CN116114791A - Microecological preparation containing modified sodium humate and preparation method thereof - Google Patents
Microecological preparation containing modified sodium humate and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/105—Aliphatic or alicyclic compounds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/28—Silicates, e.g. perlites, zeolites or bentonites
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- 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
- C12R2001/07—Bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- 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
- C12R2001/145—Clostridium
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a composite microecological preparation containing modified sodium humate and a preparation method thereof, wherein the composite microecological preparation containing the modified sodium humate is prepared by carrying out primary combined fermentation on bacillus subtilis, clostridium butyricum and bacillus coagulans bacterial liquid, then adding the modified sodium humate, corn starch, talcum powder and salt, drying and sieving.
Description
Technical Field
The invention belongs to the field of feed additives, and particularly relates to a microecological preparation containing modified sodium humate and a preparation method thereof.
Background
The microbial ecological agent is a living microbial agent prepared by utilizing normal microorganisms or substances for promoting the growth of microorganisms, and has the advantages of multiple biological activity functions, no environmental pollution, no residual influence and the like, thereby playing a great role in indirectly replacing antibiotic drugs. At present, the strains of the microecological preparation mainly comprise saccharomyces cerevisiae, bacillus subtilis, bacillus licheniformis, bacillus coagulans, clostridium butyricum, lactobacillus plantarum, lactobacillus acidophilus and enterococcus faecium, and most of the strains are simply mixed after single strain fermentation, so that nutrient substances of the culture substrate are wasted.
Sodium humate C 9 H 8 Na 2 O 4 The zinc oxide feed additive has the effects of inhibiting bacteria, relieving diarrhea and astringing, is nontoxic, odorless and corrosion-free, and is considered as one of important substitutes for the 'post-antibiotic age' of livestock and poultry cultivation and the forbidden zinc oxide for feeding. However, the prior art shows that the pH value (pH is more than 11.0) of the preparation is greatly influenced by adding 1% of common sodium humate as a carrier, so that the activity of the microecological preparation is weakened or even lost in the preparation and storage processes.
Patent CN108865935a discloses a microecological preparation with improved water quality and a preparation method thereof, which is a liquid preparation, and most of carriers are water-insoluble, so that the dispersion degree of thalli in the preparation is affected, and thalli precipitation is easy to occur, thereby leading to difficult maintenance of activity of a formed product and storage inactivation.
Patent CN109010662a discloses a digestion-promoting and growth-promoting microecological preparation, wherein the effective bacterial powder and active substances are unevenly dispersed due to the respective density difference.
In summary, there is a need in the market for a method for producing a microecological preparation that has high activity, uniform dispersion, easy storage, and can utilize a mixture of various strains of humic acid.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a composite microecological preparation containing modified sodium humate and a preparation method thereof, and the composite microecological preparation containing modified sodium humate is prepared by carrying out primary combined fermentation on bacillus subtilis, clostridium butyricum and bacillus coagulans bacterial liquid, then adding modified sodium humate, corn starch, talcum powder and salt, drying and sieving, wherein the composite microecological preparation is prepared by collocating aerobic, facultative anaerobic and anaerobic nutritional type thalli, and the aerobic synergistic combined fermentation is realized, so that the finished thalli has reasonable proportion, high total number of effective viable bacteria, uniform dispersity and long stability period, and is more resistant to storage and transportation.
In order to achieve the above effects, the present invention adopts the following technical scheme:
the invention provides a preparation method of a mixed microecological preparation, which comprises the following steps:
1) Preparing bacillus subtilis liquid, bacillus coagulans liquid and clostridium butyricum liquid by using the culture liquid respectively;
2) Preparing a fermentation medium by using molasses, medium Wen Doubing powder, ammonium sulfate, corn flour, dephenolized cottonseed protein powder, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate and water;
3) Inoculating bacillus subtilis liquid to a fermentation medium, culturing for a period of time, and then inoculating for the second time;
4) Inoculating bacillus coagulans liquid to the fermentation medium in the step 3), culturing for a period of time, and then carrying out third inoculation;
5) Inoculating clostridium butyricum liquid to the fermentation medium in the step 4), and culturing to obtain mixed bacterial liquid;
6) And uniformly mixing the mixed bacterial liquid, the modified sodium humate, the corn starch, the talcum powder and the salt, drying and sieving to obtain the mixed microecological preparation containing the sodium humate.
