CN115354005A - Compound microbial preparation and preparation method and storage method thereof - Google Patents
Compound microbial preparation and preparation method and storage method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 98
- 230000000813 microbial effect Effects 0.000 title claims abstract description 82
- 238000000034 method Methods 0.000 title claims abstract description 43
- 150000001875 compounds Chemical class 0.000 title claims abstract description 21
- 238000003860 storage Methods 0.000 title description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 71
- 230000004151 fermentation Effects 0.000 claims abstract description 71
- 241000186660 Lactobacillus Species 0.000 claims abstract description 67
- 229940039696 lactobacillus Drugs 0.000 claims abstract description 67
- 241000194033 Enterococcus Species 0.000 claims abstract description 59
- 238000004321 preservation Methods 0.000 claims abstract description 35
- 239000002131 composite material Substances 0.000 claims abstract description 32
- 241000894006 Bacteria Species 0.000 claims abstract description 24
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- 230000001105 regulatory effect Effects 0.000 claims abstract description 16
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- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 41
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 claims description 28
- 235000020661 alpha-linolenic acid Nutrition 0.000 claims description 28
- 229960004488 linolenic acid Drugs 0.000 claims description 28
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- 239000008103 glucose Substances 0.000 claims description 25
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- 239000008120 corn starch Substances 0.000 claims description 20
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- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 20
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 20
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 20
- 229960005055 sodium ascorbate Drugs 0.000 claims description 20
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- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 20
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- RZRNAYUHWVFMIP-KTKRTIGZSA-N 1-oleoylglycerol Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(O)CO RZRNAYUHWVFMIP-KTKRTIGZSA-N 0.000 claims description 18
- RZRNAYUHWVFMIP-HXUWFJFHSA-N glycerol monolinoleate Natural products CCCCCCCCC=CCCCCCCCC(=O)OC[C@H](O)CO RZRNAYUHWVFMIP-HXUWFJFHSA-N 0.000 claims description 18
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 17
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 229940032049 enterococcus faecalis Drugs 0.000 claims description 4
- 229940076230 magnesium sulfate monohydrate Drugs 0.000 claims description 4
- LFCFXZHKDRJMNS-UHFFFAOYSA-L magnesium;sulfate;hydrate Chemical compound O.[Mg+2].[O-]S([O-])(=O)=O LFCFXZHKDRJMNS-UHFFFAOYSA-L 0.000 claims description 4
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 4
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- 235000017281 sodium acetate Nutrition 0.000 claims description 4
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- 240000001046 Lactobacillus acidophilus Species 0.000 claims description 3
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- 241000218588 Lactobacillus rhamnosus Species 0.000 claims description 3
- 239000000919 ceramic Substances 0.000 claims description 3
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- 239000012528 membrane Substances 0.000 claims description 3
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- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims description 2
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- 241000186673 Lactobacillus delbrueckii Species 0.000 claims description 2
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- 239000000263 2,3-dihydroxypropyl (Z)-octadec-9-enoate Substances 0.000 description 7
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- 239000002609 medium Substances 0.000 description 7
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- 239000001963 growth medium Substances 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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Classifications
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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
-
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
-
- 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
- C12R2001/225—Lactobacillus
-
- 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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a compound microbial preparation, a preparation method and a preservation method thereof, and relates to the field of microbes. According to the invention, the pH in the fermentation process is regulated and controlled in stages, so that the mixed fermentation effect of lactobacillus and enterococcus can be improved, the number and activity of two strains are balanced, the quality of the microbial inoculum is improved, the problem of a short plate with weak stress resistance of lactobacillus is solved, and the occurrence of mixed bacteria in mixed fermentation is effectively avoided or reduced. The invention also provides a preservation reagent which can effectively prolong the preservation period of the composite microbial preparation, the bacterial quantity is not obviously reduced within the preservation time limit of 30 days and 90 days, the preservation effect is better than that of the existing composite microbial preparation of lactobacillus and enterococcus, and the production activity is greatly promoted.
Description
Technical Field
The invention relates to the field of microorganisms, and particularly relates to a compound microbial preparation, and a preparation method and a preservation method thereof.
Background
Lactic acid bacteria are a general term for a group of bacteria that can produce a large amount of lactic acid using carbohydrates and cannot form spores. The bacteria are widely distributed in nature and have abundant species diversity. Except for a few, most of the lactobacillus is a flora which is indispensable in a human body and has important physiological functions, and is widely present in intestinal tracts of the human body, so the lactobacillus is also the most widely applied bacterium in the probiotic industry at present.
