon source in thawing cultivation.
3. According to the invention, the olive oil is used for wrapping ammonium bicarbonate to form a delayed slow-release nitrogen source, so that stable supplementation of the nitrogen source is realized, meanwhile, the acid-base property of the culture medium is adjusted, and the stability of the culture medium is maintained.
Detailed Description
A specific embodiment of the present invention will be described in detail with reference to examples, but the claims of the present invention are not limited thereto.
Example 1
A fermentation culture process of strains comprises the following steps:
step 1, inoculating strains into a culture medium for thawing culture to obtain seed liquid;
step 2, inoculating the seed solution into a fermentation medium for anaerobic fermentation culture to obtain a fermentation liquor;
step 3, adsorbing and filtering the fermentation liquor to obtain filter residues, then placing the filter residues into water for ultrasonic separation, and filtering to obtain crushed prefabricated liquor;
and 4, carrying out low-temperature ultrasonic treatment on the crushed prefabricated liquid, centrifuging at a low temperature, and taking supernatant to obtain a fermentation product.
The mass ratio of the culture medium in the step 1 is as follows: 5% of skimmed milk powder, 0.3% of cyclodextrin, 1.2% of corn flour, 0.2% of glucose, 0.7% of yeast extract, 0.2% of peptone, 0.2% of lactose and 1.1% of dipotassium hydrogen phosphate; the balance being water. The cyclodextrin is beta-cyclodextrin.
The preparation method of the culture medium comprises the following steps: a1, adding glucose, peptone and iso-lactose into water, uniformly stirring, then adding cyclodextrin to form ultrasonic treatment for 10min to obtain an initial solution, wherein the stirring speed is 1000r/min, the ultrasonic frequency of ultrasonic treatment is 50kHz, and the temperature is 20 ℃; a2, adding the yeast extract into the primary solution, and performing low-temperature ultrasonic treatment for 10min to obtain a prefabricated inclusion liquid; the temperature of the low-temperature ultrasonic treatment is 5 ℃, and the ultrasonic frequency is 50kHz; and a3, adding corn flour, skimmed milk powder and potassium dihydrogen phosphate into the pre-prepared inclusion liquid, and rapidly stirring to obtain a culture medium, wherein the rapid stirring temperature is 5 ℃, and the stirring speed is 2000r/min.
The temperature of the thawing culture in the step 1 is 35 ℃ and the time is 10 hours.
The strain adopts commercial bifidobacterium.
The inoculation amount of the strain is 5% of the mass of the culture medium.
The inoculation amount of the seed liquid in the step 2 is 6% of that of a fermentation medium, and the anaerobic fermentation temperature is 38 ℃; the fermentation time was 8h and the pH was 7.1.
The saidThe mass ratio of the fermentation medium in the step 2 is as follows: glucose 2%, corn extract 1.5%, yeast powder 1.5%, beef peptone 1.0%, ferulic acid 1.5%, sodium acetate 0.5%, triammonium citrate 0.2%, ammonium bicarbonate 0.1%, tween-80.1%, mgSO4 0.05%, mnSO40.02%, potassium dihydrogen phosphate 0.05%, and the balance water; the method for wrapping the ammonium bicarbonate comprises the steps of adding micro-nano ammonium bicarbonate into olive oil, rapidly stirring, filtering and then airing at a high position to obtain an olive oil-wrapped ammonium bicarbonate material, wherein the mass ratio of the ammonium bicarbonate to the olive oil is 1:3, the rapid stirring speed is 1000r/min, and the air airing at the high position is 10 ℃; the preparation method of the fermentation medium comprises the following steps: b1, glucose, corn extract, yeast powder, beef peptone, ferulic acid, sodium acetate, tri-ammonium citrate, tween-80 and MgSO 4 、MnSO 4 Adding potassium dihydrogen phosphate into water, stirring uniformly, and sterilizing to obtain a prefabricated culture medium, wherein the stirring speed of the uniform stirring is 1000r/min, and sterilizing is performed by adopting an autoclave at 121 ℃; the pressure is 103.4kPa, and the time is 40 minutes; and b2, cooling the prefabricated culture medium to 30 ℃, then adding olive oil to wrap ammonium bicarbonate, and homogenizing and stirring to obtain the fermentation culture medium, wherein the temperature of the homogenizing and stirring is 30 ℃, and the stirring speed is 1000r/min.
The adsorption filtration in the step 3 adopts active carbon, and the void structure of the active carbon is gradually reduced from top to bottom; and the filtrate was filtered repeatedly 2 times.