Further, the culture solution in the step 1) is prepared from yeast extract powder 0.3% -0.5%, peptone 0.8% -1.0%, glucose 0.5% -0.8%, sodium chloride 0.8% -1.0%, calcium chloride 0.03% -0.05%, manganese sulfate 0.01% -0.03%, ammonium sulfate 0.05% -0.08%, pH value is regulated to 6.5+ -0.5 by sodium hydroxide and hydrochloric acid, water is supplemented to 100%, high-temperature sterilization, cooling, inoculation, cultivation and bacterial solution concentration OD 600 Is 0.6X10 8 -1.0×10 8 CFU/mL。
Further, in the fermentation medium of the step 2), 3 to 5 percent of molasses, 1.5 to 3.0 percent of medium Wen Doubing powder, 0.05 to 0.10 percent of ammonium sulfate, 2 to 5 percent of corn powder, 0.1 to 0.2 percent of dephenolized cottonseed protein powder, 0.05 to 0.15 percent of dipotassium hydrogen phosphate, 0.1 to 0.3 percent of sodium dihydrogen phosphate, 0.05 to 0.15 percent of magnesium sulfate, 0.01 to 0.05 percent of calcium chloride, 0.003 to 0.01 percent of manganese sulfate and 100 percent of water are supplemented.
Further, the step 3) is to inoculate bacillus subtilis liquid according to the proportion of 0.5% -2% (v/v); the culture condition is to use 1M phosphoric acid or ammonia water to adjust the pH of the fermentation culture medium, and the pH is controlled to be 4.5-6.5; controlling the dissolved oxygen to be 20% -50%; the culture was carried out at 37℃for 12-14 hours, and the culture temperature was slowly raised to 40℃over 1 hour.
Further, the step 4) is to inoculate bacillus coagulans seed liquid according to the proportion of 1% -2.5% (v/v); the culture condition is to use 1M phosphoric acid or ammonia water to adjust the pH of the fermentation culture medium, the pH is controlled to be 6.5-7.5, the dissolved oxygen is controlled to be 15-25%, the culture is carried out for 13-16 hours at 40 ℃, and the temperature is slowly reduced to 35 ℃ within 1 hour.
Further, the step 5) is to inoculate clostridium butyricum seed liquid according to the proportion of 1% -3% (v/v); the culture condition is to use 1M phosphoric acid or ammonia water to adjust the pH of the fermentation culture medium, the pH is controlled to be 6.0-8.0, and the fermentation culture medium is subjected to stationary culture for 20-24 hours.
Further, in the step 6), 5% -12% (w/w) of modified sodium humate, 3% -5% (w/w) of corn starch, 0.05% -0.1% (w/w) of talcum powder and 0.1% -0.2% (w/w) of salt; drying is spray drying; spray air inlet temperature is 127-138 ℃, and air outlet temperature is 68-72 ℃.
The invention also provides a mixed microecological preparation containing sodium humate, which is prepared by the preparation method.
The preparation method of the modified sodium humate used in the invention comprises the following steps:
(1) Uniformly mixing 10 parts of humic acid mineral powder, 1 part of sodium hydroxide and 10 parts of distilled water in a stirring reaction kettle, and introducing steam to react for 120 minutes to obtain a sodium humate crude product;
(2) Centrifuging the crude sodium humate at 2000rpm for 20min to obtain a centrifugate containing soluble sodium humate, and transferring the centrifugate into a stirring reaction kettle;
(3) Adjusting the sodium humate content in the centrifugate to 40-50% in a stirring reaction kettle;
(4) Adding single Ning Zhi acyl hydrolase and laccase into a stirring reaction kettle for enzymolysis; the tannic acid hydrolase accounts for 0.05 to 0.25 percent of the weight of the sodium humate; laccase accounts for 0.01-0.05% of the weight of sodium humate, the reaction temperature is 40-60 ℃, the reaction is carried out at 150-200rpm for 60-120min, and the modified sodium humate reaction solution is obtained;
(5) And drying the modified sodium humate reaction solution to obtain a modified sodium humate product.
