CN116491577A - High-activity probiotic composite microecological preparation and preparation method and application thereof - Google Patents
High-activity probiotic composite microecological preparation and preparation method and application thereof Download PDFInfo
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- CN116491577A CN116491577A CN202310508093.8A CN202310508093A CN116491577A CN 116491577 A CN116491577 A CN 116491577A CN 202310508093 A CN202310508093 A CN 202310508093A CN 116491577 A CN116491577 A CN 116491577A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 44
- 239000002131 composite material Substances 0.000 title claims abstract description 26
- 239000006041 probiotic Substances 0.000 title claims abstract description 25
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 25
- 230000000694 effects Effects 0.000 title claims abstract description 19
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 24
- 230000004151 fermentation Effects 0.000 claims abstract description 24
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 20
- 241000193749 Bacillus coagulans Species 0.000 claims abstract description 20
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 20
- 229940054340 bacillus coagulans Drugs 0.000 claims abstract description 20
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 18
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 18
- 230000004913 activation Effects 0.000 claims abstract description 4
- 238000013329 compounding Methods 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 25
- 238000012258 culturing Methods 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 7
- 230000001105 regulatory effect Effects 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 238000001994 activation Methods 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 abstract description 16
- 241000894006 Bacteria Species 0.000 abstract description 9
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 5
- 238000003860 storage Methods 0.000 abstract description 2
- 235000021050 feed intake Nutrition 0.000 description 7
- 238000001816 cooling Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 235000021052 average daily weight gain Nutrition 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000011229 interlayer Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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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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- 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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- 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
- C12R2001/07—Bacillus
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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
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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Abstract
The invention discloses a high-activity probiotic composite microecological preparation and a preparation method and application thereof, and belongs to the technical field of fermented feeds. The probiotics composite microecological preparation is prepared by using bacillus amyloliquefaciens BA, bacillus coagulans BC and lactobacillus plantarum LP as strains and performing strain activation, strain compounding, fermentation and expansion culture. The preparation method is simple in operation, low in cost, quick in preparation and safe, the prepared probiotics composite microecological preparation is rich in various active probiotics, high in active bacteria content, stable in storage, safe and reliable, can promote animal growth, remarkably improve microecological balance of animal intestinal tracts, maintain animal intestinal tracts healthy, and improve animal product quality.
Description
Technical Field
The invention belongs to the technical field of fermented feeds, and particularly relates to a high-activity probiotic composite microecological preparation, and a preparation method and application thereof.
Background
The compound microecological preparation is a biological product compounded by a plurality of beneficial microorganisms, is a novel microbial additive which rapidly rises at home and abroad in recent years, and has the advantages of pure nature, no residue, no drug resistance, environmental protection and the like. The microecological preparation has the advantages of regulating intestinal flora balance, promoting animal growth, digesting, absorbing and utilizing nutrients, improving animal immunity and body health condition, etc. The implementation of the feed inhibition policy makes the compound microecological preparation one of ideal antibiotic substitutes. The microecological preparation not only can reduce the death rate of animals, but also can improve the yield in the breeding production, and simultaneously can ensure the quality and safety of foods, and finally can well maintain the ecological safety.
At present, a plurality of microecological preparation like products exist on the market, but the following main problems exist: (1) the content and activity of viable bacteria in the product are low; (2) the synergistic effect among all strains in the microbial inoculum is poor; (3) the preparation process is complex and tedious, it is difficult to ensure the growth and propagation of all strains, and the consistency and stability of the product are poor. The quality problem of the microecological preparation affects the application effect of the microecological preparation on animals, and the wide application of the microecological preparation is limited.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide the high-activity probiotic composite microecological preparation and the preparation method and application thereof.
In order to achieve the above object, the present invention provides the following solutions:
the invention aims to provide a high-activity probiotics composite microecological preparation which is prepared by taking bacillus amyloliquefaciens (Bacillus amyloliquefaciens, BA), bacillus coagulans (Bacillus coagulans, BC) and Lactobacillus Plantarum (LP) as strains;
the bacillus amyloliquefaciens BA, the bacillus coagulans BC and the lactobacillus plantarum LP are all conventional commercial products.
The second purpose of the invention is to provide a preparation method of the high-activity probiotic composite microecological preparation, which comprises the following steps:
(1) Activating strains: respectively activating bacillus coagulans, lactobacillus plantarum and bacillus amyloliquefaciens in a culture medium;
(2) And (3) strain compounding: inoculating the activated bacillus amyloliquefaciens, bacillus coagulans and lactobacillus plantarum into a culture medium A, and culturing for 18 hours at 34 ℃; inoculating the strain into a culture medium B, and culturing for 14-16 hours at 35-37 ℃ to obtain a composite seed solution;
the culture medium A is as follows: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl and 1000mL/L distilled water, adjusting pH to 7.0-7.2, sterilizing at 121deg.C for 20min;
the culture medium B is as follows: glucose 22g/L, beef extract 2.5g/L, sterilizing at 115deg.C for 30min;
(3) Fermentation and expansion culture: adding the culture medium B into a fermentation tank, regulating the pH to 7.0+/-0.2, sterilizing at 121 ℃ for 30min, cooling or naturally cooling by tap water through a tank interlayer, and inoculating the composite seed liquid and fermenting when the temperature is reduced to 40 ℃ to obtain the high-activity probiotic composite microecological preparation.
