CN112608861A - Composite preparation containing clostridium butyricum and pediococcus acidilactici as well as preparation method and application of composite preparation - Google Patents
Composite preparation containing clostridium butyricum and pediococcus acidilactici as well as preparation method and application of composite preparation Download PDFInfo
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- clostridium butyricum
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- 241000193171 Clostridium butyricum Species 0.000 title claims abstract description 108
- 241000191998 Pediococcus acidilactici Species 0.000 title claims abstract description 74
- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 239000002131 composite material Substances 0.000 title claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims abstract description 35
- 230000004151 fermentation Effects 0.000 claims abstract description 35
- 239000001963 growth medium Substances 0.000 claims abstract description 31
- 241000894006 Bacteria Species 0.000 claims abstract description 23
- 150000001875 compounds Chemical class 0.000 claims abstract description 19
- 238000012258 culturing Methods 0.000 claims abstract description 13
- 241001465754 Metazoa Species 0.000 claims abstract description 11
- 230000004083 survival effect Effects 0.000 claims abstract description 6
- 230000004584 weight gain Effects 0.000 claims abstract description 6
- 235000019786 weight gain Nutrition 0.000 claims abstract description 6
- 238000011049 filling Methods 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 26
- 238000011081 inoculation Methods 0.000 claims description 24
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- 241000238553 Litopenaeus vannamei Species 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 11
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 8
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- 238000009472 formulation Methods 0.000 claims description 6
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- 210000002966 serum Anatomy 0.000 description 5
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- 229960001305 cysteine hydrochloride Drugs 0.000 description 4
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- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 101001077374 Oryza sativa subsp. japonica UMP-CMP kinase 3 Proteins 0.000 description 1
- 108010080032 Pediocins Proteins 0.000 description 1
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- 206010047400 Vibrio infections Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
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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
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/41—Pediococcus
- A23V2400/413—Acidilactici
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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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
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Abstract
The invention discloses a composite preparation containing clostridium butyricum and pediococcus acidilactici, a preparation method and application thereof. The composite preparation comprises the components with the bacterium content of 5-6 x 108The content of Clostridium butyricum and bacteria is 5.0-6.0 × 109The lactic acid coccus of CFU/mL, wherein the clostridium butyricum adopts clostridium butyricum GBW-N1 with the preservation number of CGMCC No. 14499. The compound preparation is inoculated by two strains at different times and is designedFermenting and culturing in a fermentation culture medium for 16-22h, and filling the fermentation liquor for use. The compound preparation can well improve the weight gain rate, the survival rate and the growth performance of aquatic animals, is beneficial to improving the yield of aquaculture, has good stability and is convenient to store, thereby having wide application value.
Description
Technical Field
The invention belongs to the field of aquaculture, and particularly relates to a composite preparation containing clostridium butyricum and pediococcus acidilactici, and a preparation method and application thereof.
Background
Clostridium butyricum (Clostridium butyricum) and Pediococcus acidilactici (Pediococcus acidilactici) are common gram-positive bacteria in environment and animal intestinal tracts, and the thallus of the Clostridium butyricum can adhere to the intestinal tracts to occupy space and inhibit pathogenic bacteria; the pediococcus acidilactici can generate probiotics such as pediocin, L-lactic acid and the like in the reproductive metabolism, and plays an important role in the balance regulation of intestinal flora. The two microbial agents which participate in the preparation mainly have the problems that the production cost of solid products is still high and the use frequency is limited; the liquid product cannot be applied in large scale due to unstable storage. Therefore, the research and development of a product which can be efficiently produced in large scale and has high stability in transportation and storage has very important significance for the application of anaerobic bacteria in aquaculture.
Disclosure of Invention
The invention provides a compound preparation containing clostridium butyricum and pediococcus acidilactici, a preparation method and application thereof.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme:
the invention provides a compound preparation containing clostridium butyricum and pediococcus acidilactici, and the compound preparationThe preparation contains 5-6 × 10 of bacteria8The content of Clostridium butyricum and bacteria is 5.0-6.0 × 109CFU/mL of Pediococcus acidilactici.
Further, the clostridium butyricum adopts clostridium butyricum GBW-N1 with the preservation number of CGMCC No. 14499.
Furthermore, the compound preparation has good stability.
