CN112385754B - Composite microbial agent for protecting liver and intestine of penaeus vannamei boone and application thereof - Google Patents

Composite microbial agent for protecting liver and intestine of penaeus vannamei boone and application thereof Download PDF

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CN112385754B
CN112385754B CN202011306543.8A CN202011306543A CN112385754B CN 112385754 B CN112385754 B CN 112385754B CN 202011306543 A CN202011306543 A CN 202011306543A CN 112385754 B CN112385754 B CN 112385754B
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penaeus vannamei
microbial inoculum
bacteroides thetaiotaomicron
vannamei boone
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CN112385754A (en
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曹爱智
娄倩倩
籍立民
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Shandong Longchang Animal Health Products Co ltd
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Abstract

The invention discloses a composite microbial inoculum for protecting liver and intestine of penaeus vannamei boone and application thereof. The compound microbial inoculum comprises bacteroides thetaiotaomicron bacterial powder, bile acid and eucalyptus globulus essential oil, and the mass ratio of the bacteroides thetaiotaomicron bacterial powder to the bile acid to the eucalyptus globulus essential oil is 2-3: 1; the bacteroides thetaiotaomicron bacterium powder is prepared by culturing, fermenting and drying bacteroides thetaiotaomicron GL-02 with the preservation number of CGMCC No.20818. The compound microbial inoculum can enhance the digestion and absorption capacity of fat of the penaeus vannamei, ensure that the liver absorbs and stores sufficient nutrient substances and energy, promote the penaeus vannamei to smoothly transform the liver, promote the liver to discharge mycotoxin, antibiotics, heavy metals and other toxins which enter the organism out of the body, reduce the damage of toxic and harmful substances to the hepatopancreas, increase the growth of the body length of the penaeus vannamei, and be beneficial to the development of the breeding industry of the penaeus vannamei.

Description

Composite microbial agent for protecting liver and intestine of penaeus vannamei boone and application thereof
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to a compound microbial inoculum for protecting liver and intestine of penaeus vannamei boone and application thereof.
Technical Field
The penaeus vannamei boone belongs to omnivorous species, is deeply popular with the masses in China due to rich nutrition, delicious taste and various preparation methods, has higher economic value, and therefore starts to be cultured in a large scale in China. However, in the process of culturing penaeus vannamei boone, hepatopancreatic diseases or yellowing phenomena are easy to occur, and the main reasons are as follows: organic matters at the bottom of the shrimp pond are increased; the metal content of the water body is too high; the water body is lack of certain trace elements, so that the prawn is indigestion and lack of nutrient substances; the nutrient metabolism is not thorough, so that the hepatopancreas burden of the prawns is caused; the penaeus vannamei does not secrete bile acid per se, has poor fat absorption capacity, and even a small stress occurs, the penaeus vannamei can influence the digestion and absorption of the hepatopancreata, and the hepatopancreata is yellow or has diseases. In addition, the problem that white feces easily appear in the rapid growth period and the middle and later culture periods of the penaeus vannamei boone, and the white feces are pathological tissues, mucus and venom, mucosa shed by intestinal tracts and a small part of normal excrement components which are actually discharged by the hepatopancreas of the penaeus vannamei boone. Therefore, in order to better promote the growth of the penaeus vannamei boone, the development of a natural, efficient and harmless preparation is necessary.
Disclosure of Invention
The invention provides a compound microbial inoculum for protecting liver and intestine of penaeus vannamei boone and application thereof. The compound microbial inoculum is safe and reliable, can promote the penaeus vannamei boone to smoothly transform the liver, and can restore and protect the liver and intestine health of the penaeus vannamei boone.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a compound microbial inoculum for protecting livers and intestines of penaeus vannamei boone, which comprises bacteroides thetaiotaomicron bacterium powder, bile acid and eucalyptus globulus oil.