The lactobacillus is applied to the fermentation industry for a long time, is widely applied to lactic acid bacteria, has the advantages of strong acid production capacity and high bacterial yield after culture expansion, can play roles in promoting digestion and improving intestinal functions, but has the problems of poor stress resistance, general competitive capacity and the like. Although enterococcus faecalis and enterococcus faecium in the enterococcus genus also belong to lactic acid bacteria, the application is not as good as lactobacillus.
At present, the lactobacillus and the enterococcus have the problems of ineffective application, such as the problems of easy pollution, poor survivability and the like during fermentation, and the conditions of low bacterial quantity, more mixed bacteria and the like easily occur in the processes of storage, transportation and application.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a compound microbial preparation, and a preparation method and a preservation method thereof.
The invention is realized by the following steps:
in a first aspect, an embodiment of the present invention provides a method for preparing a complex microbial preparation, which includes the following steps: mixing the seed liquid of lactobacillus and the seed liquid of enterococcus, and fermenting; in the fermentation process, the pH in the fermentation medium is regulated and controlled according to 3 stages: in the first stage, regulating and controlling the pH value to be 6.6-6.8; in the second stage, regulating and controlling the pH value to be 5.9-6.1; in the third stage, the pH is regulated to 5.0-5.2; wherein the first stage is 0 to (6 to 10) hours of fermentation, the second stage is (6 to 10) to (14 to 18) hours of fermentation, and the third stage is (14 to 18) to (28 to 32) hours of fermentation.
In a second aspect, the embodiments of the present invention provide a complex microbial preparation, which is prepared from the complex microbial preparation described in the previous embodiments.
In a third aspect, the present invention provides a kit for preparing a complex microbial preparation, which includes the complex microbial preparation described in the previous embodiments.
In a fourth aspect, the embodiment of the present invention provides a preservation reagent for a complex microbial preparation, where the preservation reagent mainly includes, by weight: 0.5 to 0.6 portion of glucose, 2 to 2.4 portions of corn starch, 0.2 to 0.24 portion of sodium ascorbate, 0.48 to 0.5 portion of ferrous sulfate heptahydrate, 0.4 to 0.48 portion of dipotassium phosphate, 0.08 to 0.1 portion of potassium dihydrogen phosphate, 4 to 4.8 portions of linolenic acid, 0.5 to 0.6 portion of glycerol monooleate, 0.4 to 0.5 portion of carboxymethyl chitosan and 0.85 to 0.9 portion of sodium chloride.
In a fifth aspect, embodiments of the present invention provide a complex microbial preparation, comprising: the complex microbial preparation described in the foregoing example and the preservation reagent for the complex microbial preparation described in the foregoing example.
In a sixth aspect, an embodiment of the present invention provides a method for preserving a complex microbial preparation, including: the complex microbial preparation described in the foregoing example was mixed with the preservation reagent for the complex microbial preparation described in the foregoing example.
The invention has the following beneficial effects:
the invention discovers that the mixed fermentation effect of the lactobacillus and the enterococcus can be improved by regulating and controlling the pH in the fermentation process in stages, the quantity and the activity of two strains are balanced, the quality of the microbial inoculum is improved, the problem of short plate with weak stress resistance of the lactobacillus is solved, the occurrence of mixed bacteria in the mixed fermentation is effectively avoided or reduced, and a way is provided for the application of the composite microbial inoculum.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
The embodiment of the invention provides a preparation method of a compound microbial preparation, which comprises the following steps: mixing the seed liquid of lactobacillus and the seed liquid of enterococcus and fermenting; in the fermentation process, the pH in the fermentation medium is regulated and controlled according to 3 stages:
in the first stage, regulating and controlling the pH value to be 6.6-6.8;
in the second stage, regulating and controlling the pH value to be 5.9-6.1;
in the third stage, the pH is regulated to 5.0-5.2;
wherein the first stage is 0 to (6 to 10) hours of fermentation; the second stage is the (6-10) - (14-18) th hour of fermentation; the third stage is the (14 to 18) th to (28 to 32) th hours of fermentation.
