自然青贮饲草和发酵蔬菜中产乳酸菌的分离鉴定及其特性研究

Isolation and characterization of lactic acid-producing bacteria from naturally fermented silages and vegetables

从优质构树叶和燕麦自然发酵青贮以及泡菜和盐菜等传统自然发酵蔬菜中分离鉴定产乳酸菌株,并揭示其生长特性与产酸特性等,研究其对玉米秸秆青贮饲草发酵品质的影响。采用平板划线法分离单菌落,用16S rDNA序列测序鉴定其种属名,采用分光光度计法绘制不同菌株的生长曲线和pH耐受性,pH计法绘制产酸曲线,HPLC法测定菌株发酵过程中产生有机酸种类及含量,烘干法测定干物质,凯氏定氮法测定粗蛋白质,范式法测定粗纤维、酸性洗涤纤维和酸性洗涤木质素,蒽酮比色法测定可溶性糖,灰化法测定灰分。结果表明,从不同植物发酵材料中分离的产酸菌种类有差别,共鉴定出6株植物乳杆菌(Lactobacillus plantarum)、6株短乳杆菌(Lactobacillus brevis)、27株弯曲乳杆菌(Lactobacillus curvatus)、35株布氏乳杆菌(Lactobacillus buchneri)、1株戊糖片球菌(Pediococcus pentosaceus)、1株科氏葡萄球菌(Staphylococcus cohnii)以及19株芽孢杆菌属(Bacillus sp.)和1株肠球菌属(Enterococcus sp.)。其中,弯曲乳杆菌G3、植物乳杆菌G17和Y17、短乳杆菌Y4、布氏乳杆菌PC-C1、芽孢杆菌属YC1-1-4等菌株的生长速度和产酸速度均较快。植物乳杆菌对低pH的耐受性最强,其次是布氏乳杆菌、短乳杆菌和戊糖片球菌,而弯曲乳杆菌最差。HPLC分析表明,G3、G17、Y4、YC1-1-4、PC-C1以及YC2-6和G18具有较高的有益有机酸(乳酸、乙酸和丙酸)总产量。产酸菌用于调制玉米秸秆青贮,可显著提高饲草中粗蛋白质含量,显著降低酸性洗涤纤维和酸性洗涤木质素的含量。

The lactic acid producing bacteria were isolated from the naturally fermented silages of Broussonetia papyrifera leaves and whole-crop oat as well as from the traditional fermented and salted vegetables,and their growth and acid-producing characteristics were unveiled to study their effects on fermentation quality of corn stalk silage.Single clones of acid-producing bacteria were obtained through plate streaking method and their taxa were identified based on 16S rDNA sequences.Growth curves and pH tolerance abilities of bacteria were studied using spectrophotometric method,and acid-producing curves of bacteria were measured with a portable pH meter.The organic acid kinds and concentrations produced by bacteria were determined using HPLC.Drying method for determination of DM,Kjeldahl method for determination of CP,paradigm method for determination of CF,ADF and ADL,anthrone colorimetry for determination of WSC,ashing method for determination of Ash.Results showed that a total of 96 strains were isolated and identified,including 6 strains of Lactobacillus plantarum,6 strains of Lactobacillus brevis,27 strains of Lactobacillus curvatus,35 strains of Lactobacillus buchneri,1 strain of Staphylococcus cohnii,1 strain of Pediococcus pentosaceus,19 strains of Bacillus sp.,and 1 strain of Enterococcus sp.Among them,L.curvatus G3,L.plantarum G17 and Y17,L.brevis Y4,L.buchneri PC-C1,and Bacillus YC1-1-4 possessed a higher growth rate as well as organic acid producing rate.L.plantarum had the highest tolerant ability to initial low pH in culture medium,followed by L.buchneri,L.brevis,and P.pentosaceus,and the lowest was L.curvatus.HPLC analysis indicated that the strains of G3,G17,Y4,YC1-1-4,PC-C1,YC2-6,and G18 produced the highest yield of beneficial organic acids including lactic acid,acetic acid,and propionic acid.These acid-producing bacteria were used for producing corn stalk silage,in which the content of crude protein was significantly increased and the contents of acid detergent fiber and acid detergent lignin were reduced.