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CN-121975907-A - Sorting method of corrugated plate Bao Yi raw bacteria and compound feed

CN121975907ACN 121975907 ACN121975907 ACN 121975907ACN-121975907-A

Abstract

The invention belongs to the technical field of microorganism application and aquatic feed, in particular to a method for sorting the raw bacteria of a corrugated plate Bao Yi, the method is to separate and screen probiotics with strong adaptability and comprehensive functions from the intestinal tracts of the haliotis discus hannai, the probiotics are naturally adapted to the intestinal tract environment of the haliotis discus hannai, and the probiotics have the secretion capacity of digestive enzymes and the inhibition capacity of pathogenic bacteria. On the other hand, the compound feed containing probiotics obtained by the sorting method is also disclosed, the feed can improve the growth performance and the feed utilization rate of abalones, and meanwhile, the disease resistance is also enhanced.

Inventors

  • ZHANG WENBING
  • HUANG WENCHAO
  • Ren Guiming
  • ZHANG TIANSHU
  • MAI KANGSEN
  • WU ZHENHUA
  • ZHANG ENZE
  • JIANG MINGCAN
  • LIU YONGQING
  • LIU YANG
  • QU PENG
  • Ma Daisong
  • ZHOU HONGYING

Assignees

  • 中国海洋大学深圳研究院
  • 中国海洋大学
  • 中国水产有限公司

Dates

Publication Date
20260505
Application Date
20260116

Claims (10)

  1. 1. The method for sorting the raw bacteria of the corrugated plate Bao Yi is characterized by comprising the following steps of: s1, selecting a plurality of healthy haliotis discus hannai, dissecting and taking intestinal tracts of the healthy haliotis discus hannai; S2, flushing the intestinal tract with a sterile phosphate buffer solution to remove the residue of intestinal contents, shearing the intestinal tract, adding an equal volume of 0.9% sterile NaCl solution, and homogenizing by a sterile homogenizer to obtain intestinal tract homogenate; S3, diluting the intestinal homogenate according to a gradient of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 , taking 0.1mL of each diluted bacterial liquid, uniformly coating the bacterial liquid on a 2216E agar culture medium plate, and culturing at a constant temperature of 28 ℃ for 48 hours with 3 repetitions of each dilution; S4, picking single colonies with different forms on a 2216E agar culture medium plate, inoculating the single colonies into 2216E liquid culture medium, carrying out shake culture at 28 ℃ and 150rpm for 24 hours, and repeatedly streaking and purifying for 3 times to obtain a purified strain.
  2. 2. The method for sorting bio-organisms from the haliotis discus Bao Yi according to claim 1, wherein the number of healthy haliotis discus haliotis selected in S1 is 30 and the average body weight is 4.62±0.19g.
  3. 3. The method of claim 1, wherein the step of immersing the healthy haliotis discus abalone selected for anesthesia in 75% ethanol is further included in S1 before dissecting the intestinal tract thereof under aseptic conditions.
  4. 4. The method of claim 1, wherein the step of washing the intestinal tract with sterile phosphate buffer is performed at least 3 times in S2.
  5. 5. The method for sorting the raw bacteria of the corrugated plate Bao Yi according to claim 1, which is characterized by further comprising the step of S5 after S4, screening the cellulase-producing strain, specifically, the strain obtained in S4 is inoculated on a screening medium containing 1% sodium carboxymethyl cellulose, cultured at 28 ℃ for 48 hours, dyed with 0.1% Congo red solution for 1 hour, decolorized with 1mol/L NaCl solution, and the strain capable of forming transparent circles is screened to obtain the cellulase-producing strain.
  6. 6. The method for sorting the raw bacteria of the frizzled Bao Yi according to claim 1, which is characterized by further comprising the step of S6 after S5, screening the vibrio resistant strains, specifically, uniformly coating the vibrio harveyi on an LB culture medium plate, then adding 100 mu L of strains to be screened, culturing for 24 hours at 28 ℃, and screening the strains with the diameter of a bacteriostasis zone being more than or equal to 10mm to obtain the vibrio resistant strains.
  7. 7. The method for sorting microorganisms on a corrugated disk Bao Yi according to claim 1, wherein the concentration of Vibrio harveyi in S6 is 1.0X10: 10 7 CFU/mL.
  8. 8. A compound feed comprising the strain of Pantoea fringensis Bao Yisheng obtained by the method of selecting the strain of Pantoea fringensis Bao Yi according to any one of claims 1 to 7 and a basic feed.
  9. 9. The compound feed according to claim 8, wherein the preparation method of the basic feed is characterized in that raw materials of the basic feed are respectively crushed, sieved by a 100-mesh sieve, uniformly mixed according to weight percentage, added with water and stirred to be homogeneous, the mixture is pressed into a sheet shape with the thickness of 3mm by a double-roller tablet press, then cut into a sheet shape with the thickness of 30mm by 20mm, steamed for 10min at 100 ℃, and finally dried at 60 ℃ until the moisture content is less than 8%, so that the basic sheet feed is obtained.
  10. 10. The compound feed according to claim 9, wherein the adding method of the bacteria in the wrinkle disc Bao Yi is that the bacteria in the wrinkle disc Bao Yi are inoculated to 2216E liquid culture medium, cultured for 24 hours at 28 ℃ and 150rpm, the bacteria are collected by 4500 Xg centrifugation for 15min, and are resuspended after being washed 2 times with sterile seawater, and the bacteria suspension with different concentrations is prepared by dilution; Drying the bacteria-added feed in a 38 ℃ air-blast drying oven until the moisture is less than 8%, sealing and packaging, and preserving at-20 ℃ to prepare the bacteria-added feed 1 time a week so as to ensure the bacterial activity.