The invention also provides a preparation method of the compound preparation, which comprises the following specific steps:
(1) respectively carrying out activation culture on clostridium butyricum and pediococcus acidilactici in a seed culture medium to obtain clostridium butyricum seed solution and pediococcus acidilactici seed solution;
(2) inoculating the clostridium butyricum seed solution into a fermentation tank, fermenting for 4-5h in a fermentation medium at 37 ℃, then inoculating the pediococcus acidilactici seed solution into the fermentation tank, co-culturing for 16-22h at 37 ℃ to obtain the compound preparation, and filling for later use.
Further, the inoculation amount of the clostridium butyricum seed solution is 2%; the inoculation amount of the pediococcus acidilactici seed liquid is 2%.
Further, the formula of the fermentation medium is 6-12 g/L of tryptone, 5-10 g/L of beef extract, 5-15 g/L of yeast extract, 20-30 g/L of glucose, 2-5 g/L of dipotassium phosphate, 0.1-0.3 g/L of magnesium sulfate, 1-3 g/L of sodium acetate, 0.5-1 g/L of cysteine hydrochloride, 10-20 g/L of calcium carbonate and pH 6.8-7.0.
Further, the fermentation conditions in the step (2) are as follows: the pressure of the tank body is controlled to be 0.05MPa by high-purity nitrogen, the rotating speed is 80-100r/min, and the pH value of the fermentation medium is controlled to be more than or equal to 6.0 by adopting a sodium hydroxide solution or ammonia water.
The invention also provides application of the compound preparation in preparing a microbial preparation for improving the growth performance of aquatic animals.
Further, when in use, the compound preparation is mixed with aquaculture feed of aquatic animals at the dosage of 20-30mL/kg of feed, and is continuously fed for more than 30 days.
Furthermore, the compound preparation can increase the weight gain rate and the survival rate of aquatic animals and reduce the feed coefficient.
Further, the compound preparation can increase the intestinal villus quantity, epithelial cell density and alkaline phosphatase content in serum of aquatic animals.
Further, the composite preparation can significantly reduce the number of vibrios.
Further, the aquatic animals include Penaeus vannamei Boone.
Compared with the prior art, the method has the advantages and technical effects that:
the invention prepares the composite microbial inoculum by the staggered inoculation culture method of clostridium butyricum and pediococcus acidilactici which are non-toxic to animals, and the components and pH of a culture medium in the fermentation process are designed and verified through elaborate experiments, thereby being beneficial to the growth of clostridium butyricum with weak growth in the co-culture process and leading the clostridium butyricum to obtain higher bacterial count within 22 h. The compound preparation can improve the weight gain rate, the survival rate and the content of alkaline phosphatase in serum in the culture of the penaeus vannamei boone, can reduce the bait coefficient and play a good antibacterial effect, has good stability and can be stored for a long time, so the compound preparation has the production, storage and application effects, solves the problems of cost and stability in the popularization of clostridium butyricum and pediococcus acidilactici in aquaculture, shortens the culture period by more than 50 percent compared with the condition that the compound preparation is fermented independently and then mixed, and also solves the problems of bacterial contamination, bottle expansion and the like of products.
Drawings
FIG. 1 is a diagram showing colonies, cells and spores of Clostridium butyricum GBW-N1 on a TSC medium.
FIG. 2 shows 100X 10 fold microscopic examination of crystal violet staining in 12h coculture of Clostridium butyricum and Pediococcus acidilactici.
FIG. 3 is the product appearance of the co-cultured complex formulation of Clostridium butyricum and Pediococcus acidilactici.
Figure 4 is the appearance of a plastic package with a venting cover for the composite formulation.
Fig. 5 is a circulating water aquaculture system for culturing penaeus vannamei boone.
FIG. 6 is a diagram of defecation and intestinal rupture of Penaeus vannamei Boone.
FIG. 7 shows the detection of enzyme activity in the serum of Penaeus vannamei Boone.
FIG. 8 is a tissue section of the midgut of Penaeus vannamei Boone.
Fig. 9 shows the bacterial flora structure of the intestinal tract of penaeus vannamei boone.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples.
Example 1
Screening, separation and identification of Clostridium butyricum GBW-N1
1. Separation, screening and purification of clostridium butyricum GBW-N1
And (3) coating the bacterial suspension cultured by the collected sample on a tryptone-sulfite-cycloserine agar (TSC) culture medium after gradient dilution, separating and purifying for multiple times to obtain a single bacterial colony, and storing the single bacterial colony named as GBW-N1.