Further, the activating process of the bacillus coagulans and the lactobacillus plantarum in the step (1) comprises the following steps: adding bacillus coagulans or lactobacillus plantarum into MRS liquid culture medium, and culturing at 37 ℃ for 16-18 h.
Further, the activation process of the bacillus amyloliquefaciens in the step (1) comprises the following steps: preparing an activation culture medium according to the proportion of 10g/L glucose, 20g/L beef extract and 2g/L potassium dihydrogen phosphate, regulating the pH to 7.2, sterilizing, inoculating bacillus amyloliquefaciens, and culturing at 37 ℃ for 16h.
Further, the mass ratio of the bacillus amyloliquefaciens to the bacillus coagulans to the lactobacillus plantarum in the step (2) is 1-2:1:1.
Further, the amount of the culture medium B in the step (3) accounts for 70% of the volume of the fermentation tank; the dosage of the compound seed liquid accounts for 10 percent of the volume of the fermentation tank.
Further, the fermentation conditions of step (3) are: the temperature is 37 ℃, the pH is 7.0, the aeration rate is 2:1, and in the culture process, the air is intermittently aerated and slowly stirred, and the culture is carried out for 18-24 hours.
The invention also aims to provide the application of the high-activity probiotic composite microecological preparation in the field of feed.
The invention has the beneficial effects that:
the preparation method of the probiotics composite microecological preparation is simple to operate, low in cost, quick to prepare and safe, the prepared probiotics composite microecological preparation is rich in various active probiotics, high in active bacteria content, stable in storage, safe and reliable, animal growth can be promoted, microecological balance of animal intestinal tracts can be remarkably improved, animal intestinal tracts can be maintained, and animal product quality can be improved.
Detailed Description
The present invention will be described in further detail with reference to examples for better understanding of the technical scheme of the present invention to those skilled in the art.
Example 1
(1) Activating strains: thawing frozen Bacillus coagulans and Lactobacillus plantarum bacteria liquid, respectively taking 20 μL, respectively adding into 15mL centrifuge tube filled with MRS liquid culture medium, culturing at 37deg.C in incubator for 18 hr, and measuring viable count by dilution plate coating method to obtain total viable count of 6X10 8 CFU/mL；
Preparing an activation culture medium according to the proportion of 10g/L glucose, 20g/L beef extract and 2g/L potassium dihydrogen phosphate, regulating the pH to 7.2, inoculating bacillus amyloliquefaciens with standard form and good growth state after sterilization, shake-culturing for 16h in a biochemical incubator with constant temperature of 37 ℃, and preserving at 4 ℃ for later use.
(2) And (3) strain compounding: inoculating the activated bacillus amyloliquefaciens, bacillus coagulans and lactobacillus plantarum into a culture medium A according to the mass ratio of 2:1:1, and culturing in a biochemical incubator at the constant temperature of 34 ℃ for 18 hours; then inoculating the strain into a culture medium B, and culturing for 16 hours at 35-37 ℃ to obtain a composite seed solution;
the culture medium A is as follows: peptone 10g/L, yeast extract 5g/L, naCl10g/L, distilled water 1000mL/L, pH7.0, and sterilizing at 121deg.C under high pressure steam for 20min;
the culture medium B is as follows: glucose 22g/L, beef extract 2.5g/L, sterilizing at 115deg.C for 30min;
(3) Fermentation and expansion culture: performing expansion culture by using a 50L fermentation tank and a fermentation medium B, firstly cleaning the fermentation tank until the interior of the fermentation tank is cleaned, and emptying at 121 ℃ for 30min; in the fermenter, 70% by volume of the liquid is added to the fermentation medium, the pH is adjusted to 7.0, 0.05 to 2% by volume of an antifoaming agent is added, and the inlet is closed. Sterilizing the culture medium in the fermentation tank for 30min, and cooling or naturally cooling with tap water via interlayer of the tank body. When the temperature is reduced to 40 ℃, 5L of compound seed liquid is added, inoculation is carried out through a peristaltic pump, an inoculation opening is closed, and stirring is uniform. The fermentation conditions are set as follows: the temperature is 37 ℃, the pH value is 7.0, the ventilation amount is 2:1, in the culture process, the intermittent ventilation is carried out, the slow stirring is carried out, the culture is carried out for 24 hours, the fermentation liquor, namely the high-activity probiotic composite microecological preparation, is obtained, and the viable count of the fermentation liquor is detected every 2 hours in a microscopic way.
The number of viable bacteria in the fermentation broth was determined by plate colony counting. Taking 1mL of fermentation liquor, and sequentially and gradually diluting to 10 -6 And 10 -7 The whole amount of bacteria in the fermentation broth was calculated by plating 0.1mL of the dilution onto LB agar plates (at least 3 replicates), incubating for 24h at 37℃in an inverted position, and counting.
