Screening And Functional Study Of Bacillus From Intestines Of Largemouth Bass

Largemouth bass(Micropterus salmoides)is a freshwater aquaculture species of great importance in China due to its tender and delicious meat,which is highly favored by consumers.However,the intensification of farming practices and the deterioration of water quality have increased the threat of pathogenic microorganisms to largemouth bass,which has severely limited the healthy and sustainable development of the industry.Probiotics,such as Bacillus spp.,are effective and environmental-friendly alternatives to antibiotics and are widely used in aquaculture to improve growth and water quality.Therefore,the study and application of Bacillus spp.for the healthy farming of largemouth bass is of particular importance.This study isolated and identified Bacillus spp.from the the intestines of healthy largemouth bass,screened safe and highly enzyme-producing strains based on their biological characteristics,and studied their drug sensitivity,biofilm formation,artificial gastrointestinal fluid resistance,and high-temperature tolerance.Probiotic Bacillus were added to largemouth bass feed,and their effects on growth,immune function,and resistance to Nocardia spp.were studied.The main results of this study are as follows:1.Ninety-four strains of Bacillus were isolated from the intestines of healthy largemouth bass and identified by 16S r DNA sequence analysis.Among them,22 strains of Bacillus subtilis,13 strains of Bacillus pumilus,15 strains of Bacillus altitudinis,24strains of Bacillus cereus,and 20 strains of unclassified Bacillus were identified.Based on the Catalogue of Feed Additive Varieties,Bacillus subtilis and Bacillus pumilus,which can be added to aquaculture animals,were selected for research.Four strains that were non-hemolytic and had the ability to produce protease,amylase,cellulase,and lipase were screened based on their hemolytic and enzymatic properties.These four strains were Bacillus subtilis K-3,K-12,K-13,and K-22.2.The study of the biological characteristics of four strains of Bacillus subtilis showed that all four strains were strong biofilm-forming strains and sensitive to various common antibiotics such as gentamicin,kanamycin,ofloxacin,and cefotaxime.The survival rate of the four strains in an artificial gastric juice with p H 2 for 2 hours was low(<60%),while their survival rates were higher(25.85%～100%)when cultured in an artificial gastric juice with a p H 3-4 for 2 hours.Among them,the survival rates of strains K-12 and K-13 were approximately 100%,which was higher than those of K-3 and K-22.The survival rate of the four strains in an artificial intestinal fluid after 2 hours of culture was 100%.The live bacteria counts after being placed in water bath at 60℃,70℃,80℃,and 90℃for 30 min were approximately 1×10~8 CFU/m L,1×10~7 CFU/m L,1×10~5 CFU/m L,and 1×10~4 CFU/m L,respectively.These results suggest that the four strains have some tolerance to high temperatures.3.Four strains of bacteria were prepared into endospores and added to largemouth bass puffed feed,maintaining the endospore content at 1×10~9 CFU/g for 42 d of feeding.The results showed that the body weight gain rate of the K-13 experimental group was significantly higher than that of the control group(P<0.05);the activities of lipase and protease in the intestinal tract of K-12 and K-13 experimental groups were significantly higher than those in the control group(P<0.05).Compared with the control group,the expression levels of pro-inflammatory factors in the head-kidney,spleen,and intestine of the K-13 feeding group were down-regulated,and the expression levels of anti-inflammatory factors were up-regulated,indicating that the K-13 strain has the potential to promote the growth of largemouth bass and reduce the occurrence of intestinal inflammation.4.In vitro antibacterial experiments showed that strain K-13 could antagonize Nocardia seriolae.K-13 was mixed with feed and fed to largemouth bass for 14 d,and 0.1m L of Nocardia seriolae with concentration of 1×10~5 CFU/m L was injected into the abdominal cavity.The results showed that compared with the control group,the death time of largemouth bass in the experimental group was delayed,the mortality rate was reduced,and the protective rate of strain K-13 against largemouth bass reached 37.78%,indicating that strain K-13 could antagonize Nocardia seriolae and reduce its lethality to largemouth bass.5.The feed containing endospore concentration of 1×10~9 CFU/g of K-13 was administered to largemouth bass,with conventional feed serving as the control group.The Clostridium bacterial count in the gut was monitored for seven consecutive days.Results revealed that within the first three days,the number of Clostridium bacteria in the gut remained at 1×10~4 CFU/m L,increasing to 5×10~3 CFU/m L by day five,significantly higher than the control group(<50 CFU/m L).These findings suggest that after feeding with K-13 strain-containing feed,the K-13 strain can colonize the gut for at least five days.In conclusion,this study isolated and screened Bacillus subtilis K-13,which has the characteristics of high safety,strong enzyme production ability,antagonism against Nocardia seriolae,and promotion of the growth of largemouth bass.It can be used as probiotic preparation added to largemouth bass feed and has good application prospects and promotional value.