The bacterial colony of the bacterial strain GBW-N1 cultured on the TSC solid medium for 24 hours is shown in figure 1, and the bacterial colony is as shown in figure 1a, which is round, black, smooth in surface, slightly convex in the middle and irregular in edge; the bacterial strain GBW-N1 is cultured on a TSC liquid medium for 48 hours, and is shown in figure 1b, wherein the bacterial strain is rod-shaped, 1.1-1.2 mu m is multiplied by 2.1-3.6 mu m, is singly arranged, is gram-positive, spores are fusiform, are mesogenic and are expanded.
2. Molecular characterization of Clostridium butyricum GBW-N1
The DNA of the strain GBW-N1 is used as a template, 16S rRNA universal primers are used for amplification, sequence determination is carried out on amplified fragments, the 16S rDNA sequencing result of the obtained strain GBW-N1 is compared with the sequence in GenBank, and the result shows that the strain GBW-N11 has the highest homology with Clostridium butyricum, so that the strain GBW-N1 is determined to be Clostridium butyricum.
3. Strain deposit of Clostridium butyricum GBW-N1
And (3) performing strain preservation on the screened strain GBW-N1, wherein the preservation unit of the clostridium butyricum GBW-N1 is as follows: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: year 2017, month 08, day 07; the preservation number of the Clostridium butyricum is CGMCC No. 14499.
4. Strain characteristics of Clostridium butyricum GBW-N1
The clostridium butyricum GBW-N1 can grow in the pH value range of 6-8, and the optimal growth pH value is 7.1-7.5; the growth can be normally carried out within the range of dissolved oxygen content of 1-5 mg/L, and the optimal growth dissolved oxygen concentration is 2-4 mg/L; can produce urease, beta-glucosidase and glycerol, and can degrade or decompose power-nitrate, simon citrate and semi-solid agar; can normally grow and propagate at the temperature of 30-45 ℃, and the optimal growth temperature is 35-38 ℃. The clostridium butyricum GBW-N1 is an anaerobic bacterium, can germinate and proliferate in an anaerobic environment, and has probiotic functions of enzyme production, acid production, bacteriostasis and the like.
Secondly, preparation of clostridium butyricum seed liquid
Inoculating single colony of Clostridium butyricum GBW-N1 in a Kirschner flask filled with RCM culture medium, culturing at 37 deg.C for 3d, scraping lawn with 0.8% physiological saline to obtain seed solution, and using for subsequent use. The concentration of the clostridium butyricum seed solution is 490 ten thousand CFU/mL, and is used for subsequent experiments.
Preparation of pediococcus acidilactici seed liquid
Selecting single colony of Pediococcus acidilactici cultured on MRS solid culture medium, inoculating in MRS liquid culture medium, and performing amplification culture at 37 deg.C for 16h as seed solution for subsequent use. The concentration of Pediococcus acidilactici seed solution was 23 hundred million CFU/mL for subsequent experiments.
The Clostridium butyricum used in the examples described below was Clostridium butyricum GBW-N1.
Example 2: co-culture shake flask condition groping of clostridium butyricum and pediococcus acidilactici
Test 1: configuring 220mL MRS culture medium in 250mL Shuniu screw bottle, dividing into 3 groups, and culturing together: inoculating 1% clostridium butyricum seed solution and 1% pediococcus acidilactici seed solution; control 1(CK 1): only inoculating 1% pediococcus acidilactici seed solution; control 2(CK 2): only 1% clostridium butyricum seed liquid is inoculated, and three groups are placed in an incubator at 37 ℃ for static culture for 20-28 h. The results (table 1) show that co-culture of pediococcus acidilactici and clostridium butyricum with the lactobacillus universal medium MRS has a growth advantage over clostridium butyricum, and the content of clostridium butyricum in co-culture is less than 0.3% of the bacterial content in single culture, because pediococcus acidilactici grows faster and causes a decrease in the pH of the medium, so that the pH of the medium is lower than the optimum growth pH (4.0-9.8) of clostridium butyricum, and the growth of clostridium butyricum is finally inhibited.