The probiotics compound microecological preparation with high activity prepared by the embodiment has high survival rate of 89.8 percent, wherein the number of the bacillus coagulans is 5.1 multiplied by 10 11 cfu/g, lactobacillus plantarum number of 3.90X10 10 cfu/g, bacillus amyloliquefaciens number of 4.8X10 11 cfu/g. The total number of viable bacteria of the prepared probiotic composite microecological preparation is up to 1.8x10 12 cfu/g。
Detecting mixed bacteria: 1g of the fermentation product is weighed into a sterile volumetric flask and 9mL of sterile water is added. After being uniformly mixed by a vortex instrument, the mixture is streaked and inoculated on an LB solid culture medium, and is cultured for 24 hours at 37 ℃, and microscopic examination is carried out for no bacteria.
Shelf life test: the probiotic composite microecological preparation sample is preserved at 4deg.C, and the number of detected viable bacteria is 10 every month 8 cfu/mL。
Example 2
The difference from example 1 is that the activated Bacillus amyloliquefaciens, bacillus coagulans and Lactobacillus plantarum were inoculated into the medium A in a mass ratio of 1:1:1.
Example 3
The difference from example 1 is that the activated Bacillus amyloliquefaciens, bacillus coagulans and Lactobacillus plantarum were inoculated into the medium A in a mass ratio of 1.5:1:1.
Application example
The probiotic composite microecologics with high activity of examples 1-3 are added into animal feed, and the mass of the preparation is 0.5% of the mass of the feed.
The piglets with similar weight and day-age (28 days-age) are selected to be randomly divided into 4 groups, 3 replicates of each group, and 5 replicates of each group, namely a test 1 group (example 1), a test 2 group (example 2), a test 3 group (example 3) and a blank control group (without probiotics compound microecologics). Feeding for one month, weighing on an empty stomach, recording feed intake by taking groups as units, and calculating average daily feed intake, average daily weight gain and feed-meat ratio, wherein the results are shown in Table 1.
Average daily feed intake= (total feed intake/pig head number)/test days
Average daily gain= (average last weight-average initial weight)/test day
Feed to meat ratio = average daily feed intake/average daily gain
TABLE 1
As shown in Table 1, the average daily feed intake, average daily weight gain and feed/meat ratio index of the test group were all superior to those of the blank control group, the effect of the test group 1 was the best, the average daily feed intake reached 782.55g, the average daily weight gain reached 461.33g, and the feed/meat ratio reached 1.70. The high-activity probiotics composite microecological preparation provided by the invention can promote animal growth, remarkably improve microecological balance of animal intestinal tracts and maintain intestinal health of animals.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A high-activity probiotics composite microecological preparation is characterized in that bacillus amyloliquefaciens BA, bacillus coagulans BC and lactobacillus plantarum LP are used as strains to prepare the preparation.
2. A method of preparing the high-activity probiotic composite microecological preparation according to claim 1, comprising the steps of:
(1) Activating strains: respectively activating bacillus coagulans, lactobacillus plantarum and bacillus amyloliquefaciens in a culture medium;
(2) And (3) strain compounding: inoculating the activated bacillus amyloliquefaciens, bacillus coagulans and lactobacillus plantarum into a culture medium A, and culturing for 18 hours at 34 ℃; inoculating the strain into a culture medium B, and culturing for 14-16 hours at 35-37 ℃ to obtain a composite seed solution;
the culture medium A is as follows: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, 1000mL/L distilled water, regulating pH to 7.0-7.2, and sterilizing;
the culture medium B is as follows: glucose 22g/L, beef extract 2.5g/L, sterilizing;
(3) Fermentation and expansion culture: adding the culture medium B into a fermentation tank, regulating the pH to 7.0+/-0.2, sterilizing, and inoculating the composite seed liquid and fermenting when the temperature is reduced to 40 ℃.
3. The preparation method according to claim 2, wherein the activation process of bacillus coagulans and lactobacillus plantarum in step (1) is as follows: adding bacillus coagulans or lactobacillus plantarum into MRS liquid culture medium, and culturing at 37 ℃ for 16-18 h.
4. The preparation method according to claim 2, wherein the activation process of the bacillus amyloliquefaciens in the step (1) is as follows: preparing an activation culture medium according to the proportion of 10g/L glucose, 20g/L beef extract and 2g/L potassium dihydrogen phosphate, regulating the pH to 7.2, sterilizing, inoculating bacillus amyloliquefaciens, and culturing at 37 ℃ for 16h.
5. The preparation method according to claim 2, wherein the mass ratio of the bacillus amyloliquefaciens to the bacillus coagulans to the lactobacillus plantarum in the step (2) is 1-2:1:1.
6. The method according to claim 2, wherein the amount of the medium B in the step (3) is 70% of the volume of the fermenter; the dosage of the compound seed liquid accounts for 10 percent of the volume of the fermentation tank.
7. The method according to claim 2, wherein the fermentation conditions of step (3) are: the culture is carried out for 18 to 24 hours at the temperature of 37 ℃ and the pH value of 7.0, wherein the ventilation amount is 2:1 of gas-liquid ratio.
8. Use of the high-activity probiotic composite microecological preparation according to claim 1 in the field of feed.
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