Text: Bread Clip Knowledge
Edit|Bread Clip Knowledge
«——【Preamble】】 ——»
Philippine clams are the most productive monospecies of farmed shellfish in mainland China, with an annual production of more than 4 million tons, accounting for 90% of the world's total production.
In recent years, with the increase of clam breeding density and the deterioration of seawater quality, the frequent occurrence of clam diseases, coupled with the degradation of clam germplasm resources, the low coverage rate of improved varieties, and the existence of problems such as southern seedlings and northern rearing, the survival rate of cultured clams has declined, causing economic losses to the clam breeding industry.
The traditional method of aquaculture disease prevention and control is to kill microorganisms in the water environment by applying antibiotics, using chemical disinfectants or irradiating ultraviolet rays, etc., these methods are not selective, can not fundamentally solve the problem of aquaculture diseases, and the large use of antibiotics and disinfectants will cause ecological problems.
In order to ensure the sustainable and healthy development of the aquaculture industry, aquatic probiotics, as a substitute for chemical agents, are widely used in aquaculture, which has the advantages of non-toxic, non-drug resistance, high abundance and low cost.
In the aquaculture water environment, on the one hand, probiotics can maintain the microecological balance of the water body, inhibit and prevent the breeding of harmful bacteria, and reduce the risk of aquatic animal diseases; On the other hand, the colonization of probiotics in the intestinal tract of aquatic animals can enhance the digestion and absorption capacity of aquatic animals for feed, prevent the invasion of pathogenic bacteria through the mass effect, and improve the survival rate of farmed animals.
目前，国内外应用于水产养殖的益生菌包括芽孢杆菌属（Bacillussp.）、乳球菌属（Lactococcussp.）、乳杆菌属（Lactococcussp.）、片球菌属（Pediococcussp.）、肠球菌属（Enterococcussp.）、链球菌属（Streptococcussp.）等。
So how to improve the survival rate of probiotics with probiotic effects by isolating culturable bacteria from the intestinal tract of healthy clams, and screening them through in vitro enzyme production test, safety evaluation, and breeding test?
«——【Separation Method·】——»
Samples of Philippine clams (average body weight (14.51±1.18) g, average shell length (3.74±0.15) cm) were collected from Shangpintang Breeding Base, Shicheng Township, Zhuanghe City, Liaoning Province, and the clam shell was wiped with 75% alcohol under the ultra-clean workbench for surface disinfection, the clams were dissected with a sterilized scalpel, the intestinal tissue was removed, the intestinal contents were rinsed with sterile seawater for 3 times, and then the intestinal tissue was homogenized with sterile seawater to make the clam intestinal tissue suspension.
The clam intestinal tissue suspension was diluted serial with sterile seawater at 10-fold, 100-fold, 103-fold, 104, 105, 106-fold, and 107-fold, respectively.
The homogenates diluted to 10-4, 10-5, 10-6 and 10-7 were evenly coated on 2216E solid medium with 0.1mL respectively, and 3 groups were set up in parallel, inverted at 25 °C for 72 hours and then observed.
The strains to be tested were activated on 2216E solid medium, the activated strains were inoculated into LB (2.5% NaCl) liquid medium, and the constant temperature culture shaker was 25 °C, 120r/min, and after shaking culture for 24 hours, 2.5 μL of the above bacterial solution was taken and planted on Columbia blood agar plates, and cultured at 25 °C for 24 h, and the hemolytic ring formed around the colony was observed to determine the type of hemolysis.
The enzyme production ability of the strain was determined by spot seeding method, and 2.5 μL of fresh bacterial solution was spotted on amylase selection medium, protease selection medium and lipase (Tween80) selection medium, and incubated at 25°C for 72 h.
Observe whether there is a transparent hydrolysis circle around the colonies in the protease selection medium, and record the diameter of the protease hydrolysis circle and the diameter of the colony. Observe whether there is a halo around the colony in the lipase selection medium, and record the diameter of the halo and the diameter of the colony.
Single colonies were picked from 2216E solid medium, inoculated into 2216E liquid medium, incubated on a shaker (25°C, 120rpm), 30μL of bacterial solution was taken for Gram staining, and oil microscopy was carried out.