The mass ratio of the filter residue to the distilled water in the step 3 is 1:4, the ultrasonic separation temperature is 20-30 ℃, the ultrasonic frequency is 80kHz, the secondary filter residue obtained after the filtration of the crushed prefabricated liquid is placed into the distilled water for secondary low-temperature ultrasonic treatment, the ultrasonic treatment is repeated for 2 times, the filtered filtrate is combined into the crushed prefabricated liquid, the temperature of the low-temperature ultrasonic treatment is 5 ℃, and the ultrasonic frequency is 80kHz.
The temperature of the low-temperature ultrasonic treatment in the step 4 is 1 ℃, the ultrasonic frequency is 50kHz, the time is 0.5h, and the temperature of the low-temperature centrifugation is 5 ℃. The volume of the fermentation product is 8 times that of the fermentation liquid.
The fermentation product of this example was examined to be in the form of a clear liquid and the viable count was 1.19 to 1.26 hundred million/mL.
Example 2
A fermentation culture process of strains comprises the following steps:
step 1, inoculating strains into a culture medium for thawing culture to obtain seed liquid;
step 2, inoculating the seed solution into a fermentation medium for anaerobic fermentation culture to obtain a fermentation liquor;
step 3, adsorbing and filtering the fermentation liquor to obtain filter residues, then placing the filter residues into water for ultrasonic separation, and filtering to obtain crushed prefabricated liquor;
and 4, carrying out low-temperature ultrasonic treatment on the crushed prefabricated liquid, centrifuging at a low temperature, and taking supernatant to obtain a fermentation product.
The mass ratio of the culture medium in the step 1 is as follows: 8% of skimmed milk powder, 0.5% of cyclodextrin, 1.4% of corn flour, 0.4% of glucose, 1.1% of yeast extract, 0.3% of peptone, 0.5% of lactose and 1.1% of dipotassium hydrogen phosphate; the balance being water. The cyclodextrin is beta-cyclodextrin.
The preparation method of the culture medium comprises the following steps: a1, adding glucose, peptone and iso-lactose into water, uniformly stirring, then adding cyclodextrin to form ultrasonic treatment for 20min to obtain an initial solution, wherein the stirring speed is 2000r/min, the ultrasonic frequency of ultrasonic treatment is 70kHz, and the temperature is 30 ℃; a2, adding the yeast extract into the primary solution, and performing low-temperature ultrasonic treatment for 20min to obtain a prefabricated inclusion liquid; the temperature of the low-temperature ultrasonic treatment is 10 ℃, and the ultrasonic frequency is 70kHz; and a3, adding corn flour, skimmed milk powder and potassium dihydrogen phosphate into the pre-prepared inclusion liquid, and rapidly stirring to obtain a culture medium, wherein the rapid stirring temperature is 10 ℃, and the stirring speed is 3000r/min.
The temperature of the thawing culture in the step 1 is 36 ℃ and the time is 20h.
The strain adopts commercial bifidobacterium.
The inoculation amount of the strain is 6% of the mass of the culture medium.
The inoculation amount of the seed liquid in the step 2 is 8% of that of a fermentation medium, and the anaerobic fermentation temperature is 39 ℃; the fermentation time was 10h and the pH was 7.3. The step supplies sufficient carbon to the strain in weak period, so as to greatly improve the activity of the strain and obtain long-term fermentation culture.
The mass ratio of the fermentation medium in the step 2 is as follows: glucose 4%, corn extract 2%, yeast powder 3%, beef peptone 2%, ferulic acid 3%, sodium acetate 1%, triammonium citrate 0.3%, ammonium bicarbonate 0.5%, tween-80.1%, mgSO4 0.05%, mnSO40.02%, potassium dihydrogen phosphate 0.05% and the balance water; the method for wrapping the ammonium bicarbonate comprises the steps of adding micro-nano ammonium bicarbonate into olive oil, rapidly stirring, filtering and then airing at a high position to obtain an olive oil-wrapped ammonium bicarbonate material, wherein the mass ratio of the ammonium bicarbonate to the olive oil is 1:4, the rapid stirring speed is 2000r/min, and the air airing at the high position is 20 ℃; the preparation method of the fermentation medium comprises the following steps: b1, glucose, corn extract, yeast powder, beef peptone, ferulic acid, sodium acetate, tri-ammonium citrate, tween-80 and MgSO 4 、MnSO 4 Adding potassium dihydrogen phosphate into water, stirring uniformly, and sterilizing to obtain a prefabricated culture medium, wherein the stirring speed of the uniform stirring is 2000r/min, and sterilizing is carried out by adopting an autoclave at 121 ℃; the pressure is 103.4kPa, and the time is 60 minutes; and b2, cooling the prefabricated culture medium to 31 ℃, then adding olive oil to wrap ammonium bicarbonate, and homogenizing and stirring to obtain the fermentation culture medium, wherein the temperature of the homogenizing and stirring is 30 ℃, and the stirring speed is 2000r/min.