Further, the bacteroides thetaiotaomicron bacterium powder is prepared by culturing, fermenting and drying bacteroides thetaiotaomicron GL-02 with the preservation number of CGMCC No.20818.
Further, the preparation method of the bacteroides thetaiotaomicron bacterial powder comprises the following steps: picking Bacteroides thetaiotaomicron GL-02 into GAM liquid culture medium, activating for 48h at 35 ℃ under anaerobic environment, taking 1mL of the obtained activated bacterium liquid, inoculating into 50mL of GAM liquid culture medium again, culturing for 24-76h, centrifuging the culture solution at 10000rpm/min for 5min, then resuspending the strain precipitate with sterile physiological saline with the volume 10-12 times of the mass of the strain precipitate, centrifuging at 10000rpm/min for 5min again, and secondarily resuspending with sterile physiological saline with the volume 5 times of the mass of the strain precipitate; then skim milk is selected as a protective agent, and the Bacteroides thetaiotaomicron GL-02 bacterial powder is prepared by a vacuum freeze-drying method.
Further, the Bacteroides thetaiotaomicron GL-02 bacteria heavy suspensionThe content of the bacteria is not less than 1 x 10 9 CFU/mL。
Further, the bacterium content of the compound bacterium agent is not less than 5 multiplied by 10 8 CFU/g。
Further, the mass ratio of the bacteroides thetaiotaomicron bacterial powder, the bile acid and the eucalyptus globulus bracteatum essential oil in the composite microbial inoculum is 2-3:1.
The invention provides an application of the compound microbial inoculum in preparation of a feed additive for penaeus vannamei boone.
Furthermore, the usage amount of the compound microbial inoculum is 1 kilogram of feed added with 10-25g of the compound microbial inoculum.
Further, the use method of the complex microbial inoculum comprises the following steps: the compound microbial inoculum is directly mixed into the breeding feed of the penaeus vannamei boone, and the feed is continuously fed for 20-30 days for 3 times per day.
Further, the addition amount of the compound microbial inoculum is 1 kilogram of the compound microbial inoculum added in the culture feed for the penaeus vannamei boone, and is 10-25 g.
Furthermore, the compound microbial inoculum can restore the liver and intestinal health of the penaeus vannamei boone and reduce jejunum.
Furthermore, the composite microbial inoculum can promote the prawn to smoothly transform the liver.
Furthermore, the compound microbial inoculum can increase the body length growth of the penaeus vannamei boone.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention separates a bacteroides thetaiotaomicron GL-02 from the excrement of pigs, and the bacteroides does not cause harm to animal and human bodies and does not influence the environment; the invention utilizes Bacteroides thetaiotaomicron GL-02 bacterial powder, bile acid and eucalyptus globulus oil to prepare a brand new compound microbial inoculum, and the compound microbial inoculum is directly added into the breeding feed of the penaeus vannamei, so that the digestion and absorption capacity of fat of the penaeus vannamei can be enhanced, sufficient nutrient substances and energy can be absorbed and stored by the liver of the penaeus vannamei, and the penaeus vannamei can be promoted to be smoothly transferred to the liver; bile acid, eucalyptus essential oil and bacteroides thetaiotaomicron GL-02 bacterial powder in the composite microbial inoculum have good activity, can promote prawn livers to discharge mycotoxin, antibiotics, heavy metals and other toxins which enter organisms out of the bodies, and reduces damage of toxic and harmful substances to livers and pancreas. The bacteroides thetaiotaomicron in the composite microbial inoculum can promote the decomposition of fat, reduce the accumulation of fat and increase the absorption of minerals by prawns.
Drawings
FIG. 1 is a photograph of colony of Bacteroides thetaiotaomicron GL-02.
Fig. 2 shows the problem of yellow liver on the left side, the problem of thin intestinal tract in the middle, and the problem of white feces on the right side of a penaeus vannamei boone with problems in cultivation.