采用通用引物27F（5’-AGAGTTTGATCCTGGCTCAG-3’）和1492R（5’-GGTTACCTTGTTACGACTT-3’）进行PCR扩增，30µL反应体系包含：2μL模板DNA、3μL正向引物27F(10μmol/L)、3μL反向引物1492R(10μmol/L)、15μL2×EasyTaqPCRsuperMix、7μLddH2O。
反应程序为:95°C预变性2min,95°C变性30s,55°C退火30s,72°Cstretching 90s,循环35次,72°C总体stretching 5min,20°C preserved。
The PCR amplification product was detected by 1.5% agarose gel electrophoresis, and the gel imager observed whether there were bright bands, and the successfully amplified PCR product was sent to BGI for sequencing.
The sequencing results were submitted to GenBank for Blast alignment analysis, and the sequences with high homology were selected, and the genetic distance calculated based on the Poisson model by MEGA7.0 software was used to construct a phylogenetic tree based on the Poisson model, and the species of enzyme-producing bacteria were identified.
Single colonies of enzyme-producing bacteria were selected and cultured in a liquid medium containing 2.5% LB containing NaCl at 25°C and 120r/min for 24 h to prepare a bacterial suspension.
According to the 10% inoculation amount, 15mL of bacterial suspension was inoculated into 150mL of LB liquid medium, samples were taken every 6 hours, 5mL was sampled each time, the blank culture medium was used as a control, the zero point was corrected, and the absorbance at 600 nm wavelength was determined by a spectrophotometer.
If the absorbance value exceeds 0.8, the sample needs to be diluted to make the value lower than 0.8, and the growth curve is plotted with the measurement time on the abscissa and the OD600nm value as the ordinate.
In order to quickly and accurately quantify the bacterial solution, the absorbance value-number standard curve was drawn by plate counting method, and the absorbance of the logarithmic bacterial suspension was taken and its absorbance at 600 nm wavelength was determined after serial dilution.
The diluted bacterial solution was coated onto LB agar plates containing 2.5% NaCl, 3 replicates per group, incubated at 25°C for 24h, plates with colony numbers between 30 and 300 were selected for counting and colony forming units (CFU) were calculated, and then the OD600nm of the same bacterial solution was used as the abscissa and CFU as the ordinate to draw the standard curve and obtain the regression equation.
In order to determine the acid tolerance of strain C26, the logarithmic growth phase bacterial solution was taken and diluted to 1×109cfu/mL, and inoculated into LB (containing 2.5% NaCl) liquid medium with pH of 3.5, 4.5, 5.5 and 6.5 respectively according to the inoculum amount (the pH of the medium was adjusted with 0.1mol/LHCl solution and 0.1mol/LNaOH solution), and incubated at 25 °C and 120r/min shaker for 24 h, respectively at 0h, 2h, 4h, 8h, The OD600nm value of the bacterial solution was measured for 24 hours, and three groups of replicates were set.
In order to determine the tolerance of strain C26 to bile salts, the logarithmic growth phase bacterial solution was taken and diluted to 1×109cfu/mL, and inoculated into LB (pH 8.0, containing 2.5% NaCl) liquid medium with 0, 0.1, 0.3 and 0.5 g/100mL bovine bile salt (purchased from Jinclone Biotechnology Co., Ltd.) respectively (the pH of the medium was adjusted with 0.1mol/LNaOH solution and 0.1mol/LHCl solution) respectively The OD600nm value of the bacterial solution was measured at 0h, 2h, 4h, 8h and 24h at 25°C and 120rpm shaker for 24h. Set three sets of duplicates.
Healthy larvae of clams (shell length 0.3~0.5cm) were collected from the breeding pond in Changyi, Shandong Province for the challenge test, first temporarily raised in the laboratory for 7 days, the water temperature was constant at (21±2) °C during the breeding process, chlorella was fed 3 times a day, and the water was changed every 2 days.
Take 4 8L plastic buckets, disinfect and clean them, and put 4L of sterilized filtered seawater and 100 young clam shells respectively. Four barrels of juvenile scallops were randomly divided into two groups, each group with 2 barrels, one group was the control group fed daily bait, and the other group was the challenge test group with potential probiotics.
According to the standard curve of strain cell concentration, the bacterial solution was quantitatively pipetted and placed in a sterile centrifuge tube, centrifuged at 6000r/min for 5min, and then the supernatant was removed, and the bacterial suspension was resuspended in an appropriate amount of sterile seawater to prepare the bacterial suspension, and the appropriate amount of bacterial suspension was added to the test group to make the final concentration of the water strain 5×107cfu/mL.