The adsorption filtration in the step 3 adopts active carbon, and the void structure of the active carbon is gradually reduced from top to bottom; and the filtrate was repeatedly filtered 3 times.
The mass ratio of the filter residue to the distilled water in the step 3 is 1:7, the ultrasonic separation temperature is 30 ℃, the ultrasonic frequency is 90kHz, the secondary filter residue obtained after the filtration of the crushed prefabricated liquid is placed into the distilled water for secondary low-temperature ultrasonic treatment, the ultrasonic treatment is repeated for 4 times, the filtered filtrate is combined into the crushed prefabricated liquid, the temperature of the low-temperature ultrasonic treatment is 10 ℃, and the ultrasonic frequency is 90kHz.
The temperature of the low-temperature ultrasonic treatment in the step 4 is 5 ℃, the ultrasonic frequency is 70kHz, the time is 1h, and the temperature of the low-temperature centrifugation is 10 ℃. The volume of the fermentation product is 10 times that of the fermentation liquid.
The fermentation product of this example was examined to be in the form of a clear liquid and the viable count was 1.27-1.33 hundred million/mL.
Example 3
A fermentation culture process of strains comprises the following steps:
step 1, inoculating strains into a culture medium for thawing culture to obtain seed liquid;
step 2, inoculating the seed solution into a fermentation medium for anaerobic fermentation culture to obtain a fermentation liquor;
step 3, adsorbing and filtering the fermentation liquor to obtain filter residues, then placing the filter residues into water for ultrasonic separation, and filtering to obtain crushed prefabricated liquor;
and 4, carrying out low-temperature ultrasonic treatment on the crushed prefabricated liquid, centrifuging at a low temperature, and taking supernatant to obtain a fermentation product.
The mass ratio of the culture medium in the step 1 is as follows: 7% of skimmed milk powder, 0.4% of cyclodextrin, 1.3% of corn flour, 0.3% of glucose, 0.8% of yeast extract, 0.2% of peptone, 0.3% of lactose and 1.1% of dipotassium hydrogen phosphate; the balance being water. The cyclodextrin is beta-cyclodextrin.
The preparation method of the culture medium comprises the following steps: a1, adding glucose, peptone and iso-lactose into water, uniformly stirring, then adding cyclodextrin to form ultrasonic treatment for 15min to obtain an initial solution, wherein the stirring speed is 1500r/min, the ultrasonic frequency of ultrasonic treatment is 60kHz, and the temperature is 25 ℃; a2, adding the yeast extract into the primary solution, and performing low-temperature ultrasonic treatment for 15min to obtain a prefabricated inclusion liquid; the temperature of the low-temperature ultrasonic treatment is 8 ℃, and the ultrasonic frequency is 60kHz; and a3, adding corn flour, skimmed milk powder and potassium dihydrogen phosphate into the pre-prepared inclusion liquid, and rapidly stirring to obtain a culture medium, wherein the rapid stirring temperature is 8 ℃, and the stirring speed is 2500r/min.
The temperature of the thawing culture in the step 1 is 35 ℃ and the time is 15h.
The strain adopts commercial bifidobacterium.
The inoculation amount of the strain is 6% of the mass of the culture medium.
The inoculation amount of the seed liquid in the step 2 is 7% of that of the fermentation medium, and the anaerobic fermentation temperature is 38 ℃; the fermentation time is 8-10h, and the pH is 7.2. The step supplies sufficient carbon to the strain in weak period, so as to greatly improve the activity of the strain and obtain long-term fermentation culture.