FIG. 3 shows Penaeus vannamei Boone after the compound microbial inoculum is used; the left side is the penaeus vannamei fed for 5 days, and the right side is the penaeus vannamei fed for 20 days.
FIG. 4 shows shrimp larvae cultured for 30 days without the complex microbial inoculum.
FIG. 5 shows shrimp larvae cultured for 30 days by using the complex microbial inoculum.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Example 1:
1. screening of Bacteroides thetaiotaomicron GL-02
Mixing 10g of healthy pig feces with 100mL of sterile water, filtering to remove impurities to obtain feces diluent, and adding sterile water into the diluent to obtain 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Diluting the solution; respectively take 10 -2 、10 -3 、10 -4 、10 -5 The diluted solution was applied to GAM agar medium and the content was 80% N 2 、10％CO 2 And 10% of H 2 Culturing at 35 ℃ for 48h in an anaerobic environment, selecting obvious bacterial colonies, purifying for multiple times to obtain single bacterial colonies, naming the obtained single bacterial colonies as GL-02, and freezing and preserving. The GAM agar culture medium comprises the following components: 10.0g peptone, 3.0g soytone, 10.0g monthly peptone, 13.5g serum digest powder, 2.2g of beef extract powder, 5.0g of yeast extract powder, 1.2g of beef liver extract powder, 3.0g of glucose, 3.0g of sodium chloride, 2.5g of monopotassium phosphate, 5.0g of soluble starch, 0.3g of L-cysteine, 0.15g of sodium thioglycolate, 15.0g of agar, 1mL of 0.1% vitamin K1 solution, 3mL of hemin solution, 100mg of kanamycin, 1000mL of distilled water and pH 7.1.
As shown in figure 1, the colony of the strain GL-02 on GAM agar medium is round or round-like, yellow, 0.5-2.5mm in diameter, smooth and dull in surface, slightly convex in middle, opaque, and relatively neat in edge.
2. Identification of Bacteroides thetaiotaomicron GL-02
Extracting genome DNA of the strain GL-02, using the genome DNA as a template, performing PCR amplification by using a 16S rRNA universal primer to obtain a 16S rRNA amplification sequence, sequencing, and performing Blast comparison on the sequence to show that the strain GL-02 has the highest homology with Bacteroides thetaiotaomicron in a GenBank gene library, thereby judging the strain GL-02 to be Bacteroides thetaiotaomicron.
And (3) performing strain preservation on the screened Bacteroides thetaiotaomicron GL-02, wherein the preservation unit comprises the following steps: 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: 09 month and 25 days 2020; the preservation number of the Bacteroides thetaiotaomicron is CGMCC No.20818.
3. Preparation of Bacteroides thetaiotaomicron GL-02 bacterial powder
Selecting the preserved Bacteroides thetaiotaomicron GL-02 to GAM liquid culture medium (the agar can be reduced on the formula of the GAM agar culture medium), activating for 48h under 35 ℃ anaerobic environment, inoculating 1mL of the obtained activated bacterium liquid into 50mL of GAM liquid culture medium again for culturing for 24-76h, centrifuging the culture solution at 10000rpm/min for 5min, then re-suspending the strain precipitate with sterile physiological saline with the volume 10-12 times of the mass of the strain precipitate, centrifuging at 10000rpm/min for 5min again, re-suspending with sterile physiological saline with the volume 5 times of the mass of the strain precipitate, and ensuring that the bacterium content of the Bacteroides thetaiotaomicron GL-02 in the heavy suspension is not less than 1 x 10 9 CFU/mL; then selecting skimmed milk as protectant, and freeze drying under vacuumThe Bacteroides thetaiotaomicron GL-02 bacterial powder is prepared by the method.