During the challenge test, the water temperature was kept constant at (21±2) °C, chlorella was fed 3 times a day, the water was changed every 2 days, and the bacterial solution was re-added to the challenge test group to a final concentration of 5×107cfu/mL during each water change. The mortality of young scallops was observed and recorded daily, and the challenge test cycle was 5 days.
The sensitivity of the screened enzyme-producing probiotics to antimicrobial drugs was detected by disc agar diffusion method (K-B method).
Eight antibiotics were selected for susceptibility testing, namely tetracycline, kanamycin, cephalexin, penicillin, erythromycin, chloramphenicol, cotrimoxazole, and norfloxacin.
A small amount of bacterial solution with a concentration of 1×108cfu/mL was dipped in a sterilized cotton swab and evenly coated in 2216E solid medium, the antimicrobial susceptibility paper was pasted on the surface, and the diameter of the inhibition zone was determined after incubation at 35°C for 24 hours, and the average value was calculated by repeating 3 times, and the sensitivity was determined according to the instructions of the antimicrobial susceptibility paper.
Healthy larvae of clams (body weight 0.4~0.5g, shell length 1.25~1.27cm) were collected from the breeding pond in Changyi, Shandong Province for breeding experiments, temporarily raised in the laboratory for 7 days, the water temperature was constant at (21±2) °C during the breeding process, chlorella was fed 3 times a day, and the water was changed every 2 days. Take 6 plastic buckets, disinfect and clean them, and put 2L of sterilized filtered seawater and 50 young clam scallops respectively.
The total weight of each bucket of clams was weighed separately, and the shell length, shell width and shell height of individual clams were measured with vernier calipers.
Six barrels of juvenile scallops were randomly divided into two groups, with 3 barrels in each group, one group was the control group fed daily bait, and the other group was the culture experimental group with latent probiotics.
According to the standard curve of strain cell concentration, the bacterial solution was quantitatively pipetted and placed in a sterile centrifuge tube, centrifuged at 6000r/min for 5min, and then the supernatant was removed, and the bacterial suspension was resuspended in an appropriate amount of sterile seawater to prepare the bacterial suspension, and the appropriate amount of bacterial suspension was added to the experimental group to make the final concentration of the strain in the water body 1×107cfu/mL.
During the breeding experiment, the water temperature was kept constant at (21±2) °C, chlorella was fed 3 times a day, the water was changed every 2 days, and the bacterial solution was re-added to the breeding test group to the final concentration of 1×107cfu/mL during each water change.
At the end of the 60-day culture test, the total weight of each bucket of clams was weighed again, and the shell length, shell width and shell height of a single clam were measured with vernier calipers.
The growth rate of young clam shellfish is calculated separately according to the following formula:
Weight gain rate (WGR, %) = (final weight - initial weight) / initial weight × 100%,
Daily increase shell length (μm/day) = (final shell length - initial shell length) / cultivation time × 1000,
Daily increase shell width (μm/day) = (final shell width - initial shell width) / breeding time × 1000,
Daily increase shell height (μm/day) = (final shell height - initial shell height) / cultivation time × 1000.
The trial data were expressed as mean ± standard deviation (mean±S.D.), and the one-way ANOVA was used to compare the significance of the difference between groups (P<0.05 for the significant difference, P<0.01 for the extremely significant difference). Mapping was performed using the Origin software.
«——【Results and Analysis·】——»
A total of 21 strains of cultureable bacteria were isolated from the intestinal tract of healthy clams, and the results showed that none of the 21 strains produced β-hemolytic rings, indicating that none of them had hemolytic properties.
The ability of these 21 bacterial strains to produce amylase, protease and lipase was detected, and the results are shown in Table 1.
Among the 21 strains of bacteria tested, 8 strains produced amylase, 7 strains produced protease, 8 strains produced lipase, and 6 strains did not have the ability to produce enzymes.
Among them, strain C26 had the ability to produce amylase, protease and lipase, and the ratios (R/r) of the diameter of the hydrolysis circle (R) of enzyme production to the average diameter of colony (R) were 1.91±0.25 (amylase), 1.93±0.05 (protease) and 1.48±0.03 (lipase), respectively.