TABLE 1 live bacteria content of different treatment groups in test 1
Test 2: configuring 220mL MRS culture medium in a 250mL Shuniu screw bottle, and dividing the culture medium into a co-culture group 1: inoculating 1% clostridium butyricum seed solution, culturing for 7h, and then inoculating 1% pediococcus acidilactici seed solution; co-culture group 2: inoculating 2% clostridium butyricum seed solution, culturing for 7h, and inoculating 1% pediococcus acidilactici seed solution; control 1(CK 1): only inoculating 1% pediococcus acidilactici seed solution; control 2(CK 2): only 1% clostridium butyricum seed solution is inoculated; control 2(CK 3): only 2% clostridium butyricum seed solution was inoculated; all are placed in an incubator at 37 ℃ for static culture for 20 h. Experiment 2 adopted time-phased inoculation, namely, after clostridium butyricum was cultured, pediococcus acidilactici was cultured, and the inoculation ratio of clostridium butyricum was increased, but the bacteria content of clostridium butyricum after co-culture was still very low (table 2), which indicates that even if pediococcus acidilactici was inoculated with a delay of 7 hours, clostridium butyricum hardly proliferated to a higher bacteria content when clostridium butyricum and pediococcus acidilactici were co-cultured in MRS medium, which may be related to anaerobic environment and MRS medium nutrient components.
Table 2 viable bacteria content of different treatment groups in test 2
Test 3: setting different formula culture media according to the nutritional characteristics of clostridium butyricum to carry out single-bacterium culture, respectively preparing 220mL of 3 culture media in 250mL screw bottles, respectively inoculating clostridium butyricum seed liquid and pediococcus acidilactici seed liquid according to the volume ratio of 5%, placing at 37 ℃ for culturing for 34h, shaking the screw bottles every 6h to uniformly mix the calcium carbonate at the bottom, and maintaining the stability of the pH value of the culture media. The formula of the three culture media is as follows:
culture medium 1: 10g/L of tryptone, 5g/L of beef extract, 5g/L of yeast extract, 20g/L of glucose, 2g/L of monopotassium phosphate, 1g/L of magnesium sulfate, 3g/L of sodium acetate, 1g/L of sodium bicarbonate, 1g/L of cysteine hydrochloride, 5g/L of calcium carbonate and pH 7.0.
Culture medium 2: 10g/L tryptone, 3g/L beef extract, 5g/L yeast extract, 20g/L glucose, 2g/L sodium chloride, 1g/L ammonium sulfate, 4g/L dipotassium phosphate, 1g/L ferrous sulfate, 0.5g/L magnesium sulfate, 0.2g/L manganese sulfate, 1g/L sodium bicarbonate, 5g/L calcium carbonate, 0.5g/L cysteine hydrochloride, and 800.2 mL/L Tween, and the pH value is 7.0.
Culture medium 3: 8g/L of tryptone, 5g/L of beef extract, 5g/L of yeast extract, 15g/L of glucose, 5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate, 1g/L of sodium acetate, 1g/L of cysteine hydrochloride, 10g/L of calcium carbonate and pH 7.0.
The results show (Table 3) that the bacterial load of Clostridium butyricum cultured for 34 hours in Medium 1 and Medium 3 was amplified nearly 391-fold and 434-fold compared to the initial bacterial load, and was amplified about 10-fold in Medium 2. The bacterial load of Pediococcus acidilactici was not increased slightly in medium 1, but amplified about 6-fold in medium 2 and 16-fold in medium 3.
TABLE 3 live bacteria content of different treatment groups in test 3
Test 4: medium 1 and Medium 3 were selected as co-cultured media for the experiments, and 220mL of each of the two media was prepared in a 250mL screw bottle, control 1(CK 1): inoculating clostridium butyricum seed liquid according to the inoculation proportion of 5 percent; control 2(CK 2): inoculating 5% of pediococcus acidilactici seed liquid; and (3) co-culture group: inoculating a mixed solution of clostridium butyricum seed solution and pediococcus acidilactici seed solution according to an inoculation ratio of 5% (the ratio of the clostridium butyricum seed solution to the pediococcus acidilactici seed solution is 1: 1); culturing the three groups at 37 deg.C for 34h, shaking the screw bottle every 6h to mix the calcium carbonate at the bottom uniformly, and maintaining the pH of the culture medium stable. The results show (table 4): the pure culture of the clostridium butyricum is more suitable for being cultured by adopting a culture medium 1, the bacterial load is 0.99 hundred million CFU/mL, the pure culture of the pediococcus acidilactici is more suitable for being cultured by adopting a culture medium 3, and the bacterial load is 9.9 hundred million CFU/mL. However, the bacterial load of clostridium butyricum and pediococcus acidilactici is reduced during co-culture, and No. 3 is adopted as a culture medium for co-culture of the two strains by integrating the bacterial load of the two strains.
