传统发酵牦牛酸乳中细菌素产生菌的筛选与鉴定

从西藏羊八井地区、云南香格里拉的传统牦牛酸乳中分离出26株乳酸菌,以枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌以及酿酒酵母为指示菌。采用牛津杯双层琼脂扩散法检测菌株的抑菌谱大小,并经过有机酸排除、过氧化氢排除、蛋白酶水解检测试验,最终筛选得到5株产细菌素的菌株,编号分别为3、23、24、21和25。通过微生物形态学与16SrDNA序列同源性分析,鉴定这些菌株分别为干酪乳杆菌、植物乳杆菌和乳酸乳球菌乳球亚种。抑菌谱试验表明,这些细菌素能够抑制部分食源性革兰氏阳性菌和阴性致病菌,对真菌无抑菌活性。     

1株产细菌素植物乳杆菌的筛选及其细菌素抑菌性质研究

从广西巴马地区取样的米粉中共分离出31株乳酸菌,以单核细胞增生李斯特菌为指示菌,采用牛津杯平板扩散法检测抑菌物质,筛选得到1株产细菌素的乳酸菌M23。通过微生物形态学与16S rRNA基因序列分析,鉴定该菌株为植物乳杆菌。植物乳杆菌M23代谢产生的乳酸菌细菌素在p H 2.0~10.0范围内具有良好的抑菌活性,121℃处理15 min的条件下依然具有抑菌活性,表明其具有良好的酸碱稳定性和热稳定性。发酵试验表明,该乳酸菌素的最佳收获时间为乳酸菌培养14 h。抑菌谱试验表明,该乳酸菌素能够有效抑制部分食源性革兰氏阳性、阴性致病菌。     

清香酒醅中高产细菌素乳酸菌的筛选及其细菌素特性研究

目的:从清香酒醅中筛选高产细菌素乳酸菌,分析其在酿酒环境中应用的可能性。方法:本研究采用牛津杯法筛选清香酒醅中具有抑菌活性的乳酸菌,结合形态学、16S rDNA法鉴定高产细菌素乳酸菌,并对酒醅乳酸菌细菌素的稳定性及对酿酒微生物的作用进行研究。结果:从清香白酒酒醅中分离的146株乳酸菌中,筛选到对指示菌金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)具有明显抑制效果的4株菌,经鉴定YP36为植物乳杆菌(Lactobacillus plantarum),MC15-1和MC49-1是短乳杆菌(Lactobacillus brevis),SL28为布氏乳杆菌(Lactobacillus buchneri)。细菌素稳定性方面,菌株YP36和SL28的细菌素在4~121℃范围内热稳定性无显著变化(P>0.05),菌株MC15-1和MC49-1抑菌能力在4~60℃范围内较稳定(P>0.05),121℃处理后的抑菌活性分别是4℃下的抑菌活性的68.4%和56.38%;4菌株细菌素的酸碱稳定性非常相似,在pH2.0~4.0范围内,抑菌活性无显著变化(P>0.05),pH为6.0时略有下降,当pH为8.0时抑菌活性消失。4株菌的细菌素对清香酒醅中酵母菌无抑制作用。结论:菌株YP36、MC15-1、SL28以及MC49-1理化性质良好,对乳酸菌在酒醅环境中应用具有重要的意义。     

筛选用于四川泡菜生物保鲜的产细菌素乳酸菌

从8 种中国传统四川泡菜中筛选产细菌素的乳酸菌,以用于四川泡菜的生物保鲜。从四川泡菜样品中共分离出227 株乳酸菌,并筛选出8 株能够产生细菌素类化合物抑制英诺克李斯特氏菌（Listeria innocua）、金黄色葡萄球菌（Staphylococcus aureus）、枯草芽孢杆菌（Bacillus subtilis）和蜡样芽孢杆菌（Bacillus cereus）生长的乳酸菌。其中Lactobacillus harbinensis B22、屎肠球菌E6（Enterococcus faecium E6）和植物乳杆菌E11（Lb. plantarumE11）能够产生抑制革兰氏阴性菌大肠杆菌（Escherichia coli）生长的细菌素类化合物。进一步研究表明,这3 株乳酸菌耐酸、耐高盐,能够在四川泡菜模拟培养基中生长并产生耐酸耐高温的细菌素类化合物。结果表明,Lactobacillus harbinensis B22、屎肠球菌E6和植物乳杆菌E11可用于四川泡菜的生物保鲜。     

