A probiotic for aquatic feed and preparation method thereof
A technology of bacterial agents and aquatic animals, applied in the field of microorganisms, can solve problems such as inability to exert the best beneficial effect
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Embodiment 1
[0057] Example 1. Isolation and identification of Bacillus velezensis T23
[0058] In this study, Bacillus velezensisT23 was isolated and used as an indigenous strain of fish gut. Specifically, under sterile conditions, the intestinal contents were slowly extruded from the hindgut of the fish intestine to the anterior intestine, placed in a 50 mL centrifuge tube containing 10 mL of PBS buffer, mixed well and allowed to stand for 1 minute, and then removed. The serum was serially diluted, taking 10 -5 ~10 -9The dilution gradient of 100μL was coated with TSA medium (15g / L casein trypsin digest, 5g / L soybean meal papain digest, 5g / LNaCl, 15g / L agar), after culturing for 24 hours, pick colonies and streak Purification, the purified single colony was inoculated into the liquid medium, and after culturing for 15-16 hours, an appropriate amount of bacterial liquid was taken and sent to a sequencing company for 16S rDNA sequencing. The sequence similarity to 16S rDNA of Bacillus ve...
Embodiment 2
[0060] Example 2. Bacillus velesi T23 promotes the growth of zebrafish and reduces the feed coefficient
[0061] 1. Preparation of Bacillus velesi T23 inoculum
[0062] 1. Activation of strains: Bacillus velesi T23 was stored in an ultra-low temperature refrigerator, streaked on LB solid plates, and cultured at 30°C overnight;
[0063] 2. First-class seed culture: pick a single colony in LB (Luria-Bertani) medium, cultivate at 30°C, 180r / min for 24h;
[0064] 3. Secondary seed liquid culture: inoculate in LB medium with 1% inoculum, and cultivate for 12h at 30°C and 180r / min;
[0065] 4. Solid fermentation: the secondary seed liquid is inoculated into the solid medium at 5% inoculation amount; the culture of T23 obtained by shaking culture at 30°C for 48 hours, namely the T23 fermentation product, is the Bacillus velesi T23 inoculum. In the bacterial agent of Bacillus velesi T23, the content of Bacillus velesi T23 is 8×10 8 cfu / g.
[0066] Preparation method of solid ferme...
Embodiment 3
[0073] Example 3. Bacillus velesi T23 improves the intestinal morphology of zebrafish
[0074] After the culturing test, the zebrafish treated with Bacillus group starved for 24 h in Example 2 and the zebrafish treated with control group were dissected on ice, and the intact intestine was taken out and placed in a centrifuge tube filled with 4% paraformaldehyde. Fixation, HE staining was done by Beijing Zhongke Wanbang Biotechnology Co., Ltd., and photography was done in our laboratory (software is ZEN). Intestinal villus length and muscle thickness were measured by image J software.
[0075] The results showed that the addition of Bacillus velesi T23 to the Bacillus group significantly increased zebrafish intestinal villus length and muscle layer thickness compared with the control group ( figure 2 ) (P<0.01), indicating that Bacillus velesi T23 can improve the intestinal health of fish by improving the intestinal morphology.
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