| Deoxynivalenol(DON)is a secondary metabolite mainly produced by Fusarium graminearum and Fusarium oxysporum.It is widely distributed in wheat,maize,barley,oat and other cereals.It not only reduces the grain yield and quality,but also causes huge economic losses.Moreover,intake of DON contaminated grains will do great harm to human and animal health.Currently,several physical,chemical and biological methods have been used to remove DON.Compared with traditional physical and chemical methods,biological detoxification has become the hotspot of food safety research due to its mild processing conditions,high efficiency,high specificity,and no pollution to feed and environment.In recent years,more and more studies have showed that lactic acid bacteria have the ability to remove the mycotoxins,and lactic acid bacteria are also recognized as a group of food-grade microorganisms which have many physiological functions,To ensure food safety and detoxify animal feeds,it is necessary to study the removal ability of the lactic acid bacteria on DON removal and the corresponding mechanism.In this study,a lactic acid bacteria which is capable of removing deoxynivalenol was selected through the co-culture.The tolerance of lactic acid bacteria to DON was evaluated through studying the effects of reaction time,cell concentration,temperature,and pH.At the same time,the mechanism to remove DON by lactobacillus paracasei was investigated.On this basis,the solid state fermentation of Lactobacillus paracasei was carried out using wheat bran as the medium.The optimum fermentation conditions were determined by single factor and orthogonal experiments,which provided a basis for its application in the feed and the detoxification of grains.The main results are shown as follows:1.Screening of lactic acid bacteria capable of removing deoxynivalenol and study its reaction conditions.The lactic acid bacteria isolated from pickled fish,pickled cabbage,koumiss,and the strains preserved by our laboratory,were co-cultured with DON,so as to select a strain capable of removing deoxynivalenol efficiently.The results showed that Lactobacillus paracasei with the highest removal ability of DON was obtained from 150 strains of lactic acid bacteria.When the concentration of bacteria exceeded 1010 CFU/mL,the reaction temperature was 37?,and pH was 7,the removal rate was 40.4%in the co-culture system with a final concentration of 50?g/mL DON.Lactobacillus paracasei has considerably high tolerance to DON.No significant influence on the growth of Lactobacillus paracasei was observed when DON concentration ranged from 20 to 50 ?g/mL.2.The mechanism to remove deoxynivalenol by lactobacillus paracasei.To start with,the results showed that the main removal position on lactobacillus paracasei was the cell wall,rather than fermentation supernatant and intracellular fluid.The removal rate of cell wall reached 40.7%.In comparison,the removal rates of DON by the fermentation supernatant and intracellular liquid were less than 10%.Then,after thermal or acid treatment,the removal rate increased to 45.50%and 42.34%respectively.Thus,it was assumed that that the removal rate was irrelevant to the activity of the strain.Instead,the effect of inactivated strains on DON removal was better.After urea treated,the DON removal efficiency decreased to 18.80%,implying that the interaction between lactic acid bacteria and mycotoxins involve hydrophobic reactions.The DON removal rate of lactobacillus paracasei treated by lysozyme decreased to 13.99%,indicating that the integrity of peptidoglycan structure was important for bacteria to adsorb toxin.The removal rate of the bacteria treated by 10%TCA increased to 41.16%.which indicated that the removal rate increased with the cell wall permeability.The photograph captured by Confocal Laser Scanning Microscopy showed that there was obvious blue fluorescence on the outer-layer of cell wall of Lactobacillus paracasei,whereas the signal inside cell was weak.This result demonstrated that cell wall played a major role on DON removal.Moreover,the adsorption of DON by Lactobacillus paracasei reached equilibrium after 18 h adsorption.The adsorption thermodynamics study showed that the adsorption process was nonlinear and the isothermal regression equation fitted to the Langmuir equation well.According to the Langmuir equation,the maximum adsorption capacity of lactic acid bacteria to DON was 50.5 ?g/mL.Finally,through the adsorption stability test,it was found that after three washing treatments,the content of DON was hardly detected.The stability of the living bacteria was stronger than that of the inactivated strain.It was found that the adsorption process of Lactobacillus paracasei to DON was reversible.3.The optimization of the solid-state fermentation by Lactobacillus paracasei.In this study,solid-state fermentation by Lactobacillus paracasei using bran as the culture medium was carried out.Single factor experiment and orthogonal experiment were conducted to optimize the fermentation conditions.Crude protein content,viable bacteria count and DON removal rate were used as the indicators for optimization.The optimum fermentation conditions were shown as follows;the bran content was 40 mg/250 mL,the ratio of material to water was 1:0.6,inoculum concentration was 9%,cultivation time and temeprature were 48 h and 37?.At the aforementioned conditions,the DON removal rate was 51.04%,which was 30.8%higher than that before the optimization.After solid-state fermentation,the crude protein content increased from 9.26%to 12.06%,and the soluble dietary fiber increased from 28.10 mg/g to 34.45 mg/g.Moreover,the pH value dropped from 6.23 to 4.36 and the number of living bacteria was 2.92 ×108 CFU/mL.In pig intestinal tract environment simulated in vitro,the removal rate of DON was reduced to 27.13%and 15.91%,respectively.Aftert solid-state fermentation,the removal rate of DON was 31.25%and 36.37%respectively.In.the gastrointestinal tract,the adsorption capacity of both Lactobacillus paracasei and fermented bran decreased. |
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