The mass ratio of the fermentation medium in the step 2 is as follows: glucose 3%, corn extract 1.8%, yeast powder 2%, beef peptone 1%, ferulic acid 1.8%, sodium acetate 0.7%, citric acid triammonium 0.2%, ammonium bicarbonate 0.3%, tween-80.1%, mgSO4 0.05%, mnSO40.02%, potassium dihydrogen phosphate 0.05% and the balance water; the method for wrapping the ammonium bicarbonate comprises the steps of adding micro-nano ammonium bicarbonate into olive oil, rapidly stirring, filtering and then airing at a high position to obtain an olive oil-wrapped ammonium bicarbonate material, wherein the mass ratio of the ammonium bicarbonate to the olive oil is 1:3, the rapid stirring speed is 1500r/min, and the air airing at a high position is 15 ℃; the preparation method of the fermentation medium comprises the following steps: b1, glucose, corn extract, yeast powder, beef peptone, ferulic acid, sodium acetate, tri-ammonium citrate, tween-80 and MgSO 4 、MnSO 4 Adding potassium dihydrogen phosphate into water, stirring uniformly, and sterilizing to obtain a prefabricated culture medium, wherein the stirring speed of the uniform stirring is 1500r/min, and sterilizing is performed in an autoclave at 121 ℃; the pressure is 103.4kPa, and the time is 50 minutes; and b2, cooling the prefabricated culture medium to 30 ℃, then adding olive oil to wrap ammonium bicarbonate, and homogenizing and stirring to obtain the fermentation culture medium, wherein the temperature of the homogenizing and stirring is 30 ℃, and the stirring speed is 1500r/min.
The adsorption filtration in the step 3 adopts active carbon, and the void structure of the active carbon is gradually reduced from top to bottom; and repeatedly filtering the filtrate for 2-3 times.
The mass ratio of the filter residue to the distilled water in the step 3 is 1:6, the ultrasonic separation temperature is 25 ℃, the ultrasonic frequency is 85kHz, the secondary filter residue obtained after the filtration of the crushed prefabricated liquid is placed into the distilled water for secondary low-temperature ultrasonic treatment, the ultrasonic treatment is repeated for 3 times, the filtered filtrate is combined into the crushed prefabricated liquid, the temperature of the low-temperature ultrasonic treatment is 8 ℃, and the ultrasonic frequency is 85kHz.
The temperature of the low-temperature ultrasonic treatment in the step 4 is 4 ℃, the ultrasonic frequency is 60kHz, the time is 0.5h, and the temperature of the low-temperature centrifugation is 8 ℃. The volume of the fermentation product is 9 times of that of the fermentation liquid.
The fermentation product of this example was examined to be in the form of a clear liquid and the viable count was 1.23-1.31 hundred million/mL.
Actual measurement of Properties
Example 1 actual measurement of broiler chickens
The fermentation product of example 3 was used as a test example. The test time was 15 days of broiler until slaughter.
White feather broilers of the same batch and same day age were selected and randomly divided into 2 groups: control group and test group, each group contains 2.6 ten thousand; wherein the control group normally drinks water, and the test group puts 100mL of the fermentation product into 1000L of water.
The experimental data are as follows:
| comparative example | Test examples | |
| Number of columns (Only) | 26520 | 26520 |
| Number of columns (Only) | 25460 | 25558 |
| Death (only) | 1060 | 962 |
| Feed amount (kg) | 104780 | 105062 |
| Dry weight of chicken (jin) | 143540 | 147250 |
| Survival rate (%) | 96 | 96.4 |
| Average weight (jin) | 5.63 | 5.76 |
| Feed to meat ratio | 1.46 | 1.43 |
| Age of day | 41 | 42 |
From the above data, it is shown that: each chicken in the test group is 0.13 jin longer than that in the control group, the feed meat ratio is 0.033 lower than that in the control group, and the death rate is reduced by 0.4 points; the input-output ratio of the test group was 1:4 in terms of cost. Therefore, from the data, the fermentation product prepared by the invention can promote growth, reduce death rate and increase economic benefit.
Example 2 actual measurement of river crab
The test objects of the crabs and the feed of the crabs are divided into two groups: control and test groups, wherein the control group used commercial feed and the control group sprayed 100mL of fermentation product onto 500 jin of feed.
The nutrient levels of the commercial feed are as follows:
| nutrient level | Percentage (%) |
| Crude protein | ≥36 |
| Crude fat | ≥4.5 |
| Coarse fibers | ≤6.5 |
| Moisture content | ≤12 |
Experiment 1, selecting 120 crabs with strong physique and neat specification and initial weight of 0.3+/-0.05 g, randomly dividing the crabs into 2 groups, setting 3 repetitions in each group, and setting 20 repetitions in each group; the test period was fed 1 time per day for 19:00 a day, and the following morning 7:00 a day was cleaned of residual bait and changed in water. The cultivation period is 7 weeks. During the test, the water temperature is between 27 and 30 ℃, the pH is between 7.2 and 7.4, and the dissolved oxygen content is higher than 6.0mg/L.
The results of the production performance of the crabs are shown in the following table: the initial weight of the crabs is 0.3g, after 7 weeks of culture test, the final average weight and the weight gain rate of the crabs in the control group are respectively 4.89g and 1531.6%, the final average weight and the weight gain rate of the crabs in the test group are respectively 5.68g and 1794.7%, the weight gain rate is obviously higher than that of the control group, and the survival rate (65%) is obviously higher than that of the control group (55%).