Table 4 viable bacteria content of different treatment groups in test 4
Test 5: the inoculation amount of the culture medium 3 and the inoculation amount of the culture medium is continuously optimized, and the design of the inoculation amount is shown in a table 5:
TABLE 5 inoculum design
| Grouping | Clostridium butyricum inoculation amount | Pediococcus acidilactici inoculation amount |
| Group A | 2％ | 2％ |
| Group B | 3％ | 3％ |
| Group C | 5％ | 5％ |
220mL of the culture medium 3 is prepared into 250mL of screw bottles, the inoculation is carried out according to the test groups, the mixture is cultured for 34h at 37 ℃, the screw bottles are shaken every 6h, the calcium carbonate at the bottom is uniformly mixed, and the pH value of the culture medium is maintained to be stable. By adjusting the inoculation volume ratio of the pediococcus acidilactici to the clostridium butyricum, the result (table 6) shows that when the inoculation volume ratio of the pediococcus acidilactici to the clostridium butyricum inoculation bacterial quantity is 2%, the bacterial quantity of the clostridium butyricum and the pediococcus acidilactici co-cultured by the pediococcus acidilactici and the clostridium butyricum bacterial quantity can reach a highest quantity, so that the inoculation quantity of the pediococcus acidilactici to the clostridium butyricum is determined to be 2%.
TABLE 6 live bacteria content of different treatment groups in test 5
In conclusion, when the clostridium butyricum and the pediococcus acidilactici are co-cultured in a shake flask, the selected culture medium formula comprises 8g/L tryptone, 5g/L beef extract, 5g/L yeast extract, 15g/L glucose, 5g/L dipotassium hydrogen phosphate, 0.1g/L magnesium sulfate, 1g/L sodium acetate, 1g/L cysteine hydrochloride, 10g/L calcium carbonate and 7.0 pH; the inoculation amount of the clostridium butyricum seed liquid and the pediococcus acidilactici seed liquid is 2 percent.
Example 3: investigation of 50L fermentation tank for co-culture of clostridium butyricum and pediococcus acidilactici
Unable accurate control pH of culture in-process in the screw bottle, and can not nitrogen gas pressurize, can not form strict anaerobic environment, have certain influence to clostridium butyricum, can only tentatively confirm the fermentation medium of coculture. To further optimize the process, the optimization experiments were continued in a 50L fermentor.
Test 1: inoculating single colony of clostridium butyricum GBW-N1 in a Kirschner flask filled with RCM culture medium, culturing at 37 ℃ for 3 days, and scraping lawn with 0.8% physiological saline to obtain seed solution; picking single colony of pediococcus acidilactici, inoculating in MRS liquid culture medium, and performing amplification culture at 37 deg.C for 16h to obtain seed solution.
The formula of the fermentation medium comprises 8g/L of tryptone, 5g/L of beef extract, 5g/L of yeast extract, 15g/L of glucose, 5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate, 1g/L of sodium acetate, 1g/L of cysteine hydrochloride and 10g/L of calcium carbonate. The fermentation tank is filled with 30L of liquid, the pH value of a culture medium is adjusted to 7.0, autoclaving is carried out at the temperature of 121 ℃ for 30min, the culture medium is cooled to 37 ℃, inoculation is started, after clostridium butyricum seed liquid is inoculated firstly, pediococcus acidilactici seed liquid is inoculated after 8h, the inoculation amount of the clostridium butyricum seed liquid and the pediococcus acidilactici seed liquid is respectively 2%, the rotating speed is 80r/min, high-purity nitrogen is filled, the pressure is maintained at 0.05MPa, the pH value is controlled to be not less than 6.0 by adding ammonia in a flowing mode in the process, the pH value of; and after 8h, measuring the content of reducing sugar and the content of viable bacteria every 2h, wherein the pot stopping standard is that the spore falling rate is more than or equal to 50 percent when the pot is observed by microscopic examination. The results (table 7) show that: the content of clostridium butyricum after 20h can stop is 6.3 hundred million CFU/mL, while the content of pediococcus acidilactici after can stop is only 0.35 hundred million CFU/mL, which is mainly because the content of reducing sugar after the pediococcus acidilactici is inoculated for 8.36mg/mL after 8h, the carbon source is not enough for the pediococcus acidilactici to grow, and the stop of pH control after the clostridium butyricum produces spores has no influence on the output spores of the clostridium butyricum.