Therefore, strain C26 was taken as the research object, and the follow-up clam safety test and culture test were carried out to test its potential as a probiotic of clams.
The colonies of strain C26 on 2216E solid medium were round, with neat edges, slightly convex middle, smooth surface, and opaque light yellow.
The logarithmic growth phase cells were stained with Gram, and the results were shown in Figure 1, and the cells of strain C26 were red, rod-shaped, and short-rod-shaped, and they were identified as Gram-negative bacilli.
The 16S rDNA sequence of the screened C26 strain was sequenced, the sequence was aligned in the GenBank database (www.ncbi.nlm.nih.gov), and the phylogenetic tree was made by MEGA7.0 software (see Figure 2).
检测结果表明：菌株C26与假交替单胞菌属（Pseudoalteromonas）同源性较高，在98%覆盖度下一致性最高为99.44%，包括P.sp.BSi20396（EU330365.1）、P.sp.BSi20139（DQ492721.1）、P.sp.AC-144（AJ519790.1）等，在99%覆盖度下与P.sp.LP621（EU849123.1）一致性为99.24%。
Eleven strains of different species of Pseudoalteromonas were selected, including P.sp.BSi20396, P.sp.BSi20139, P.sp.AC-144 with the highest homology, and P.sp.LP621 and strain C26 with the highest homology at 99% coverage.
Combined with the colony morphology, micromorphology and sequence alignment analysis of strain C26, the potential probiotic C26 was preliminarily identified as Pseudoalteromonas spp.
The growth curve of strain C26 at room temperature in LB liquid medium (containing 2.5% NaCl) is shown in Figure 3, and its growth delay period is long, which only enters the logarithmic growth period at 24 h and enters the stable phase at 36 h, and the OD600nm value of the bacterial solution in the logarithmic growth period is between 1.0~2.5.
Different concentrations of strain C26 in LB liquid medium (containing 2.5% NaCl) were taken (logarithmic growth phase), diluted with blank medium and determined OD600nm, and the same batch of diluted bacteria was coated onto LB agar plate (containing 2.5% NaCl), and the number of colonies was observed and recorded after 24 hours, and the colony-forming unit (CFU) was calculated.
The standard curve was drawn with OD600nm as the abscissa and CFU as the ordinate, as shown in Figure 4, and the regression equation y=109x-9×106 (R2=0.9974) was obtained, which was used to calculate the concentration of C26 solution.
In order to test the tolerance of strain C26 to the intestinal environment of animals, acid resistance test and bile salt resistance test were carried out.
The growth of strain C26 in LB (with 2.5% NaCl) liquid medium at pH 3.5, 4.5, 5.5 and 6.5 at room temperature was determined, respectively, and the results are shown in Figure 5.
During the 24-h incubation cycle, the C26 cultured in the medium of pH 6.5 and pH 5.5 grew well, and the OD600nm of the bacterial solution showed an upward trend and was stable to 8 h.
After 8 h and 24 h of incubation, the OD600nm of C26 solution reached about 1.0 in the medium of pH6.5, and the OD600nm of C26 solution reached between 0.9 and 1.0 in the medium of pH5.5.
However, the growth of C26 was inhibited in the medium of pH3.5 and pH4.5, and the OD600nm of the bacterial solution was maintained at the initial value during the 24-hour culture cycle, and no growth was observed.
The results showed that strain C26 grew well in an acidic environment with a pH of no less than 5.5, and its growth was inhibited when the pH was lower than 4.5.
The growth status of strain C26 in LB (pH 8.0, containing 2.5% NaCl) liquid medium containing bile salts at concentrations of 0, 0.1, 0.3 and 0.5 g/100mL was determined at room temperature, and the results were shown in Figure 6.
During the 24-hour incubation cycle, the OD600nm of the bacterial solution in the blank control group (excluding bile salts) showed an overall upward trend, and increased significantly after 4 hours of incubation, reaching 0.48±0.10 and 0.73±0.05 at 8 h and 24 h, respectively.
In the medium containing 0.1g/100mL bile salt, the OD600nm of the bacterial solution showed a trend of increasing (2h), decreasing (4h) and re-increasing (8h, 24h), and the values were 0.16±0.02 and 0.12±0.02 at 2h and 4h, respectively, reaching 0.39±0.06 at 8h, and 0.48±0.05 at 24h, respectively, and its peak value was significantly lower than that of the blank control group (P<0.05).