The beneficial effects are that:
1. according to the invention, the aerobic bacillus subtilis is inoculated firstly, a large amount of oxygen is consumed, a proper low-oxygen environment is provided for secondary inoculation and fermentation of the facultative anaerobic bacillus coagulans, a proper anaerobic environment is provided for tertiary inoculation and fermentation of the anaerobic clostridium butyricum after the primary two bacterial strains are inoculated and fermented for a period of time, the three bacterial strains realize aerobic synergistic fermentation, the primary competitive inhibition can be avoided by the tertiary inoculation, the proliferation can be promoted mutually, and the respective fermentation period is shortened.
2. According to the invention, bacillus subtilis, clostridium butyricum and bacillus coagulans are inoculated for three times through the collocation of aerobic, facultative anaerobic and anaerobic nutritional type thalli, and the combined fermentation is carried out once, so that the effective utilization of secondary metabolites of strains and the full utilization of nutrient components of a culture medium are realized.
3. The modified sodium humate is added, so that the pH of the microecological preparation is reduced, and the effective viable bacteria retention rate of the microecological preparation is improved.
4. When the modified sodium humate-containing microecological preparation prepared by the invention is used as a feed additive, the beneficial effects of beneficial bacteria in animal digestive tracts can be maintained and the toxic effects of harmful bacteria are limited by improving the number of living bacteria and the effective survival rate of the living bacteria, so that the anti-stress capability of organisms is improved, the feed conversion rate is further improved, and the weight of animals is increased.
Detailed Description
The invention will be further illustrated with reference to specific examples, but the invention is not limited to these examples only. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
The reagents used in the invention are all commercially available and have no special requirements; the fermentation equipment used in the invention is a main body of a commercial equipment with conventional volume and pressure requirements, and is modified by partially connecting pipelines and accessories according to actual culture requirements.
The strain sources of the invention are as follows: the collection number of the bacillus subtilis is CCTCC CB 20081704, the collection number of the clostridium butyricum is CCTCC CB 20081668, and the collection number of the bacillus coagulans is CCTCC AB 92066.
The preparation method of the liquid mixed microecological preparation refers to CN107699527, a probiotics microecological preparation, and the preparation method and application implementation thereof;
the preparation method of the solid mixed microecological preparation refers to CN107699527, a probiotics microecological preparation, and the preparation method and application implementation thereof;
common sodium humate refers to CN109081924, which is sodium humate for feeding, and a preparation method and application implementation thereof;
preparation of modified sodium humate refers to CN201810462099.5 modified sodium humate products, and a preparation method and application implementation thereof;
the measurement of the number of living bacteria is carried out by referring to the measurement of the total number of microbial colonies in the food of GB 4789.2-2016;
the reducing sugar content was determined with reference to the reducing sugar content of the food of GB 5009.7-2016.
Example 1:
a preparation method of a microecological preparation containing modified sodium humate comprises the following steps:
1) Preparation of mixed fermentation broth of bacillus subtilis, clostridium butyricum and bacillus coagulans
(1) Preparing seed liquid: taking materials according to 0.5% of yeast extract powder, 1.0% of peptone, 0.5% of glucose, 1.0% of sodium chloride, 0.03% of calcium chloride, 0.01% of manganese sulfate and 0.05% of ammonium sulfate, regulating the pH to 6.5 by using hydrochloric acid, supplementing 100% of water, sterilizing at high temperature, and cooling; respectively inoculating Bacillus subtilis, bacillus coagulans and Clostridium butyricum, preparing strain seed solution, and culturing to obtain strain solution concentration (OD) 600 ) 0.7X10 respectively 8 CFU/mL、0.85×10 8 CFU/mL、0.9×10 8 CFU/mL。
(2) Preparing a fermentation medium: according to the components of 4 percent of molasses, 2.0 percent of medium Wen Doubing powder, 0.07 percent of ammonium sulfate, 3 percent of corn meal, 0.15 percent of dephenolized cottonseed protein powder, 0.1 percent of dipotassium hydrogen phosphate, 0.2 percent of sodium dihydrogen phosphate, 0.1 percent of magnesium sulfate, 0.03 percent of calcium chloride, 0.007 percent of manganese sulfate and 100 percent of water, and the materials are taken and uniformly mixed.