Although lactobacillus grows at a slower rate than enterococcus, lactobacillus is more tolerant to low pH; wherein, in the first stage of fermentation, the pH is controlled to be 6.6-6.8, mainly for the purpose of enabling enterococcus to be greatly amplified to play a role in inhibiting infectious microbes; in the second stage, the pH is controlled to be 5.9-6.1, mainly because the enterococcus and the lactobacillus in the stage are scaled, the growth of the enterococcus is slowed down by reducing the pH, meanwhile, the pH has no influence on the growth of the lactobacillus, and the lactobacillus can be largely expanded and cultured in the stage; and in the third stage, the pH is controlled to be 5.0-5.2, mainly, although the growth of the lactobacillus is also inhibited to a certain extent under the pH, the enterococcus hardly grows under the pH, and the ratio of the enterococcus to the lactobacillus in the fermentation liquid is adjusted through the fermentation in the third stage, so that the ratio of the lactobacillus is increased.
The preparation method of the lactobacillus and enterococcus compound microecological preparation provided by the embodiment of the invention is simple to operate, bacteria are not easily infected in the fermentation process, the ratio of the lactobacillus to the enterococcus after fermentation is close, the total bacteria content is high, and the preparation method is easy to apply.
Alternatively, the first stage is 0 to (a range between any one or any two of 6, 7, 8, 9 and 10) hours of fermentation. The second stage is the second hour of fermentation (any one or a range between any two of 6, 7, 8, 9 and 10) to (any one or a range between any two of 14, 15, 16, 17 and 18). The third stage is the (any one or range between any two of 14, 15, 16, 17 and 18) th hour of fermentation to (any one or range between any two of 28, 29, 30, 31 and 32).
In some preferred embodiments, the first stage is 0 to (7-9) hours of fermentation; the second stage is the (7-9) - (15-17) th hour of fermentation; the third stage is the (15-17) - (29-31) th hour of fermentation.
In some preferred embodiments, the first stage is from 0 to 8 hours of fermentation, the second stage is from 8 to 16 hours of fermentation, and the third stage is from 16 to 30 hours of fermentation.
In some embodiments, the method of making comprises modulating the pH in the fermentation medium with a pH modulating agent.
In some embodiments, the pH modulating agent comprises: 1-20% of sodium hydroxide and/or 1-20% of hydrochloric acid. Alternatively, the volume fractions of hydrochloric acid and sodium hydroxide may be independently any one of 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, or a range between any two.
In some embodiments, the ratio of viable bacteria of the seed fluid of lactobacillus to the seed fluid of enterococcus is: (0.5-2): (0.5 to 2), specifically, (any one or a range between any two of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2): (any one or a range between any two of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2).
In some embodiments, the lactobacillus is selected from at least one of lactobacillus rhamnosus, lactobacillus casei, lactobacillus delbrueckii, lactobacillus plantarum, lactobacillus reuteri, and lactobacillus acidophilus.
In some embodiments, the enterococcus includes at least one of enterococcus faecalis and enterococcus faecium.
In some embodiments, the conditions of the fermentation further comprise: at the temperature of 36-38 ℃, the rotating speed is 40-50 rpm, and ventilation is not performed; specifically, the temperature may be in the range of any one or two of 36 ℃, 37 ℃ and 38 ℃.
In some embodiments, the initial pH of the fermentation medium is 7.0 to 7.2. The pH may specifically be any one of 7.0, 7.1 and 7.2 or a range between any two.
In some embodiments, the fermentation medium employed for the fermentation comprises the following components, in parts by weight: 2 to 2.4 percent of glucose, 1 to 1.2 percent of maltose, 1 to 1.2 percent of bran, 1 to 1.2 percent of peptone, 1 to 1.2 percent of yeast extract powder, 0.5 to 0.6 percent of sodium acetate, 0.02 to 0.24 percent of magnesium sulfate monohydrate, 0.02 to 0.24 percent of manganese sulfate monohydrate, 0.2 to 0.24 percent of dipotassium hydrogen phosphate and the balance of water.
In some embodiments, the preparation method further comprises filtering the fermented bacteria liquid.
In some embodiments, the step of filtering comprises: filtering the fermented bacterial liquid by using a vibrating screen, wherein the volume ratio of the obtained filtrate to water is (1-3): (1-3), and then concentrating the mixture to the volume same as that of the original bacterium solution through a ceramic membrane filter. The volume ratio may be specifically in the range of any one or two of 1.
Preferably, in the composite microbial preparation, the ratio of the viable count of lactobacillus to the viable count of enterococcus is (1-1.5): 1.
the embodiment of the invention also provides a compound microbial preparation, which is prepared from the compound microbial preparation in any embodiment.