一株产广谱细菌素乳酸菌菌株的分离鉴定及其细菌素特性研究

目的:从母乳婴儿粪便筛选获得产广谱细菌素的乳酸菌菌株,并对其所产细菌素的理化及生物学特性进行研究分析,预测其在食品工业中的应用前景。方法:以课题组从母乳婴儿粪便分离得到的23株乳酸菌为试材,采用牛津杯琼脂扩散法,选取单增李斯特氏菌(Listeria monocytogenes)和大肠杆菌(Escherichia coli)为指示菌筛选产广谱细菌素的优良菌株。通过菌体形态观察、生理生化特征及16S r DNA序列分析对菌株进行分类学鉴定,并从耐酸、耐热、蛋白酶敏感性、抑菌谱等角度分析菌株所产细菌素生物学特性。结果:分离得到1株产广谱细菌素菌株BL-1,发现该菌株所产细菌素具有较好的热稳定性和酸耐受性,在pH为2、121℃条件下细菌素活性仍然保持稳定,酶敏感性强,胃蛋白酶可将其完全分解,使用安全性较高,且抑菌谱较广,对受试的李斯特菌(Listeria monocytogenes)、金黄色葡萄球菌(Staphylococcus aereu)等革兰氏阳性菌和大肠杆菌阴性菌均表现出较强抑菌活性。最后该菌株经分类学鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)。结论:从母乳婴儿粪便中分离出一株产广谱细菌素鼠李糖乳杆菌BL-1,该菌所产细菌素具有优良的食品加工特性,有作为天然食品防腐剂的巨大应用前景。     

植物乳杆菌C8-1产类细菌素的初步研究

从发酵2周的泡菜中分离到66株乳杆菌,以其中4株乳杆菌作为出发菌株进行紫外诱变处理得到一株产类细菌素的突变株C8-1。基于细胞形态、生理生化和16S rDNA测序数据,菌株C8-1被鉴定为植物乳杆菌(Lactobacillus plantarum)。菌株C8-1所产的类细菌素具有较宽的抑菌谱,能够抑制大肠杆菌和金黄色葡萄球菌等多种食品腐败菌和致病菌;有较好的热稳定性,在4℃冷藏5 d和-20℃冷冻5d以及60,80,100℃和121℃分别加热15 min,活性损失均不超过6%;且抑菌活性随pH值增大逐步降低,当pH≥6时,抑菌活性消失,对胃蛋白酶、木瓜蛋白酶耐受性较强,对胰蛋白酶较为敏感。这些数据表明,产类细菌素的植物乳杆菌C8-1在食品加工中具有进一步地潜在应用价值。     

产乳酸菌素菌株的筛选鉴定及其特性分析

本实验对广西巴马村天然发酵米粉中的乳酸菌进行分离鉴定,筛选出一株具有抑菌活性的植物乳杆菌M44,并研究了其所产乳酸菌素的基本性质。采用微生物形态学和16S r DNA序列分析法鉴定菌株M44为植物乳杆菌,其发酵上清液具有抑菌活性,抑菌物质确定为蛋白类。采用盐析法粗提所产乳酸菌素,确定最佳硫酸铵沉淀浓度为60%。由乳酸菌素产量与菌株生长的关系实验得出乳酸菌素的最佳收获时间为发酵培养12 h。管碟法抑菌实验表明,该乳酸菌素在p H范围2~8时有良好的抑菌活性;121℃处理15 min后仍具有抑菌活性;胃蛋白酶、胰蛋白酶处理后抑菌性消失;对单核增生李斯特细菌、大肠杆菌、沙门氏菌,金黄色葡萄球菌均有抑制作用。该乳酸菌素为天然生物防腐剂的开发与应用提供了研究基础。     

生物保鲜乳酸菌的筛选及抑菌物质性质的初步研究

从收集保藏的乳酸菌中,筛选出对大肠杆菌、荧光假单胞菌、蜡状芽孢杆菌单核细胞增生李氏杆菌和金黄色葡萄球菌这些常见食品腐败菌都表现出良好抑菌效果,具有生物保鲜价值的5株乳酸菌,其中3株植物乳杆菌(ACCC10171、ACCC11095和ACCC11118),1株干酪乳杆菌(ACCC11052)和1株类干酪乳杆菌(ACCC11050)。在排除酸性末端产物和过氧化氢的干扰后,用蛋白酶K等多种蛋白酶处理这5株菌的代谢产物粗品,其抑菌活性都受到一定影响,甚至完全丧失抑菌活性,因此可以推断其代谢物质中含有对蛋白酶敏感的细菌素类物质。     

