Studies On Breeding Of Acid Protease Producing Probiotics And Molecular Mechanism By N～+ Implantation

Breeding of probiotic Aspergillus niger and the mutation mechanisms of induced byN⁺ implantation were studied. Chapter 1; The study on character of acid protease excreted byAspergillus niger ANSO₁. The study on character of acid protease excreted by Aspergillus niger ANSO₁ indicated:The optimum reaction pH of acid protease is among 2.5-4.0, the relative enzyme activityreached 100% in PH 5.0; The optimum reaction temperature was among 40℃-50℃, 100%in 45℃; The acid protease can keep better stability in high temperature. The solid enzymecan hold 51.4% activity in 80℃ for 5 minutes, the liquid enzyme can hold 54.6% activityin 80℃ for 60 seconds, the solid enzyme can better resist heat destroy than liquid enzyme;Some mental ion can influence acid protease activity. The acid protease can be notablyactivated by Mn⁺⁺ and Cu⁺⁺, restrained by Fe⁺⁺⁺ in 2.0mmol/L. The result indicates acidprotease excreted by Aspergillus niger ANSO₁ is fit to feed as additive. Chapter 2; The study on parameter of N⁺ implantation into Aspergillusniger and screening of high producing mutant The study on parameter of N⁺ implantation into Aspergillus niger indicated: Thevacuum can notably influence survival of spore of Aspergillus niger; The survival of sporeof Aspergillus niger was decreased rapidly with the implantation dose increasing firstly,then the survival appeared to rise when dose added to 50×2.6×10¹³ ions/cm², the survivaldecreased again when 100×2.6×10¹³ ions/cm² was applied; The more implantation dose,the more mutation. The medium implantation dose (30-80)×2.6×10¹³ N⁺/cm² wasprefer to induce positive mutation. The activity of protease of mutated strain Aspergillusniger ANSO₂ was raised from 75.8IU.g^-1 to 602.6 IU.g^-1; Characteristics of enzymesproduction in Aspergillus niger mutant ANS02 kept stable by 5 times subculture. The resultsuggested that N+ implantation is an effective means in screening probiotics. Chapter 3; The study on optimum enzyme producing factor of mutantstrain The single factor was firstly applied to search notably factors, then the optimummedium of mutated strain Aspergillus niger ANSO₂ was tested by the theory of centercombination. The medium included H₂O 1000mL, bran 496.6g, bean cake473.6g,(NH₄)₂SO₄ 29.8; The optimum fermentation temperature is 30℃,time, 66h; pH, 5.5.Fermented under optimum factors, the acid protease of mutant strain ANSO₂ activityreached 642.5 IU.g-1。 Chapter 4; The study on the molecule mechanism of mutation inducedby N+ implantation The mechanism of the DNA and base mutation induced by N+ implantation intomicroorganism were firstly studied by randomly amplified polymorphic DNA (RAPD)analysis and the heredity stability of mutant strain was verified. The results indicated thatproduction of enzymes production in mutant strain ANO₂ kept stable and N+ implantationcould make base mutation. The base mutation frequency of the DNA fragment sequencedwas 1.09%. Among them, base substitution and base deletion accounted for 87.5% and12.5% respectively. Of the base substitution, base transition and base transversionaccounted for 62.5% and 25% respectively. The result suggested that N⁺ implantation wasan effective means in screening probiotics.