Example 2: preparation of complex microbial inoculum
1. Preparation of bile acids
The preparation process of the bile acid comprises the following steps:
(1) Saponification: adding 1kg of pig gall powder crushed and sieved by a 40-mesh sieve into a reaction kettle, adding 10L of sodium hydroxide solution with the mass concentration of 10%, heating and stirring until the mixture is boiled, keeping the boiling state and stirring for 14 hours, then cooling saponification liquid generated by the reaction until solid and liquid are layered, removing supernatant, and obtaining residual solid which is bile acid saponification product;
(2) And (3) decoloring: adding 6L of water into the bile acid saponified product, heating to 80 ℃ to completely dissolve the bile acid saponified product, then putting the bile acid saponified product into a decoloring tank, adding 300mL of hydrogen peroxide, uniformly stirring, reacting at normal temperature for 24 hours, and then filtering the reaction solution into an acidification tank to obtain a filtrate;
(3) Acidifying: cooling the filtrate to room temperature, slowly adding 10% hydrochloric acid solution while stirring, stopping adding acid when the pH value of the solution is 3, and centrifugally filtering to obtain a white solid;
(4) And (3) purification: continuously adding water to wash the white solid matter, removing water-soluble impurities attached to the surface of the white solid matter, and finishing the water washing after the filtered water is detected to be neutral; placing the white solid after centrifugal filtration into a drying oven, and drying at 100 ℃ until the moisture is less than 10% to obtain a bile acid crude product;
(5) And (3) recrystallization: putting the bile acid crude product into an extraction tank, adding ethyl acetate with the volume 10 times of the weight of the bile acid crude product, stirring until the bile acid crude product is completely dissolved, then adding 0.08g of anhydrous sodium sulfate for dehydration, filtering and collecting filtrate, carrying out reduced pressure concentration on the filtrate, and recovering ethyl acetate to obtain liquid bile acid;
(6) And (3) drying: and (3) putting the liquid bile acid into an oven, and drying at 100 ℃ until the water content is not more than 1% to obtain the finished product bile acid.
The finished bile acid contains 78.6% of hyocholic acid and hyodeoxycholic acid and 20.0% of chenodeoxycholic acid.
2. Preparation method of eucalyptus globulus oil
Washing Australian multi-bud eucalyptus leaves in a washing tower, putting the Australian multi-bud eucalyptus leaves in a dryer for drying, crushing the Australian multi-bud eucalyptus leaves in a crusher, then respectively putting the crushed Australian multi-bud eucalyptus leaves into a steam distillation kettle, a condenser and an oil-water delayer for separating an oil phase and a water phase, putting the water phase into an ether extractor for separating wastewater, putting liquid containing ether into an ether recovery tower for recovering the ether, adding the recovered ether and oil phase liquid into a solid bed adsorption dryer for further removing water in the oil phase, and finally adding the oil phase liquid into a reduced pressure distillation tower for rectification so as to remove impurity oil, thereby obtaining the multi-bud eucalyptus essential oil. The prepared eucalyptus globulus labill essential oil is stored in a sealed and lightproof container and is placed away from light.
3. Preparation of complex microbial inoculum
Uniformly mixing the prepared bile acid, eucalyptus globulus oil and bacteroides thetaiotaomicron GL-02 bacterial powder in a mass ratio of 2: 1: 2 to obtain a composite microbial inoculum; the bacterium content in the composite bacterium agent is not less than 5 multiplied by 10 8 CFU/g。
Example 3
15 mu of southern white shrimps were carefully cultured in the Shandong Weifang pond, and during the culture period, the shrimps had a slow feeding problem, and the liver of the shrimps turned yellow, the intestinal tract was thin and white feces appeared (fig. 2). The feed is mixed with 1kg of feed and 15g of composite microbial inoculum, and the mixture is fed for 3 times a day for 20 days continuously, and the rest is carried out according to the daily culture. As shown in figure 3, after the compound microbial inoculum is fed for 5 days, the yellow color of the liver of the prawn is reduced, the intestinal tract becomes thick, the liver and pancreas are clear in outline, and the color is normal; after feeding for 20 days, the liver and intestinal tract of the prawn are recovered to be healthy, the liver and intestinal tract are cleaned to be full and bright, no white feces are produced, the intestinal tract of the prawn is coarse, and the ingestion is good; and the intestinal tract of the prawns fed with the common feed has jejunum phenomenon.
