Compared Study On The Probiotics Between Bacillus Licheniformis HN318 And Its Fermentation Broth To Hybrid Grouper(Epinephelus Fuscoguttatus ♀ × Epinephelus Polyphekadion ♂)

Hybrid grouper（Epinephelus polyphekadion♂×Epinephelus fuscoguttatus♀）is a new hybrid of Camouflage grouper and brown spotted grouper.As one of the most commonly used probiotics in aquatic products,Bacillus licheniformis improves the growth of aquatic animals,inhibits the growth of pathogenic bacteria,and enhances host immunity.In this paper,we concentrated on the Hybrid grouper and studied the growth capacity,non-specific immunity and disease resistance in fish fed on Bacillus licheniformis（HN318）.Main results are as follows:1.Artificial gastrointestinal juice tolerance and adhesion ability of B.licheniformis HN318 were systematically evaluated.The results showed that Bacillus licheniformis HN318 could survive in gastric fluid with p H of 3 and 4 and intestinal fluid with p H of 6.8and 8.0.After treated with gastric juice at p H 3 and 4 for 1 h,survived cell number of HN318 were（0.91±0.03）×10⁸ CFU/mL and（0.88±0.03）×10⁸ CFU/mL seperately,and for the next 3 h we tested,total number didn’t vary significantly.After treated with intestinal juice with p H of 6.8 and 8.0,survived cell number of HN318 were（0.18±0.02）×10⁸CFU/mL and（0.41±0.08）×10⁸CFU/mL respestively,and for the next 3h we tested,total number didn’t change significantly,either.Auto-aggregation activity of HN318gradually reached 84%at the 8th h.Cell surfacehydrophobic rates were 69.5,85.8 and64.7%in chloroform,xylene and ethyl acetate individually.Given that,there exist strong possibilities that Bacillus licheniformis HN318 could survive and colonize in the intestine.2.Hemolytic activity,safety and drug resistance of Bacillus licheniformis HN318were tested individually.The results were:Bacillus licheniformis HN318 wasγ-type and non-hemolytic;Bacillus licheniformis HN318 was rather safe(there was no deaths of hybrid grouper after injection with HN318(OD₆₀₀=1));The results of drug resistance tests showed that Bacillus licheniformis HN318 was sensitive to 20 antibiotics such as tetracycline,gentamicin and oxytetracycline,so that certain risks to transfer resistant genes are extremely low,which implied its safety and non-toxicity to serve as protential probiotics.3.Enzyme activities and anti-pathogen activities of the fermentation broth of HN318and its bacteria body were compared,and the results showed that:the protease hydrolysis circle of fermentation broth was 9.21±0.52 mm;the amylase hydrolysis circle was 19.42±0.78 mm;the protease hydrolysis circle of bacteria body was 4.84±0.49 mm;the amylase hydrolysis zone was 12.97±0.37 mm.Inhibition zones against Vibrio Harveyi,Edwardsiella tarda,Vibrio alginolyticus and Staphylococcus aureus of fermentation broth were 8.43±0.43,6.7±0.37,5.7±1.6 and 11.6±0.2 mm,respectively,while for the bacteria body,they were 4.43±0.44,5.53±0.49,3.93±0.16 and 8.6±0.74 mm,respectively,which indicated better enzyme activity and anti-pathogen activity of fermented broth than that of the bacteria body.4.By adding HN318 fermentation broth and HN318 bacteria body to the basal feed,we measured the growth index,non-specific immunity and anti-disease capability to determine their probable impact on hybrid grouper.The results displayed as follows:after fed for 8 weeks,gained weight rate and feed conversion ratio in fermentation broth groups and bacteria body groups were both palpably higher than those in the control groups（P<0.05）;Non-specific immune indexes showed that,compared with the control group,AKP and ACP activities in serum were distinctly increased in the fermentation broth group at the1st and 4th week,while there was no clear difference in the bacteria body group;SOD activities in both fermentation broth group and bacteria body group were profoundly higher than that in the control group at week 8（P<0.0 5）;The activity of LZM in fermentation broth group was strongly higher than that in the control group at week 1,4 and 8,but there was no significant difference（P>0.0 5）;At the 4th and 8th week,compared with the control group,bactericidal activity of serum in the fermentation broth group was overtly increased（P<0.05）,which was demonstrablely higher than that in the bacteria body group（P<0.05）,and the bactericidal activity of serum in the bacteria body group was substantially increased compared with the control group at the 8th week;At the 1st and 4th week,respiratory burst activities of macrophages in both the fermentation broth group and the bacteria body group were higher than that in the control group,and there was significant difference between the fermentation broth group and the control group（P<0.05）;At the 4th and the 8th week,hybrid grouper of each group were challenged with Vibrio Harveyi,the results showed as follows:at the 4th week,cumulative mortality of the fermentation broth group and the bacteria body group were 25%and 53.6%,respectively,which were lower than that of the the control group（75%）,with cumulative mortality of the fermentation broth group lower than that of the bacteria body group and the control group（P<0.05）.At week 8,cumulative mortality of fermented broth and bacteria body group were 28%and64%respectively,which were lower than that of the control group（80%）,suggesting their protective role in resistance against Vibrio harveyi in hybrid grouper.Between the two groups,fermentation broth had lower mortality than bacteria body,indicating its higher efficacy against Vibrio harveyi than that of the bacteria body.In conclusion,Bacillus licheniformis HN318 had strong tolerance against artificial gastrointestinal fluid and potent adhesion properties,which possessed no danger and toxicity to the hybrid grouper.Both HN318 fermentation broth and its bacteria body could secrete protease and amylase,and had strong antibacterial activity,with stronger enzyme activity and antibacterial ability in fermentation broth than that of its bacteria body.After fed by fermentatioon broth and bacteria body seperately,non-specific immunity and disease resistance were both highly enhanced in the hybrid grouper.Given all the experiments above,both fermentation broth of Bacillus licheniformis HN318 and its bacteria body both held the potential to serve as a brand new immunopotentiator for the hybrid grouper,with fermentation broth better than bacteria body.