In the medium containing 0.3g/100mL and 0.5g/100mL bile salts, the OD600nm of the bacterial solution also showed a trend of increasing (2h), decreasing (4h) and increasing (8h, 24h), with 24h peaks of 0.16±0.04 (including 0.3g/100mL bile salt) and 0.13±0.04 (including 0.5g/100mL bile salt), respectively, which were significantly lower than those of the blank control group and the group containing low concentration of bile salts.
The results showed that the concentration of bile salt at 0.1g/100mL could inhibit the growth of strain C26, and the concentration of bile salt above 0.3g/100mL could significantly inhibit the growth of strain C26.
In order to test the safety of strain C26 on clams, young clams were selected as the test objects, and the challenge test was carried out by bacterial liquid immersion bath method, and the test period was 5 days.
C26 was taken in the logarithmic growth phase and applied to the challenge test group at a dose of 5×107cfu/mL at the final concentration of cells, and the water was changed once every two days, and the fresh bacterial solution was re-applied during each water change, and a total of 3 times (0d, 2d, 4d) were applied during the test cycle.
The results of the challenge test are shown in Table 2, the 5-day survival rate of the two groups was 93%, while the 5-day survival rate of the two control groups was 96% and 95%, respectively, and the survival rate of the young scallops in the experimental group was slightly lower than that of the control group, but higher than 90%, so it was inferred that strain C26 was safe and non-toxic to the young clam scallops at a bath concentration of 5×107cfu/mL, and could be used as a potential probiotic for follow-up experiments.
In order to detect the antibiotic sensitivity of strain C26, a total of 8 antibiotics from seven categories, including tetracyclines, β-lactams, macrolides, aminoglycosides, sulfonamides, quinolones, and chloramphenicols, were selected to determine the sensitivity of strain C26 to these antibiotics, and the results are shown in Table 3.
Strain C26 is resistant to tetracycline, kanamycin, and cephalexin, moderately susceptible to erythromycin and penicillin, and susceptible to cotrimoxazole, norfloxacin, and chloramphenicol.
The effect of strain C26 on the growth of clams is shown in Figure 7.
The initial total weight of young clam scallops in the blank control group was 22.40±0.33g, and the total weight at the end of the test cycle was (24.00±0.40) g, and the average weight gain rate was 7.10%±0.71%.
The initial total weight of larval clam scallops in the experimental group was (23.38±0.20) g, and the total weight was (25.57±0.09) g after 60 days of immersion test, and the average weight gain rate was 9.37% ± 0.53%. The weight gain rate of larvae clam in the experimental group was significantly higher than that in the control group (P<0.05).
在养殖试验周期内，对照组蛤仔幼贝日增壳长、日增壳高、日增壳宽分别为（14.68±1.02）µm/d、（11.96±1.01）µm/d和（10.47±0.45）µm/d，试验组蛤仔幼贝日增壳长、日增壳高、日增壳宽分别为（15.01±0.47）µm/d、（11.99±0.94）µm/d和（11.10±0.50）µm/d。
Compared with the control group, there was no significant difference in the daily growth of clam shells in the experimental group (P>0.05). The results showed that strain C26 bathing treatment could significantly increase the weight gain rate of young clam scallets.
«——【·Conclusion·】——»
A strain of bacterium C26, which can produce amylase, protease and lipase, was isolated and screened from the intestinal tract of healthy Philippine clams, and the strain was identified as Pseudoalteromonas bacteria.
The results showed that the number of viable bacteria of strain C26 did not decrease and still showed an upward trend under the conditions of strong acid and high concentration of bile salts, indicating that strain C26 had strong acid tolerance and bile salt tolerance.
The results showed that the 5-day survival rate of larvae clams was still higher than 90% under the condition that the concentration of strain C26 was as high as 5×107cfu/mL, indicating that strain C26 was safe and non-toxic to larvae clams.
After the 60-day culture experiment, the weight gain rate of the C26 treatment group was 9.37% ±0.53%, which was significantly higher than that of the blank control group (7.10% ± 0.71%) (P<0.05), indicating that the growth of strain C26 had a promoting effect on the growth of young clam scallops.