The embodiment of the invention also provides a kit of a compound microbial preparation, which comprises the compound microbial preparation as described in any of the preceding embodiments.
The embodiment of the invention also provides a preservation reagent of the compound microbial preparation, which comprises the following main components in parts by weight: 0.5 to 0.6 portion of glucose, 2 to 2.4 portions of corn starch, 0.2 to 0.24 portion of sodium ascorbate, 0.48 to 0.5 portion of ferrous sulfate heptahydrate, 0.4 to 0.48 portion of dipotassium phosphate, 0.08 to 0.1 portion of potassium dihydrogen phosphate, 4 to 4.8 portions of linolenic acid, 0.5 to 0.6 portion of glycerol monooleate, 0.4 to 0.5 portion of carboxymethyl chitosan and 0.85 to 0.9 portion of sodium chloride.
Glucose is used as monosaccharide and is one of the most easily-utilized quick-acting carbon sources for microorganisms, corn starch is a product formed by processing corn through multiple steps, mainly consists of a large amount of starch, a small amount of fat and protein, can provide a large amount of slow-acting carbon sources, can be slowly decomposed under the action of enzyme to provide microorganism nutrition, can also provide partial slow-acting nitrogen sources, and the joint growth efficiency of the glucose and the corn starch ensures the activity of the microorganisms in the initial and long-term storage processes; the sodium ascorbate is used as an antioxidant, has better solubility in water than ascorbic acid, has higher clearance rate for oxygen free radicals, can reduce the harm of the sodium ascorbate to lactobacillus and enterococcus, can also reduce the oxidation of partial effective components in a liquid microbial inoculum, and improves the storage time limit; ferrous sulfate heptahydrate plays a role in reducing the oxidation-reduction potential of a liquid microbial inoculum, both lactobacillus and enterococcus are facultative anaerobes, the lactobacillus and the enterococcus can be inhibited under high oxidation-reduction potential, and aerobic bacteria are easy to breed; dipotassium hydrogen phosphate and potassium dihydrogen phosphate are used as a pH buffer pair, play a role in stabilizing the pH of the liquid microbial inoculum and prevent the pH from changing greatly in the preservation process; linolenic acid is a polyunsaturated fatty acid and has the function of forming an oily protective film to wrap a complex of lactobacillus and a protective agent, and glyceryl monooleate is used as an emulsifier to ensure that the linolenic acid and a water-based liquid microbial inoculum form uniform emulsion which is acted together with the linolenic acid in excessive water to form a uniform micro-droplet structure with an oily protective film on the outer layer and a lactobacillus and protective agent complex on the inner part; the carboxymethyl chitosan is a water-soluble chitosan derivative, has good water solubility, mainly plays roles in thickening and film forming, enables lactic acid and a protective agent to form a stable complex, enables a uniform micro-droplet structure formed by linolenic acid and glycerol monooleate to be more stable, and has broad-spectrum antibacterial action as the chitosan derivative, so that the metabolism of lactic acid bacteria can be slowed down, the survival time of the lactic acid bacteria can be prolonged, and the breeding of mixed bacteria can be inhibited; the concentration of sodium chloride is controlled to the concentration of normal saline and the main function is to maintain the osmotic pressure of the solution.
The storage reagent with the specific components and the specific proportion can effectively prolong the storage life of the composite microbial preparation of the lactobacillus and the enterococcus, the bacterial amount is not obviously reduced within the storage life of 30 days and 90 days, the storage effect is better than that of the conventional composite microbial preparation of the lactobacillus and the enterococcus, and the production activity is greatly promoted.
The embodiment of the invention also provides a compound microbial preparation, which comprises the following components: a preservation reagent for a complex microbial preparation according to any of the preceding embodiments and a complex microbial preparation according to any of the preceding embodiments.
In some embodiments, the mass ratio of the complex microbial preparation to the preservation agent is 100: (1-30). The mass ratio may be specifically in a range of any one or any two of from 100.
Preferably, the mass ratio of the compound microbial preparation to the preservation reagent is 100: (11.56-15.12). Specifically, the ratio of 100.
In addition, the embodiment of the invention also provides a preservation method of the compound microbial preparation, which comprises the following steps: mixing the complex microbial preparation described in any of the preceding examples with a preservation reagent for the complex microbial preparation described in any of the preceding examples.