2) Fermentation process control
(1) Inoculating bacillus subtilis seed liquid according to the proportion of 1% (v/v), controlling the pH of an initial culture medium to 5.0 (1M phosphoric acid adjustment), controlling the rotating speed to 100rpm-200rpm, ventilating to 0.6-0.7:1, controlling dissolved oxygen to 30% -40%, culturing for 20 hours at 37 ℃, and detecting the number of viable bacteria to 5.0X10% 9 CFU/mL, the incubation temperature was slowly raised to 40℃over 1h, and a second inoculation was performed (see step (2)).
(2) Inoculating Bacillus coagulans seed solution at a ratio of 1.5% (v/v), controlling pH of culture medium to 7.0 (1M ammonia water adjustment), rotating at 70rpm-80rpm, ventilating at 0.25:1, controlling dissolved oxygen to 18% -22%, culturing at 40deg.C for 22 hr, and detecting viable count to 8.9X10) 9 CFU/mL, stopping ventilation, stirring for 30s (50-100 rpm) every 30min, slowly cooling to 35deg.C within 1 hr, and performing third timeInoculation (see step (3)).
(3) Clostridium butyricum seed solution was inoculated at a ratio of 4% (v/v), the pH of the medium was controlled to 7.0 (1M phosphoric acid adjustment), and the culture was allowed to stand for 26 hours. Sampling and detecting total number of living bacteria 1.2×10 10 CFU/ml, microscopic spore rate 98.5%, and wet weight of thallus 300g/L.
3) Preparing the compound microecological preparation containing the modified sodium humate
The mixed fermentation bacteria liquid of bacillus subtilis, clostridium butyricum and bacillus coagulans is obtained by primary combined fermentation, large-particle fermentation residues in the mixed fermentation bacteria liquid are removed by primary filtration (40 meshes), 8% (w/w) modified sodium humate, 4% (w/w) corn starch, 0.07% talcum powder (w/w) and 0.15% salt (w/w) are added into the filtrate, and spray drying and temperature control are carried out after uniform stirring: the inlet air temperature is 132 ℃, and the outlet air temperature is 70 ℃. And (5) sieving the dried powder with a 60-mesh sieve, detecting the powder to be qualified, and packaging and warehousing the powder.
Example 2
Comparing the consumption condition of the two culture processes on nutrient substances and the growth condition of thalli when the fermentation period is 70 hours by the bacillus subtilis, clostridium butyricum and bacillus coagulans combined fermentation and duplex strain fermentation, respectively setting an experimental group and a control group:
1) Experimental setup
Experimental group: three bacteria, bacillus subtilis, bacillus coagulans and clostridium butyricum were co-cultured in accordance with example 1, and the number of viable bacteria and the content of reducing sugar were measured.
Control group 1: bacillus subtilis+clostridium butyricum
Control group 2: bacillus subtilis+bacillus coagulans
Control group 3: bacillus coagulans + clostridium butyricum,
the control groups 1-3 were fermented and cultured respectively, and the concentration of the strains used by bacillus subtilis, clostridium butyricum and bacillus coagulans, the fermentation medium and the fermentation process were all the same as those in example 1, and the viable count and the reducing sugar content were detected respectively when the fermentation period was 70 h.
3) The test results are shown in Table 1:
TABLE 1 viable count and reducing sugar content of control and experimental formulations
Table 1 shows that: under the same fermentation period and nutrient composition conditions, the three-bacterium combined fermentation (bacillus subtilis, bacillus coagulans and clostridium butyricum) is superior to the two-bacterium combined fermentation in thallus proliferation and nutrient utilization, so that the production cost is saved, and the production efficiency is improved.