TABLE 7 run 1 fermentation Process parameter tracking
Test 2: the fermentation medium is the medium of test 1 for removing sodium carbonate, sodium hydroxide is adopted to control the pH to be more than or equal to 6.0, the pH is stopped when the spore rate is 50 percent, clostridium butyricum is inoculated firstly, pediococcus acidilactici is inoculated after 4 hours, and other conditions are completely consistent with those of the test 1. The test results (Table 8) show that the bacterial amount of the clostridium butyricum co-cultured is high and can reach 7.75 hundred million CFU/mL, but the spore rate is only 30%, and when the combined fermentation is carried out for 16 hours, the spore rate of the clostridium butyricum is 95% and almost 100% of the clostridium butyricum falls off, so that the fermentation period can be shortened to 16 hours according to the bacterial amount and the spore state. In addition, the maximum amount of Pediococcus acidilactici was 5.95 hundred million CFU/mL. And filling the final fermentation liquor into a 1L cola bottle, standing at room temperature for 2 days, and finding that the bottle is seriously expanded, which indicates that the stability of the fermentation liquor is poor.
Table 8 run 2 fermentation process parameters tracking
Test 3: the medium and culture conditions were the same as in experiment 2 above. Co-cultivation was performed in two batches, one: inoculating clostridium butyricum for 4 hours, inoculating pediococcus acidilactici for another batch: the clostridium butyricum is inoculated firstly, and the pediococcus acidilactici is inoculated after 6 hours. After fermentation is finished, the bacteria content of the two batches of clostridium butyricum and pediococcus acidilactici is slightly different, and the bacteria amount inoculated at an interval of 4h (the bacteria amount of clostridium butyricum is 5.4 hundred million CFU/mL, and the bacteria amount of pediococcus acidilactici is 55 hundred million CFU/mL) is higher than that inoculated at an interval of 6h (the bacteria amount of clostridium butyricum is 3.2 hundred million CFU/mL, and the bacteria amount of pediococcus acidilactici is 45 hundred million CFU/mL).
Test 4: the fermentation scale of the medium was increased to 6 tons and the liquid loading was 60% as in test 3. Respectively preparing clostridium butyricum and pediococcus acidilactici seed solutions by adopting a 150L fermentation tank, respectively culturing the clostridium butyricum and pediococcus acidilactici seed solutions for 16h, respectively carrying out switching according to 2% inoculation amount, firstly inoculating the clostridium butyricum and then inoculating the pediococcus acidilactici, wherein the interval time is 4 h; the rotating speed is 100r/min, high-purity nitrogen is filled, the pressure is maintained at 0.05MPa, the pH value of sodium hydroxide solution flowing in the process is controlled to be more than or equal to 6.0, and the spore falling rate is observed to be more than or equal to 50% by microscopic examination according to the tank stopping standard. When the fermentation is finished, the bacterium content of clostridium butyricum is 6.0 hundred million CFU/mL, and the bacterium content of pediococcus acidilactici is 60 hundred million CFU/mL. The fermentation broth obtained after the fermentation is finished is earthy yellow (figure 3), and then is filled in a plastic bottle with a ventilating cover (figure 4) by an automatic filling machine in a dust-free workshop, and the stability is good within the shelf life of 360 days.
In conclusion, when Clostridium butyricum and Pediococcus acidilactici were co-cultured in a fermenter,inoculating clostridium butyricum seed liquid into a fermentation tank by using an inoculation amount of 2%, fermenting for 4 hours at 37 ℃, then inoculating pediococcus acidilactici seed liquid into the fermentation tank by using an inoculation amount of 2%, co-culturing for 16-22 hours at 37 ℃, controlling the pH to be more than or equal to 6.0 by using a sodium hydroxide solution or ammonia water in the fermentation process, controlling the pressure of the tank to be 0.05MPa by using high-purity nitrogen, and fermenting at 80-100 r/min. The composite microbial inoculum prepared by the step has the clostridium butyricum content of 5 multiplied by 108～6×108CFU/mL, the spore rate is more than 90%, and the shedding rate is more than 50%; the content of Lactobacilli is 5.0 × 109～6.0×109CFU/mL, the compound microbial inoculum has good stability and is convenient to store.