In some embodiments, the mass ratio of the complex microbial preparation to the preservative agent is as described in any of the preceding examples.
In some embodiments, the storage conditions after mixing are: at normal temperature (5-30 ℃), and is protected from light.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
A preparation method of a composite microbial preparation of lactobacillus and enterococcus comprises the following steps.
(1) Rejuvenation and activation of strains
Lactobacillus (Lactobacillus rhamnosus ATCC7469, from Kyork, guangdong, inc.) and enterococcus (enterococcus faecalis ATCC29212, from Kyork, inc.) preserved on the slant were inoculated into shake flask culture medium, respectively, cultured for 20-24 hours (after 5-fold dilution, OD660= 0.4-0.6), and streaked onto agar plates for seed liquid preparation after culture.
Wherein, the shaking culture condition is 36-38 ℃, the static culture is carried out, the culture medium is MRS broth, and the plate is an MRS agar plate;
(2) Preparation of seed liquid
The seed solution is cultured by adopting a second-level seed solution, the colony on the rejuvenated flat plate is inoculated to a first-level seed solution shake flask, after the culture, the bacterial solution is absorbed from the first-level seed solution and transferred to the second-level seed solution shake flask, and the cultured bacterial solution is used as a workshop seed solution;
wherein the volume of the primary seed liquid is 100mL, and the culture time is 24 hours; the volume of the secondary seed solution was 1500mL, and the culture time was 30 hours (after culture until the culture solution was diluted 5 times, OD660= 0.4-0.6); the amount of the transfer inoculum when the primary seed liquid is transferred to the secondary seed liquid is 10mL.
Wherein, the shake flask culture conditions are static culture at 37 ℃, and the culture medium is MRS broth;
(3) Fermenting in a fermentation tank
2 bottles of seed liquid (viable cell count about 1) were inoculated into a sterilized fermenter, and fermentation was terminated after 30 hours.
Wherein the initial parameters are: the pH is 7.2, the temperature is 37 ℃, the rotating speed is 40-50 rpm, and the ventilation is not carried out.
The formula of the fermentation medium is as follows: 2% of glucose, 1.2% of maltose, 1% of bran, 1% of peptone, 1.2% of yeast extract powder, 0.5% of sodium acetate, 0.024% of magnesium sulfate monohydrate, 0.02% of manganese sulfate monohydrate, 0.24% of dipotassium hydrogen phosphate and the balance of water.
In the fermentation process, the pH regulation and control process comprises the following steps:
controlling the pH value to be 6.8 within 0-8 hours;
controlling the pH value to be 5.9 within 8-16 hours;
the pH value is controlled to be 5.2 within 16 to 30 hours.
(4) Preservation of
After fermentation is finished, filtering the obtained bacterial liquid by using a vibrating screen, adding tap water with the same volume into the obtained filtrate, then concentrating the filtrate by using a ceramic membrane filter to the volume of the filtrate which is the same as that of the original bacterial liquid, transferring the obtained filtrate to a liquid storage tank, and adding a protective agent into the liquid storage tank.
Wherein the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of potassium dihydrogen phosphate, 4.8 percent of linolenic acid, 0.5 percent of glycerol monooleate, 0.5 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
In addition, "%" in the addition amounts of the components means: the mass of a certain component/the mass of the filtrate (before addition of the protective agent), and so on in the subsequent examples.
Wherein, 10% sodium hydroxide and 10% hydrochloric acid are used for adjusting the pH.
Example 2
A method for preparing a complex microbial preparation of Lactobacillus and enterococcus substantially the same as in example 1 except that in step (2), the volume of the secondary seed solution is 1200mL.
Example 3
The difference between this example and example 1 is that in step three, the fermentation medium formula is: 1% of glucose, 2.4% of maltose, 1.2% of bran, 1.2% of peptone, 1% of yeast extract powder, 0.6% of sodium acetate, 0.02% of magnesium sulfate monohydrate, 0.24% of manganese sulfate monohydrate, 0.2% of dipotassium hydrogen phosphate and the balance of water.
Example 4
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (3), the pH is controlled as follows:
controlling the pH value to be 6.6 after 0-8 hours;
controlling the pH value to be 5.9 within 8-16 hours;
the pH was controlled at 5.2 for 16 to 30 hours.
Example 5
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (3), the pH is controlled as follows:
controlling the pH value to be 6.8 after 0-8 hours;
controlling the pH value to be 6.1 within 8-16 hours;
the pH was controlled at 5.2 for 16-30 hours.
