弱化F_1F_0-ATP酶植物乳杆菌的突变分析及其作为益生添加物在四川泡菜中的应用探索

Reduced F_1F_0-ATPase Mutation Analysis of Lactobacillus Plantarum and Its Application in Sichuan Pickle as Probiotics Adjunct

从退化的植物乳杆菌中筛选到弱化F1F0-ATP酶突变株LPS08、LPS21和LPS24。在LPS08的F1F0-ATP酶β亚基中,Phe-91、Ser-380和Leu-436分别被Leu、Pro和Pro替换,导致F1F0-ATP酶的活性降低了5.61%;LPS21中,Leu-268突变改变了β亚基的构像,导致F1F0-ATP酶活性降低43.44%;LPS24中Asn-205、Ser-380和Pro-419分别被Ser、Leu和Leu所取代,降低F1F0-ATP酶活性19.46%。LPS08、LPS21和LPS24分别接入初始p H6.5的MRS液体培养基中37℃培养72 h后,p H值分别为3.73、3.82和4.25,酸度(乳酸计)分别为2.0、1.56和0.95 g/100m L。按106CFU/m L盐卤量分别将3株突变株接入p H4.6的无菌泡菜中,30°C孵育7 d后,仅有LPS21的泡菜盐卤p H维持在4.2以上,满足四川泡菜对酸味口感的要求。同时,盐卤中LPS21菌体的含量(7.27 log CFU/m L)满足国际上益生菌食品中关于活菌量的要求。因此,弱化F1F0-ATP酶LPS21突变株作为益生添加物添加到泡菜中生产益生菌泡菜时,能够从根本上解决益生菌泡菜在常温下贮存、运输和销售过程中的后酸化问题。

Three mutation strains L. plantarum LPS08,LPS21 and LPS24 with reduced F1F0- ATPase activity were screened from acid production degeneration L. plantarum CCTCC 207202. In F1F0- ATPase β- subunit of LPS08,Phe- 91,Ser- 380 and Leu-436 were replaced by Leu,Pro and Pro,respectively. But these mutations do not markedly affect F1F0- ATPase activity,only lead to reduction of activity of 5. 61%. In mutant strain LPS21,Leu- 268 mutation has changed the F1F0- ATPase β- subunit,and thus it triggered a great change with 43. 44% F1F0- ATPase activity reduction. In LPS24,the Asn- 205,Ser- 380 and Pro- 419 were respectively replaced by Ser,Leu and Leu. For these mutations,F1F0- ATPase activity decreased 19. 46%. In initial p H 6. 5 MRS liquid medium,the p H values respectively mediated by mutation strain LPS08,LPS21 and LPS24 were 3. 73,3. 68 and 4. 25,and their acidities were 2. 0 g /100 ml,1. 56 g /100 ml and 0. 95 g /100 ml after cultured at 37 °C for 72 h. Compared with parent strain CCTCC207202,only the mutation strain LPS21 could overcome post- acidification when their cultures were incorporated into aseptic Sichuan pickle products at 6 log CFU / ml salt brine level and kept at 30 °C up to 7 days,and its viable count in products( 7. 27 log CFU / ml)also satisfied the criteria for a probiotic food product. Therefore,the F1F0- ATPase mutant strain LPS21 is attractive as probiotics cultures adjunct in Sichuan pickle products to prevent post- acidification during the product handing,transportation and storage at ambient temperature.