| The resource of straw is abundant and the content of cellulose is high,but the utilization rate is low because of the complex structure of cellulose which is difficult to degrade.Microbial fermentation has the advantages of high digestibility,good palatability,no toxicity and easy operation.It has become the most economical and effective means to degrade cellulose and improve the nutritional value of straw.In ruminant rumen,there are a large number of fibrinolytic bacteria,which is helpful to the degradation and absorption of cellulose and the improvement of immunity.In this study,the rumen microorganism of cattle in Dabie Mountain was used as the research object to isolate and identify the strains with high-yield cellulase,and to study the clinical effect by optimizing the optimum conditions of producing enzyme The aim is to develop straw resources into a new type of feed with low cellulose content,high protein content,good taste,easy digestion and absorption,and to lay a foundation for improving the immunity of cattle in Dabieshan Mountain,improving the microbial environment of its digestive tract and developing local economic varieties.This research is mainly carried out:(1)Primary screening of cellulose decomposing bacteria.The first screening of cellulose decomposing bacteria by sodium carboxymethyl cellulose plate method,and the ability of strain to decompose cellulose was preliminarily determined by Congo red dyeing method and filter paper decomposition rate.(2)Re-screening of cellulose decomposing bacteria.By measuring the filter paper enzyme activity(FPA)and carboxymethyl cellulose activity(CMC)of the primary screening strains,the ability of the strains to degrade cellulose was further determined.(3)Identification of cellulolytic bacteria.Morphology,growth characteristics under different temperatures and different p H conditions and identification of 16 S r DNA of selected cellulolytic bacteria were performed on the selected cellulose-decomposing bacteria to determine the negative and positive species of the strain.(4)Optimization of enzyme production conditions of high cellulose-producing strains.The cultivation temperature and initial p H value of the strains with high cellulose production were optimized to observe the ability of enzyme production.(5)Study on the degradation effect of rumen fibrinolytic bacteria.The high-yield cellulolytic bacteria were screened to ferment the corn stalk with the optimized optimum cultivation temperature and initial p H value.The reducing sugar and soluble protein components of the corn stalk were determined after fermentation to verify its applicability.The results showed that:1.A total of 6 cellulose-decomposing bacteria were isolated from the rumen fluid of Dabieshan cattle,numbered 1,2,3,4,5,and 6 respectively.Among them,No.1 and No.2strains had the largest diameter of transparent circles,17 mm and 18 mm,respectively,D/d> 5.0,and their decay degree of filter paper were "+++",indicating that they had ability to degrade cellulose;The diameters of the transparent circles of strains No.4 and No.5 was15 mm,respectively,both D/d were 4.5,and the degree of filter paper ulceration were "+".The diameters of transparent circles of strains No.3 and No.6 was 14 mm and 10 mm,respectively,but both D/d were < 4.5,the degree of the filter paper was "++",respectively.2.The FPA enzyme activity of different strains are different.Among them,Strain No.1and strain No.2 had the strongest FPA enzyme activity,>9.0 U/h.Other bacteria of FPA enzyme activity size is No.3,4,5 And 6 strain,between 5.0-6.5 U/ h.In addition,The CMC enzyme activities of different strains were different.The CMC enzyme activity of No.1 and No.2 strains are> 12.0 U/h,which are the strongest.The CMC enzyme activity of other bacteria is 3,4,5,and No.6 strain,6.0-8.0 U/ h.Therefore,No.1 and No.2 are high-yield cellulose strains.3.Strains No.1 and No.2 form round ridges,regular edges,sticky,opaque,and middle protruding yellow colonies,and microscopic examination are G-brevis;No.3 and No.6strains form round ridges,regular edges,smooth,wet,translucent and opaque yellow-green colonies,microscopic examination is G-bractobacillus;No.4 and No.5 strains form round bumps,irregular edges,wet,translucent and opaque yello colonies,microscopic examination is G+ bacillus bacterium,with obtuse round ends and spores;The six cellulolytic bacteria all grow well under the conditions of 25℃~37℃,p H5.0~p H7.0;No.1and No.2 were in the same branch with Stenotrophomonas sp.M11 and Stenotrophomonas maltophilia,respectively,with similarities of 99.69% and 99.47%,the above bacterias were determined to be Stenotrophomonas;No.3 and No.6 were in the same branch with Sphingobacterium sp.P6 and Sphingobacterium alimentarium,with similarities of 99.88%and 99.69%,respectively.The above bacteria were determined to be Sphingobacterium;Both No.4 and No.5 were in the same branch with Empedobacter brevis,similarity 100%,respectively,the above bacteria were determined to be Empedobacter.4.When the culture temperature of both No.1 and No.2 strains is 37°C,and the initial p H is 6.0 and 5.5,respectively,the FPA enzyme activity and CMC enzyme activity are the highest,so it is the optimal culture temperature and initial p H value.5.The contents of reducing sugar and soluble protein after fermentation of corn stalks by No.1 and No.2 strains were higher than those of before fermentation,and reached the highest on the third day.In conclusion,this study isolated and identified 6 strains of cellulolytic bacteria from the rumen fluid of Dabieshan cattle,numbered 1,2,3,4,5,and 6.Among them,No.1 and No.2 are cellulolytic bacteria with high cellulose production.The optimum enzyme production conditions of them were p H value 6.0,temperature at 37℃ and p H value 5.5,temperature at 37℃,respectively,and both of them can increase the content of reducing sugar and soluble protein in the fermentation of corn stalks. |
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