豆粕发酵用高产蛋白酶芽孢杆菌的筛选及鉴定

Screening and selecting of protease high-yield bacillus for fermented soybean meal

试验旨在从生境中筛选适用于高肽含量发酵豆粕的高产蛋白酶芽孢杆菌。初筛:样品于无菌的8.5%生理盐水中均质后80℃水浴10 min,10倍梯度稀释至一定倍数,取后三个梯度涂布于ND双层平板(上层为营养琼脂,下层为脱脂牛奶),37℃培养24 h,通过革兰氏染色、镜检以及菌落周围水解透明圈的情况对菌株进行筛选;复筛:初筛得到的菌株接种于豆粕发酵培养基,180 r/min、37℃培养48 h,根据上清液中蛋白酶活性进行复筛;菌种鉴定:运用16S rDNA和GyrA部分序列扩增测序对筛选得到的菌株进行鉴定。通过初筛,从样品中得到了7株产蛋白酶丰富的G+芽孢杆菌;复筛的条件下T1发酵上清液蛋白酶活性达1 547.49 U,极显著高于其它6株(P<0.01);经分子鉴定得T1为解淀粉芽孢杆菌。由此,从豆豉中筛选得到一株适用于豆粕发酵、产蛋白酶丰富的解淀粉芽孢杆菌T1,它在37℃、180 r/min条件下于豆粕发酵培养基中培养42 h后上清液蛋白酶活性可达1 683.99 U。

This study was aimed at screening and selecting protease high-yield bacillus from environmerit for fermented soyhean meal. Preliminary screening： after homogenized in 8.5% （w/v） sterile salted water, the samples were bathed in 80 ℃ water for 10 minutes, then, diluted with slerile water by 10-fold and coated onto the ND-hilayer plates （with nutrient agar in the upper layer and defattedmilk agar in the lower）. Seleeting based on the transparent zone around colony, Grant＇s method and microscopy after incubate at 37℃ for 24 h; Re-screening： the proteinase activities of the supernalant of the soybean meal mediums, which inoculated the strains isolated from the samples, were measured. The euhtural conditions were 37 ℃, 180 r/rain and 24 h; Identification： the selected strain was identified hy 16S rDNA and GyrA partial gene sequence analysis.We got 7 strains of G＋ haeilli yiehled a great quantity of proteases through preliminary screening. Then, after the re-screening, we gnl T1 standing out of these strains （P〈0.01）. With the molecular identification, we identified T1 is one of the strains of Bacillus amyloliquefaeiens. Therefore, we isolated, T1, a protease high-yield stain of Bacillus amyloliquetaeiens from lobster sauce. Its proteinase aetivity of the supernatant in the soybean meal medium reached 1 683.99 U while the fermentative conditions are 37 ℃, 180 r/min and 42 h.