Screening Of Bacillus Subtilis Detoxification For Aflatoxin B₁ And Application Study

The toxicity of AFB₁is very strong and it can pollute a wide range of area.Consequently,the negative impact it has on human and animals is very serious.Thus,the adoption of effective measures to detoxificate is more than a necessity.Probiotic bacteria has not only a probiotic effect but also a strong detoxification function,which has developed into the most promising detoxifier of mycotoxin poison.This study successfully screened one powerful Bacillus subtilis from the probiotic strains,focusing on studying and verifying its characteristics of detoxification,for the purpose of providing support for animal research and development of toxicity-free probiotics.Hopefully,contributions on the sustainable development of animal husbandry can be made.1.Selection and evaluation of probiotic strainsBy using Coumarin as the sole carbon source to preliminarily screen 42 probiotic strains preserved in laboratory,superior strains,which can survive with the presence of AFB₁,will be screened out.With AFB₁ forsubstratios,by using ELISA method to rescreen,three strains with superior performancein detoxification are selected.Based on a comprehensive evaluation of its biological characteristics（growth curve,antimicrobial properties,tolerance）,HDR-02 strains are successfully screened out for its strong detoxification function and superior probiotic performance.Through morphology observation,biochemical index appraisal,16S r RNA identification,HDR-02 is proved to be Bacillus subtilis.2.Study on detoxification characteristics of HDR-02The findings of the study are as follows:firstly,the impact of disparition time on the detoxification effect.The longer the course is,the higher detoxification ratio of AFB₁there will be.Secondly,the influence of different p H to the detoxification effect.Under the condition of p H 2～9,detoxification ratio shows no obvious change.Thirdly,the effect of different components on the detoxification effect.HDR-02 strains inactivated bacteria suspension has strongest detoxification capacity against AFB₁,detoxification ratio of which is 87.1%.Live bacteria suspension takes second place,and fermented liquid and intracellular content of AFB₁ has lowest removal ratio.Fourthly,assessment of stability of bacteria-AFB₁ complexes.Evaluation showed that bacteria-AFB₁ complex has high stability.Research findings suggest that detoxification ratio is related not to environment p H but to time duration.HDR-02 extracellular products can degrade the AFB₁.Inactivated bacteria suspension results ruled out the hypothesis that HDR-02 bacteria can detoxicate through biological metabolite,which indicates that HDR-02 bacteria detoxicate by closely adsorbing AFB₁.3.Verification of research findings by animal testsAnimal testing scheme:group A（blank control）;Group B（probiotics）;Group C（probiotics+AFB₁ poisoned）;Group D（AFB₁ poisoned）.Samples were taken on the 7th day and the 10th day respectively,when ducklings’production performances,serum biochemical indexes and liver pathological changes were evaluate.Accordingly,the effect of animal testing was analyzed.Production performances of ducklings’from different groups and different dates show that after taking HDR-02,the overall weight and average daily-gained weight of unpoisoned ducklings improved significantly（P<0.05）,the production performances of ducklings were improved as well.The overall weight and average daily-gained weight of AFB₁ poisoned group dramatically reduced（P<0.05）,which even resulted in the loss of lives of ducklings.Irrigation of HDR-02 bacteria suspension can noticeably reduce the influence AFB₁ poisoning had on ducklings’overall weight and average daily-gained weight（P<0.05）.Blood indexes results showed that there was no distinguishing differences between probiotic group and control group（P>0.05）.Serum biochemical indexes of ALT,AST index in AFB₁ poisoned group is obviously higher than that of control group（P<0.05）,while HDR-02 addition can significantly alleviate this phenomenon（P<0.05）.Liver slice observation indicated that HDR-02 strains and feeding management do not cause damage to liver tissue structure;AFB₁can damage liver’s structure,which leads to fatty degeneration,inflammatory cell infiltration and small bile duct proliferation pathological changes.Adding of HDR-02 strains can alleviate the damage AFB₁caused to the liver structure.The above data show that the HDR-02 not only can improve the production performance of ducklings but also has the ability of detoxification,which can effectively ease the AFB₁ damage in ducklings.