Example 6
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (3), the pH is controlled as follows:
controlling the pH value to be 6.8 after 0-8 hours;
controlling the pH value to be 5.9 within 8-16 hours;
the pH was controlled at 5.0 for 16 to 30 hours.
Example 7
A method of preparing a complex microbial preparation of L.lactis and enterococcus is substantially the same as in example 1, except that the L.lactis is L.acidophilus and the enterococcus is enterococcus faecium.
Example 8
A complex microbial preparation of Lactobacillus and enterococcus is prepared in substantially the same manner as in example 1, except that the Lactobacillus is Lactobacillus plantarum and the enterococcus is enterococcus faecalis.
Example 9
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.6% of glucose, 2% of corn starch, 0.24% of sodium ascorbate, 0.48% of ferrous sulfate heptahydrate, 0.4% of dipotassium hydrogen phosphate, 0.1% of potassium dihydrogen phosphate, 4% of linolenic acid, 0.6% of glycerol monooleate, 0.4% of carboxymethyl chitosan and 0.9% of sodium chloride.
Comparative example 1
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (3), the pH is controlled as follows:
controlling the pH value to be 5.9 within 0-16 hours;
the pH was controlled at 5.2 for 16-30 hours.
Comparative example 2
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (3), the pH is controlled as follows:
controlling the pH value to be 6.8 within 0-16 hours;
the pH was controlled at 5.2 for 16-30 hours.
Comparative example 4
A method for preparing a complex microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (3), the pH is controlled as follows:
the pH was controlled to 6.8 for 0 to 30 hours.
Comparative example 5
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium phosphate, 0.08 percent of monopotassium phosphate, 4.8 percent of linolenic acid, 0.5 percent of glycerol monooleate, 0.5 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 6
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of monopotassium phosphate, 4.8 percent of linolenic acid, 0.5 percent of glycerol monooleate, 0.5 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 7
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, wherein in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of potassium dihydrogen phosphate and 0.85 percent of sodium chloride.
Comparative example 8
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of potassium dihydrogen phosphate, 4.8 percent of linolenic acid, 0.5 percent of glycerol monooleate and 0.85 percent of sodium chloride.
Comparative example 9
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of potassium dihydrogen phosphate, 4.8 percent of linolenic acid and 0.85 percent of sodium chloride.
Comparative example 10
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5% of glucose, 2.4% of corn starch, 0.2% of sodium ascorbate, 0.5% of ferrous sulfate heptahydrate, 0.48% of dipotassium hydrogen phosphate, 0.08% of potassium dihydrogen phosphate, 0.5% of glycerol monooleate and 0.85% of sodium chloride.
Comparative example 11
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium phosphate, 0.08 percent of potassium dihydrogen phosphate, 0.5 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 12
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, wherein in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium phosphate, 0.08 percent of potassium dihydrogen phosphate, 3.0 percent of linolenic acid, 0.5 percent of glycerol monooleate, 0.5 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 13
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, wherein in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium phosphate, 0.08 percent of potassium dihydrogen phosphate, 6.0 percent of linolenic acid, 0.5 percent of glycerol monooleate, 0.5 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 14
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of potassium dihydrogen phosphate, 4.8 percent of linolenic acid, 0.4 percent of glycerol monooleate, 0.5 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 15
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, wherein in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of potassium dihydrogen phosphate, 4.8 percent of linolenic acid, 0.7 percent of glycerol monooleate, 0.5 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 16
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of potassium dihydrogen phosphate, 4.8 percent of linolenic acid, 0.5 percent of glycerol monooleate, 0.3 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 17
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, except that in the step (4), the addition amount of each component of the protective agent is as follows: 0.5 percent of glucose, 2.4 percent of corn starch, 0.2 percent of sodium ascorbate, 0.5 percent of ferrous sulfate heptahydrate, 0.48 percent of dipotassium hydrogen phosphate, 0.08 percent of potassium dihydrogen phosphate, 4.8 percent of linolenic acid, 0.5 percent of glycerol monooleate, 0.6 percent of carboxymethyl chitosan and 0.85 percent of sodium chloride.
Comparative example 18
A method for preparing a composite microbial preparation of lactobacillus and enterococcus, which is substantially the same as in example 1, wherein in the step (4), the addition amount of each component of the protective agent is as follows: 0.6 percent of glucose, 0.4 percent of dipotassium phosphate, 0.1 percent of monopotassium phosphate, 15 percent of glycerol and 0.85 percent of sodium chloride.