Description

Sorting method of corrugated plate Bao Yi raw bacteria and compound feed Technical Field The invention relates to the technical field of microorganism application and aquatic feed, in particular to a method for sorting bacteria growing on a corrugated plate Bao Yi and compound feed. Background The culture of the haliotis discus hannai abalone faces two main limiting factors, namely slow growth, the traditional compound feed can meet basic nutrition requirements, but the feed conversion rate of the haliotis discus hannai abalone is higher, generally more than 2.0, the culture period is long, the cost is high, the disease resistance is low, pathogenic vibrio such as vibrio parahaemolyticus (Vibrio parahaemolyticus) in the culture environment easily causes the death of the haliotis discus hannai in a large scale, and particularly in high-temperature seasons, the disease death rate is high, and the culture income is seriously influenced. In order to solve the problems, the prior art mostly adopts exogenous probiotics separated from soil, sea water or intestinal tracts of other aquatic animals to be added into feed, but has two defects that firstly, the exogenous probiotics and the intestinal microenvironment of the haliotis discus abalone are poor in adaptability and weak in field planting capability, and are difficult to survive in intestinal tracts for a long time, so that the effect is unstable, and secondly, most probiotics only have a single function of bacteriostasis or digestion promotion, and cannot achieve the dual aims of improving the digestibility and resisting diseases. Disclosure of Invention In order to solve the technical problems in the background art, the invention provides a sorting method of the strain of the haliotis discus Bao Yi, which is used for separating and screening probiotics with strong adaptability and comprehensive functions from the intestinal tracts of haliotis discus, wherein the probiotics are naturally adapted to the intestinal environment of abalone, and simultaneously have the secretion capacity of digestive enzymes and the inhibition capacity of pathogenic bacteria, and have important significance for promoting the high-quality development of abalone culture industry. Meanwhile, the invention also provides a compound feed containing probiotics obtained by the sorting method and application of the compound feed in culture of haliotis discus hannai. As one aspect of the present invention, there is provided a method for sorting raw fungi from a corrugated plate Bao Yi, comprising the steps of: s1, selecting a plurality of healthy haliotis discus hannai, dissecting and taking intestinal tracts of the healthy haliotis discus hannai; S2, flushing the intestinal tract with a sterile phosphate buffer solution to remove the residue of intestinal contents, shearing the intestinal tract, adding an equal volume of 0.9% sterile NaCl solution, and homogenizing by a sterile homogenizer to obtain intestinal tract homogenate; S3, diluting the intestinal homogenate according to a gradient of 10 -1、10-2、10-3、10-4、10-5、10-6, taking 0.1mL of each diluted bacterial liquid, uniformly coating the bacterial liquid on a 2216E agar culture medium plate, and culturing at a constant temperature of 28 ℃ for 48 hours with 3 repetitions of each dilution; S4, picking single colonies with different forms on a 2216E agar culture medium plate, inoculating the single colonies into 2216E liquid culture medium, carrying out shake culture at 28 ℃ and 150rpm for 24 hours, and repeatedly streaking and purifying for 3 times to obtain a purified strain. In the above method for selecting the strain of the haliotis yprickle Bao Yi, the number of healthy haliotis yprickle selected in S1 is 30, and the average weight is 4.62.+ -. 0.19g. In the above method for sorting the bio-fungus from the haliotis discus Bao Yi, the step of immersing the healthy haliotis discus abalone selected for anesthesia with 75% ethanol is further included before dissecting the intestinal tract under aseptic operation in S1. In the above method for sorting the raw bacteria of the corrugated plate Bao Yi, the number of times of washing the intestinal tract with the sterile phosphate buffer solution in S2 is at least 3. The sorting method of the strain producing cellulase of the frizzled Bao Yi further comprises the step of S5 after S4, specifically, the pure strain obtained in S4 is inoculated on a screening culture medium containing 1% of sodium carboxymethyl cellulose, cultured for 48 hours at 28 ℃, dyed for 1 hour by 0.1% of Congo red solution, decolorized by 1mol/L of NaCl solution, and the strain capable of forming transparent circles is screened to obtain the strain producing cellulase. The method for sorting the bacterial strains of the frizzled Bao Yi further comprises the step of S6 and screening the anti-vibrio bacterial strains after the step of S5, and specifically comprises the steps of uniformly coating the vibrio harveyi on an L