Example 3
The storage stability of the modified sodium humate-containing composite microecologics prepared in comparative example 1 and conventional liquid and solid microecologics (bacterial powder is independently mixed) is respectively set as an experimental group and a control group:
1) Experimental setup
Experimental group: the modified sodium humate-containing composite microecological preparation obtained in example 1
Control group 1: liquid mixed microecological preparation
Control group 2: solid mixed microecological preparation
Setting a group A and a group B respectively, standing for 30d under the treatment condition of the group A at 35 ℃; group B was left for 30d with reciprocal shaking (50 times/min) at 35℃to observe the appearance and to detect the total number of viable bacteria.
2) The test results are shown in Table 2:
table 2 viable count of formulations of control and experimental groups under different conditions
Table 2 shows that: standing for 30d at 35 ℃, wherein the loss of the viable bacteria of the composite microecological preparation containing the modified sodium humate is within 0.2%, the loss of the viable bacteria of the liquid mixed microecological preparation is 27.7%, the loss of the solid mixed microecological preparation is 9.9%, and meanwhile, the liquid mixed microecological preparation starts to generate obvious bacterial precipitation from 1 d; and (3) standing for 30 days at 35 ℃ in a reciprocating way (50 times/min), wherein the loss of the viable bacteria of the composite microecological preparation containing the modified sodium humate is within 0.5%, the loss of the viable bacteria of the liquid mixed microecological preparation reaches 40.2%, and the loss of the solid mixed microecological preparation reaches 14.4%. Experiments prove that the modified sodium humate-containing composite microecological preparation has better storage stability.
Example 4
The modified sodium humate-containing composite microecologics prepared in comparative example 1 and conventional liquid and solid microecologics (bacterial powder is independently mixed) are uniformly dispersed, and an experimental group and a control group are respectively arranged:
1) Experimental setup
Samples of the experimental group and the control group at the 7 th day of the test in example 3 were sampled at different positions to detect the total number of viable bacteria.
2) The test results are shown in table 3:
TABLE 3 viable count of control and experimental formulations under different conditions
Table 3 shows that: under the standing condition, the liquid mixed microecological preparation is extremely uneven in dispersion, the liquid mixed microecological preparation is related to bacterial precipitation, the solid mixed microecological preparation is good in uniformity degree, and the modified sodium humate-containing composite microecological preparation is best in dispersion uniformity degree, and is related to initial mixing uniformity degree; under the condition of reciprocating oscillation, the dispersion uniformity of the micro-ecological preparation mixed by the single fungus powder in the later stage is the worst, the micro-ecological preparation mixed by the liquid is better in dispersion degree relative to the respective densities of the carrier and the fungus powder, and the compound micro-ecological preparation containing the modified sodium humate is the best in dispersion uniformity degree.
Example 5
Effective viable bacteria stability in a spray drying process of the modified sodium humate-containing compound microecological preparation and the common sodium humate-containing compound microecological preparation prepared in comparative example 1 is respectively provided with an experimental group and a control group:
1) Experimental setup
Experimental group: the modified sodium humate-containing composite microecological preparation prepared in example 1
Control group: the modified sodium humate added in the example 1 is replaced by common sodium humate, and the pH value of the mixed solution of the modified sodium humate and the common sodium humate and the effective viable bacteria retention rate in the preparation process are compared.
2) The test results are shown in table 4:
TABLE 4 pH and effective viable bacteria retention rate during formulation of control and experimental formulations
| Group of | pH | Effective viable bacteria retention rate |
| Experimental group | 6.45 | 99.8％ |
| Control group | 11.93 | 84.3％ |
Table 4 shows that: compared with the high pH environment caused by common sodium humate, the composite microecological preparation containing the modified sodium humate is more beneficial to improving the effective viable bacteria retention rate of the microecological preparation from the perspective of changing the pH of the environment where the microecological preparation is located.
The above examples are intended to illustrate the explanation and specific embodiments and implementations of the present invention and are not intended to limit the present invention, and those skilled in the art, after having read the present specification, may make modifications as necessary without creative contribution to the invention, but are protected by patent laws within the scope of the claims of the present invention.