Test example 1
The amounts of bacteria after fermentation in examples 1 to 4 and comparative examples 1 to 4 were measured by the dilution plate count method, and the results are shown in Table 1.
TABLE 1 measurement results of bacterial count
As can be seen from examples 1 to 8, the preparation method of example 1 is most effective, and the range of variation in the formulation and process and the variation in the strain of examples 2 to 8 do not greatly vary with respect to the fermentation results. As can be seen from comparative example 1, the lower pH at the initial stage of fermentation results in a substantial decrease in the amount of enterococcus bacteria after fermentation; as can be seen from comparative examples 2, 3 and 4, the bacterial load of the lactobacillus after fermentation is obviously reduced under the condition that the pH is not reduced in the middle or later period of the fermentation, particularly, the influence of the pH not reduced in the middle period of the fermentation is more obvious, and the bacterial load of the lactobacillus after fermentation is greatly reduced under the condition that the pH is not reduced in the middle and later periods of the fermentation.
Test example 2
The total bacterial count of lactic acid bacteria was measured by sampling for 30 days and 90 days in the same manner as in test example 1, and the results are shown in Table 2.
TABLE 2 test results
As can be seen from Experimental example 1 and examples 7-9, the effect of the storage method in example 1 is the best, and the range of variation in examples 7-9 is not much changed with respect to the storage effect. As can be seen from comparative examples 5 and 6, the lack of corn starch or the lack of sodium ascorbate and ferrous sulfate heptahydrate in the protective agent can cause the storage effect of the microecological agent to be obviously reduced; as can be seen from comparative example 7, the lack of linolenic acid, glyceryl monooleate and carboxymethyl chitosan in the protective agent can cause great influence on the preservation effect; as can be seen from the comparison among the comparative examples 7, 9 and 10, the preservation effect of the linolenic acid and the glyceryl monooleate can be improved to a certain extent, and the preservation effect of the linolenic acid is improved more obviously; meanwhile, as can be seen from comparison of comparative example 7 with comparative example 8, comparative example 7 with comparative example 9, and comparative example 7 with comparative example 10, when linolenic acid and glyceryl monooleate are simultaneously added into the microecological bactericide, the improvement of the preservation effect is much larger than that when the linolenic acid and the glyceryl monooleate are added separately, because the glyceryl monooleate is used as an emulsifier, the linolenic acid and the aqueous liquid bactericide can form stable emulsion; as can be seen from the comparison between the comparative examples 7 and 11, the carboxymethyl chitosan can improve the preservation effect to a certain extent, and as can be seen from the comparison between the comparative examples 7 and 8, between the comparative examples 7 and 11, and between the comparative examples 7 and 1, when the carboxymethyl chitosan is used as a thickening agent to act together with linolenic acid and glycerol monooleate, the preservation effect can be greatly improved, because the carboxymethyl chitosan is used as the thickening agent, a uniform complex can be formed between lactic acid and a protective agent, and the uniform droplet structure formed by the linolenic acid and the glycerol monooleate is more stable; as can be seen from comparative examples 12 to 17, too much or too little addition of linolenic acid, glyceryl monooleate and carboxymethyl chitosan causes a significant decrease, especially when the addition is too small; as can be seen from comparative example 18, the protective agent of the present invention has a greatly improved preservation effect on lactic acid bacteria compared to conventional protective agents.
In conclusion, the preparation method of the lactobacillus and enterococcus composite microecologics is simple to operate, bacteria are not easily infected in the fermentation process, the ratio of the lactobacillus and the enterococcus is close to that after fermentation, the total bacteria content is high, the bacteria content of the lactobacillus and enterococcus composite microecologics prepared after fermentation is not obviously reduced within the preservation time limit of 30 days and 90 days, the preservation effect is better than that of the conventional lactobacillus and enterococcus composite microecologics, and the production activity is greatly promoted.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for preparing a composite microbial preparation, which is characterized by comprising the following steps: mixing the seed liquid of lactobacillus and the seed liquid of enterococcus, and fermenting; in the fermentation process, the pH in the fermentation medium is regulated according to 3 stages:
in the first stage, regulating and controlling the pH value to be 6.6-6.8;
in the second stage, regulating and controlling the pH value to be 5.9-6.1;
in the third stage, the pH is regulated to 5.0-5.2;
wherein the first stage is 0 to (6 to 10) hours of fermentation; the second stage is the (6-10) - (14-18) th hour of fermentation; the third stage is the (14 to 18) th to (28 to 32) th hours of fermentation.