Experiment 2, namely selecting 48 adult crabs with strong physique and regular specification and initial weight of 69+/-0.5 g, randomly dividing the adult crabs into 2 groups, and setting 3 repetitions in each group, wherein 8 repetitions are each repeated; the test period was fed 1 time per day for 19:00 a day, and the following morning 7:00 a day was cleaned of residual bait and changed in water. The cultivation period is 7 weeks. During the test, the water temperature is between 27 and 30 ℃, the pH is between 7.2 and 7.4, and the dissolved oxygen content is higher than 6.0mg/L.
The results of the production performance of the adult crabs are shown in the following table: after 7-week cultivation test, the final average cultivation of the control group was 94.1g, and the final average weight of the test group was 98.7g. The weight gain rate of the test group is 43.2%, which is obviously higher than that of the control group. In addition, the survival rate of the test group was equal to that of the control group.
The experiment shows that: the fermentation product prepared by the invention obviously promotes the growth of young crabs and adult crabs, and can also improve the survival rate of the stage of the crabs.
It is to be understood that the foregoing detailed description of the invention is merely illustrative of the invention and is not limited to the embodiments of the invention. It will be understood by those of ordinary skill in the art that the present invention may be modified or substituted for elements thereof to achieve the same technical effects; as long as the use requirement is met, the invention is within the protection scope of the invention.
Claims (10)
1. A fermentation culture process of strain is characterized in that: the method comprises the following steps:
step 1, inoculating strains into a culture medium for thawing culture to obtain seed liquid;
step 2, inoculating the seed solution into a fermentation medium for anaerobic fermentation culture to obtain a fermentation liquor;
step 3, adsorbing and filtering the fermentation liquor to obtain filter residues, then placing the filter residues into water for ultrasonic separation, and filtering to obtain crushed prefabricated liquor;
and 4, carrying out low-temperature ultrasonic treatment on the crushed prefabricated liquid, centrifuging at a low temperature, and taking supernatant to obtain a fermentation product.
2. The fermentation culture process of the strain according to claim 1, wherein: the mass ratio of the culture medium in the step 1 is as follows: 5-8% of skimmed milk powder, 0.3-0.5% of cyclodextrin, 1.2-1.4% of corn flour, 0.2-0.4% of glucose, 0.7-1.1% of yeast extract, 0.2-0.3% of peptone, 0.2-0.5% of lactose and 1.1% of dipotassium hydrogen phosphate; the balance being water.
3. The fermentation culture process of the strain according to claim 1, wherein: the temperature of the thawing culture in the step 1 is 35-36 ℃ and the time is 10-20h.
4. The fermentation culture process of the strain according to claim 1, wherein the inoculation amount of the strain is 5-6% of the mass of the culture medium.
5. The fermentation culture process of the strain according to claim 1, wherein the seed liquid in the step 2 is inoculated in an amount of 6-8% of a fermentation medium, and the anaerobic fermentation temperature is 38-39 ℃; the fermentation time is 8-10h, and the pH is 7.1-7.3.
6. The fermentation culture process of the strain according to claim 1, wherein the mass ratio of the fermentation medium in the step 2 is as follows: 2-4% of glucose, 1.5-2% of corn extract, 1.5-3% of yeast powder, 1.0-2% of beef peptone, 1.5-3% of ferulic acid, 0.5-1% of sodium acetate, 0.2-0.3% of triammonium citrate, 0.1-0.5% of ammonium bicarbonate, 0.1% of tween-80, 0.05% of MgSO4, 0.02% of MnSO4, 0.05% of monopotassium phosphate and the balance of water.
7. The fermentation culture process of the strain according to claim 1, wherein: and the adsorption filtration in the step 3 adopts active carbon, and the void structure of the active carbon is gradually reduced from top to bottom.
8. The fermentation culture process of the strain according to claim 1, wherein: the mass ratio of the filter residue to distilled water in the step 3 is 1:4-7, the ultrasonic separation temperature is 20-30 ℃, and the ultrasonic frequency is 80-90kHz.
9. The fermentation culture process of the strain according to claim 1, wherein the low-temperature ultrasonic treatment in the step 4 is carried out at a temperature of 1-5 ℃, ultrasonic frequency of 50-70kHz, time of 0.5-1h and low-temperature centrifugation temperature of 5-10 ℃.
10. The fermentation culture process of the strain of claim 1, wherein the ratio of the fermentation product to water is 1:10000 in the use process.
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