Claims (9)
1. The preparation method of the mixed microecological preparation is characterized by comprising the following steps:
1) Preparing bacillus subtilis liquid, bacillus coagulans liquid and clostridium butyricum liquid by using the culture liquid respectively;
2) Preparing a fermentation medium by using molasses, medium Wen Doubing powder, ammonium sulfate, corn flour, dephenolized cottonseed protein powder, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate and water;
3) Inoculating bacillus subtilis liquid to a fermentation medium, culturing for a period of time, and then inoculating for the second time;
4) Inoculating bacillus coagulans liquid to the fermentation medium in the step 3), culturing for a period of time, and then carrying out third inoculation;
5) Inoculating clostridium butyricum liquid to the fermentation medium in the step 4), and culturing to obtain mixed bacterial liquid;
6) And uniformly mixing the mixed bacterial liquid, the modified sodium humate, the corn starch, the talcum powder and the salt, drying and sieving to obtain the mixed microecological preparation containing the sodium humate.
2. The preparation method of claim 1, wherein the culture solution in the step 1) is yeast extract powder 0.3% -0.5%, peptone 0.8% -1.0%, glucose 0.5% -0.8%, sodium chloride 0.8% -1.0%, calcium chloride 0.03% -0.05%, manganese sulfate 0.01% -0.03%, ammonium sulfate 0.05% -0.08%, pH is regulated to 6.5+ -0.5 by sodium hydroxide and hydrochloric acid, water is supplemented by 100%, and the culture solution is sterilized at high temperature; the concentration OD of the bacterial liquid 600 Is 0.6X10 8 -1.0×10 8 CFU/mL。
3. The preparation method according to claim 1, wherein in the fermentation medium in step 2), molasses 3% -5%, medium Wen Doubing meal 1.5% -3.0%, ammonium sulfate 0.05% -0.10%, corn meal 2% -5%, dephenolized cottonseed protein powder 0.1% -0.2%, dipotassium hydrogen phosphate 0.05% -0.15%, sodium dihydrogen phosphate 0.1% -0.3%, magnesium sulfate 0.05% -0.15%, calcium chloride 0.01% -0.05%, manganese sulfate 0.003% -0.01% and water are supplemented by 100%.
4. The preparation method according to claim 1, wherein the inoculation in the step 3) is to inoculate bacillus subtilis liquid in a proportion of 0.5% -2% by volume; the culture condition is pH4.5-6.5, dissolved oxygen is controlled to be 20-50%, the culture is carried out for 12-14 hours at 37 ℃, and the culture temperature is slowly increased to 40 ℃ within 1 h.
5. The preparation method according to claim 1, wherein the inoculation in the step 4) is to inoculate bacillus coagulans seed liquid in a proportion of 1% -2.5% by volume; the culture condition is pH6.5-7.5, dissolved oxygen is controlled to be 15-25%, and the culture is carried out for 13-16 hours at 40 ℃ and slowly cooled to 35 ℃ within 1 h.
6. The method according to claim 1, wherein the inoculation in step 5) is to inoculate clostridium butyricum seed liquid in a proportion of 1% -3% by volume; the culture condition is pH6.0-8.0, and the stationary culture is carried out for 20-24 hours.
7. The preparation method of claim 1, wherein in step 6), the modified sodium humate accounts for 5% -12%, the corn starch accounts for 3% -5%, the talcum powder accounts for 0.05% -0.1% and the salt accounts for 0.1% -0.2% by weight.
8. The method of claim 1, wherein the drying of step 6) is spray drying; the spray air inlet temperature is 127-138 ℃, and the air outlet temperature is 68-72 ℃.
9. A mixed microecological preparation containing sodium humate prepared by the preparation method according to any one of claims 1 to 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211694601.8A CN116114791A (en) | 2022-12-28 | 2022-12-28 | Microecological preparation containing modified sodium humate and preparation method thereof |
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| CN116649464A (en) * | 2023-07-27 | 2023-08-29 | 山东昊日农牧生物技术有限公司 | Environment-friendly type fermented feed with tiepiece based on complex flora fermentation and preparation method thereof |
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