2. The method for producing a complex microbial preparation according to claim 1, wherein the first stage is from 0 to (7 to 9) hours of fermentation, the second stage is from (7 to 9) to (15 to 17) hours of fermentation, and the third stage is from (15 to 17) to (29 to 31) hours of fermentation; preferably, the first stage is 0 to 8 hours of fermentation, the second stage is 8 to 16 hours of fermentation, and the third stage is 16 to 30 hours of fermentation;
preferably, the preparation method comprises regulating the pH in the fermentation medium by using a pH regulating agent;
preferably, the pH modifier comprises: sodium hydroxide with the volume fraction of 1-20 percent and/or hydrochloric acid with the volume fraction of 1-20 percent.
3. The method for preparing a composite microbial preparation according to claim 1 or 2, wherein the ratio of viable bacteria of the seed solution of lactobacillus to the seed solution of enterococcus is as follows: (0.5-2): (0.5-2);
preferably, the lactobacillus is selected from at least one of lactobacillus rhamnosus, lactobacillus casei, lactobacillus delbrueckii, lactobacillus plantarum, lactobacillus reuteri, and lactobacillus acidophilus; the enterococcus comprises at least one of enterococcus faecalis and enterococcus faecium;
preferably, the conditions of the fermentation include: at the temperature of 36-38 ℃, the rotating speed is 40-50 rpm, and ventilation is not performed;
preferably, the initial pH of the fermentation medium is between 7.0 and 7.2.
4. The method for preparing a compound microbial preparation according to claim 3, wherein the fermentation medium used for the fermentation comprises the following components in parts by weight: 2 to 2.4 percent of glucose, 1 to 1.2 percent of maltose, 1 to 1.2 percent of bran, 1 to 1.2 percent of peptone, 1 to 1.2 percent of yeast extract powder, 0.5 to 0.6 percent of sodium acetate, 0.02 to 0.24 percent of magnesium sulfate monohydrate, 0.02 to 0.24 percent of manganese sulfate monohydrate, 0.2 to 0.24 percent of dipotassium hydrogen phosphate and the balance of water.
5. The method for preparing a complex microbial preparation according to claim 1 or 2, further comprising filtering the fermented broth;
preferably, the step of filtering comprises: filtering the fermented bacterial liquid by using a vibrating screen to obtain filtrate, wherein the volume ratio of the filtrate to water is (1-3): (1-3), and then concentrating the mixture to the volume same as that of the original bacterial liquid through a ceramic membrane filter.
6. A complex microbial preparation produced from the complex microbial preparation according to any one of claims 1 to 5.
7. A kit of complex microbial preparation comprising the complex microbial preparation of claim 6.
8. The preservation reagent for the compound microbial preparation is characterized by comprising the following main components in parts by weight: 0.5 to 0.6 portion of glucose, 2 to 2.4 portions of corn starch, 0.2 to 0.24 portion of sodium ascorbate, 0.48 to 0.5 portion of ferrous sulfate heptahydrate, 0.4 to 0.48 portion of dipotassium phosphate, 0.08 to 0.1 portion of potassium dihydrogen phosphate, 4 to 4.8 portions of linolenic acid, 0.5 to 0.6 portion of glycerol monooleate, 0.4 to 0.5 portion of carboxymethyl chitosan and 0.85 to 0.9 portion of sodium chloride.
9. A complex microbial preparation, characterized in that it comprises: a preservation agent for the complex microbial preparation of claim 6 and the complex microbial preparation of claim 8;
preferably, the mass ratio of the compound microbial preparation to the preservation reagent is 100: (1-30);
preferably, the mass ratio of the compound microbial preparation to the preservation reagent is 100: (11.56-15.12).
10. A method for preserving a complex microbial preparation, comprising: mixing the complex microbial preparation of claim 6 with a preservation reagent for the complex microbial preparation of claim 8;
preferably, the mass ratio of the compound microbial preparation to the preservation reagent is 100: (1-30);
preferably, the mass ratio of the compound microbial preparation to the preservation reagent is 100: (11.56-15.12).
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| CN113462620A (en) * | 2021-08-26 | 2021-10-01 | 福建傲农生物科技集团股份有限公司 | Preparation method and application of composite microbial agent for feed |
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