Example 4
100 mu of penaeus vannamei boone is cultured in a pond outside the Qinzhou Guangxi city, 1 mu of pond is randomly selected, 15-20g of the compound microbial inoculum is mixed and added into 1kg of prawn feed after the seedlings of the penaeus vannamei boone are released, the penaeus vannamei boone is fed for 30 days and 3 times a day, and the rest is carried out according to a conventional culture method.
The results are shown in fig. 4 and fig. 5, the average body length of the penaeus vannamei boone using the composite microbial inoculum is 4.1cm, the whole body is transparent, and the activity is good, while the average body length of the penaeus vannamei boone not using the composite microbial inoculum is 3.4cm, and a white film is occasionally formed on the surface of the penaeus vannamei boone. The length of the penaeus vannamei boone using the compound microbial inoculum is 0.7cm longer than that of the unused penaeus vannamei boone, which shows that the compound microbial inoculum can promote the growth of the penaeus vannamei boone. And after the prawn seedlings are released for about 20 days, the prawn starts to enter a liver-turning period, and compared with the prawn without the compound microbial inoculum, the prawn with the compound microbial inoculum has clear and full liver and intestinal tract, thick intestinal tract and no jejunum, so that the compound microbial inoculum can promote the prawn to turn the liver smoothly and protect the liver and the intestinal tract of the prawn.
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 modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims (7)

1. A composite microbial inoculum for protecting liver and intestine of Penaeus vannamei Boone is characterized by comprising Bacteroides thetaiotaomicron bacterial powder, bile acid and eucalyptus globulus oil; the bacteroides thetaiotaomicron bacterium powder is prepared by culturing, fermenting and drying bacteroides thetaiotaomicron GL-02 with the preservation number of CGMCC No. 20818; the mass ratio of the bacteroides thetaiotaomicron bacterial powder to the bile acid to the eucalyptus globulus essential oil in the composite microbial agent is 2-3.
2. The complex microbial inoculum of claim 1, which has a microbial content of not less than 5 x 10 8 CFU/g。
3. The use of the complex microbial inoculum of claim 1 in the preparation of a feed additive for penaeus vannamei boone.
4. The application of the complex microbial inoculum is characterized in that the complex microbial inoculum is used by the method comprising the following steps: the compound microbial inoculum is directly mixed into the breeding feed of the penaeus vannamei boone, and the feed is continuously fed for 20-30 days for 3 times per day.
5. The application of claim 4, wherein the addition amount of the compound microbial inoculum is 10-25g of the compound microbial inoculum added in 1kg of the culture feed of the penaeus vannamei boone.
6. The use of claim 3, wherein the complex microbial inoculant can restore liver and intestinal health of Penaeus vannamei Boone and reduce jejunum.
7. The use of claim 3, wherein the complex microbial inoculum can increase the body length growth of Penaeus vannamei Boone.
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Publication number Priority date Publication date Assignee Title
WO2000041576A1 (en) * 1999-01-14 2000-07-20 Food Technology Innovations Pty Limited Improved microbial preparations
CN103750341A (en) * 2014-01-13 2014-04-30 苏州万生源生物科技有限公司 Inactivated bacteroid micro-ecological preparation and preparation method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000041576A1 (en) * 1999-01-14 2000-07-20 Food Technology Innovations Pty Limited Improved microbial preparations
CN103750341A (en) * 2014-01-13 2014-04-30 苏州万生源生物科技有限公司 Inactivated bacteroid micro-ecological preparation and preparation method thereof

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对虾弧菌病的分类与防治;周英;《热带农业工程》;20200426;第44卷(第02期);第109-112页 *

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