Isolation And Identification Of β-glucosidase-producing Strains And Study On Detoxification Technology Of Flaxseed Cake

Linum usitatissimum is oil crops,the seeds are called flaxseed.The residual from flaxseed after pressing is flaxseed cake,flaxseed cake was widely used in food additives,animal breeding and crops cultivation field,because it was rich in protein,unsaturated fatty acids,cellulose and many other active components.It can be seen that the deep processing of flaxseed series is very promising.Although flaxseed cake has a variety of beneficial ingredients,it also contains other anti-nutritional factors,such as VB6 antagonists,flaxseed gum,linen kiosks,crude fiber,trypsin inhibitors,phytic acid,cyanogenic glycoside.Especially cyanogenic glycoside,it will be metabolism into hydrocyanic acid and causing intracellular poisoning in the animal body under the action of related hydrolase,thus greatly limiting the scope of application of flaxseed cake and can only be added to HCN-tolerant ruminant feed.Therefore,to make the application of flaxseed cake to further expand and deepen,the removal of cyanogenic glycoside has become an urgent problem to be solved.Studies have shown that β-glucosidase has the effect of degrading cyanogenic glycoside and can convert cyanogenic glycoside into cyanideions.Studies have been reported using naturally occurring,beta-D-glucosidase-producing bacteria to degrade cyanogenic glycoside from cassava,it has also been reported that the human β-glucosidase gene（Glu）is introduced into yeast using genetic engineering technology to obtain engineering bacteria,and the bacteria is used to degrade cyanogenic glycoside from flaxseed and achieve good detoxification effect.In addition,compared with the physical and chemical methods,the biological treatment of cyanogenic glycoside in plants with mild conditions,low energy consumption,easy operation,no reagent residue advantages and expected to have a very broad development prospects.The untreated flaxseed cake can be used as feed additives added to the ruminant diet（cattle）and there were no adverse reactions.So we infer that the cattle’s intestinal tract may exist β-glucosidase-producing strains after the process of domestication culture.In addition,β-glucosidase can degrade esculin into escin,which gives a black color when treated with Fe³⁺.In this study,the stool of cattle,which had not been treated with flaxseed meal,was used as the source of the flora,the culture medium containing esculin and Fe³⁺ was selected as the selection medium for the target strain,after screening and re-screening process,five strains of β-glucosidase-producing were isolated.Molecular biology combined with morphology,physiological and biochemical methods,the five strains were identified as Geotrichum candidum,Lichtheimia ramose,Citrobacter freundii,Providencia rettgeri,Proteus vulgaris.The activity of β-glucosidase in five strains was quantitatively determined by p-NPG method,the results showed that the activity of Geotrichum candidum（M-1）was the highest in the five strains,which could reach 3.55 U.ml-1.On the whole,the enzyme activity of the fungus was significantly higher than the bacteria producing enzyme activity.The results showed that the strain Lichtheimia ramose（M-2）had the highest virus-free rate by comparison fermentation detoxification.M-2 was used as the initial fermentation strain and the detoxification rate and crude protein incremental were used as the evaluation indexes,the single and orthogonal experiments were used to optimize the fermentation process,the optimum conditions for the fermentation of flaxseed cake were inoculum size 3%,temperature 32℃,water content 60%,time 144 h.Under this fermentation parameter,cyanogen glycoside removal rate was 89%,crude protein increment reaches 38%-42%.Studies have proofed the domestication of flaxseed cake to cattle intestinal microbes,five strains that can degrade cyanogen glycoside were obtained and while optimized the detoxification process of fermented flaxseed cake.The aim of this study was to provide a theoretical basis for the removal of cyanogen glycoside and the comprehensive utilization of flaxseed cake.