Example 4: application of composite preparation co-cultured by clostridium butyricum and pediococcus acidilactici in penaeus vannamei culture
In a recirculating aquaculture system (figure 5), 400 litopenaeus vannamei with tail length of 4-5 cm is taken as a research object and divided into 2 groups, each group is 4 in number, 20 litopenaeus vannamei are repeated, a control group is fed with feed, the feed of a test group is mixed with feed containing 20mL/kg of the compound microbial inoculum prepared in the example 3, and the test period is 30 days. Observing the defecation condition and death condition of the prawns every day, and recording the food intake; after the culture test is finished, measuring the total weight of each jar of prawns, and calculating the weight gain rate, the survival rate and the bait coefficient; and analyzing the diversity of intestinal microorganisms through high-throughput sequencing, analyzing the change of intestinal tissues through an HE stained section, and measuring the change of nonspecific indexes by taking serum.
The results show that: the test group feed is mixed with 20mL/kg of the compound microbial inoculum, so that the shrimp intestines are clearer, the shrimp bodies are fuller, and the phenomenon of defecation is avoided, while the control group has the phenomena of intestine breaking and defecation (figure 6); the weight gain rate of the test group is improved by 8.2 percent compared with that of the control group in 30 days, the survival rate is improved by 13.6 percent, and the feed coefficient is reduced by 0.08 (Table 9); the serum alkaline phosphatase AKP level was significantly increased (fig. 7). HE results (fig. 8) show that the midgut epithelial cells of prawns are more densely arranged, the number of villi is increased, and the height of villi/depth of crypts is increased. High-throughput analysis (fig. 9) shows that the ratio of vibrio in total flora is obviously reduced with the use of the composite microbial inoculum, which indicates that the composite microbial inoculum has good bacteriostatic effect, can recover and improve the intestinal function and intestinal microbial flora structure of penaeus vannamei boone, and reduces the occurrence probability of enteritis.
TABLE 9 difference in growth indexes between control group and test group
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A composite preparation containing Clostridium butyricum and Pediococcus acidilactici, which comprises bacteria with a content of 5-6 x 108The content of Clostridium butyricum and bacteria is 5.0-6.0 × 109CFU/mL of Pediococcus acidilactici.
2. The complex preparation as claimed in claim 1, wherein the Clostridium butyricum is Clostridium butyricum GBW-N1 with the collection number of CGMCC No. 14499.
3. The method for preparing the composite preparation according to claim 1 or 2, wherein the method comprises the following specific steps:
(1) respectively carrying out activation culture on clostridium butyricum and pediococcus acidilactici in a seed culture medium to obtain clostridium butyricum seed solution and pediococcus acidilactici seed solution;
(2) inoculating the clostridium butyricum seed solution into a fermentation tank, fermenting for 4-5h in a fermentation medium at 37 ℃, then inoculating the pediococcus acidilactici seed solution into the fermentation tank, co-culturing for 16-22h at 37 ℃ to obtain the compound preparation, and filling for later use.
4. The method according to claim 3, wherein the inoculum size of the Clostridium butyricum seed solution is 2%; the inoculation amount of the pediococcus acidilactici seed liquid is 2%.
5. The preparation method according to claim 3, wherein the fermentation medium comprises 6-12 g/L tryptone, 5-10 g/L beef extract, 5-15 g/L yeast extract, 20-30 g/L glucose, 2-5 g/L dipotassium hydrogen phosphate, 0.1-0.3 g/L magnesium sulfate, 1-3 g/L sodium acetate, 0.5-1 g/L cysteine hydrochloride, 10-20 g/L calcium carbonate, and has a pH of 6.8-7.0.
6. The method according to claim 3, wherein the conditions of the fermentation in the step (2): the pressure of the tank body is controlled to be 0.05MPa by high-purity nitrogen, the rotating speed is 80-100r/min, and the pH value of the fermentation medium is controlled to be more than or equal to 6.0 by adopting a sodium hydroxide solution or ammonia water.
7. Use of a complex formulation according to claim 1 or 2 for the preparation of a microbial formulation for enhancing growth performance of aquatic animals.
8. The use as claimed in claim 7, wherein the complex formulation is administered by mixing with feed at a rate of 20-30mL/kg of feed, and feeding for more than 30 days.
9. The use according to claim 7, wherein the complex formulation is capable of increasing weight gain and survival rate of aquatic animals and decreasing the feed factor.
10. The use according to claim 7, wherein the aquatic animal comprises Penaeus vannamei.
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