产细菌素乳酸菌的筛选、鉴定及抑菌特性研究

从市售豆腐乳中筛选并获得4株具有抑制常见食品致病菌的乳酸菌。经分子、生理生化鉴定,这4株乳酸菌分别为乳酸乳球菌(DH2002和DH2004)、粪肠球菌(DH2001)和肠系膜明串珠菌(DH2003)。4株乳酸菌抑菌产物具有细菌素的一般特征(即耐高温和蛋白酶处理后失活),同时4株产细菌素乳酸菌对革兰氏阳性菌具有抑菌能力,主要可以抑制厚壁菌门的相关种属和常见的食品致病菌,对革兰氏阴性菌无抑制作用。分析乳酸菌细菌素初提物对金黄色葡萄球菌的抑制作用,结果表明:DH2001和DH2002可完全抑制/消除金黄色葡萄球菌,而DH2003在一定时间内可以阻止金黄色葡萄球菌生长。     

新疆酸驼乳中细菌素乳酸菌的筛选及其抑菌性

通过双层琼脂扩散打孔法,从新疆哈萨克族传统发酵酸驼乳中分离的23株乳酸菌中,筛选出9株对金黄色葡萄球菌、枯草芽孢杆菌和大肠杆菌具有抑菌活性的菌株(其中茵株MLS5抑菌活性最强).通过有机酸、过氧化氢抑茵的排除试验和蛋白酶敏感性试验,初步确定MLS5产生的抑茵物质为蛋白质类细菌素.温度和pH耐受试验表明,该细菌素具有良好的热稳定性(121℃,20 min),pH 3～6具有抑茵活性.菌株MLS5的发酵曲线表明,菌株在37℃培养条件下具有良好的产细菌素和产酸能力,该细茵素在茵体生长对数中期产生,在生长稳定期抑茵活性达到最大.经抑茵谱试验表明,MLS5所产细菌素不仅能抑制革兰氏阳性细茵,而且对大肠杆菌和毛霉菌也有抑制作用,具有较广的抑茵谱.     

一株从泡菜中分离的产细菌素乳杆菌的鉴定及细菌素特性研究

从泡菜中分离的329 株乳酸菌中,筛选出一株产细菌素的乳酸菌,编号为SD-22。经生理生化实验鉴定为短乳杆菌(Lactobacillus brevis)。在排除过氧化氢干扰和有机酸抑菌作用后,brevicin SD-22 对革兰氏阳性细菌和革兰氏阴性细菌具有较好的抑制作用,对部分真菌也有抑制作用,但对酵母菌和青霉无抑制作用,具有良好的热稳定性和pH 值稳定性,经胰蛋白酶、胃蛋白酶和蛋白酶K 处理后抑菌活性完全消失,对α- 淀粉酶不敏感。因此初步认为该菌株是一株产广谱细菌素的乳酸菌。     

筛选自泡菜的发酵乳杆菌细菌素纯化及抑菌特性分析

从中国传统发酵蔬菜中筛选鉴定对食源性致病菌有广谱抗性的细菌素（命名为BLF52）产生菌株并对细菌素分离纯化和抑菌特性分析。分子筛凝胶过滤层析和半制备高效液相色谱（RP-HPLC）纯化细菌素并用Tricine-SDS-PAGE法测定其分子量。筛选得到产细菌素的发酵乳杆菌（Lactobacilus fermentum） CH-16。MRS培养基中34℃发酵至稳定期前期即24~30 h对S.aureus抑制活性最大（875 AU/mL）。BLF52纯化至单一组分,分子量5.6 kDa,40~80℃（30 min）、pH2.0~7.0（2 h）有良好稳定性。胃蛋白酶、木瓜蛋白酶、胰蛋白酶和蛋白酶K处理后,抑菌活性完全丧失,α-淀粉酶对其无影响,表明BLF52有典型的细菌素特性。BLF52对食源性腐败菌和致病菌如单增李斯特菌、金黄色葡萄球菌、蜡状芽孢杆菌、鼠伤寒沙门氏菌、铜绿假单胞菌、大肠杆菌和副溶血性弧菌均有抑制活性,通过杀菌模式抑制培养中的S.aureus生长。细菌素BLF52有潜力开发为适合酸性或中性食品使用的食品防腐剂。     

Characterization and antimicrobial activity of bacteriocin 217 produced by natural isolate Lactobacillus paracasei subsp. paracasei BGBUK2-16

The strain Lactobacillus paracasei subsp. paracasei BGBUK2-16. which was isolated from traditionally homemade white-pickled cheese, produces bacteriocin 217 (Bac217; approximately 7 kDa). The onset of Bac217 biosynthesis was observed in the logarithmic phase of growth, and the production plateau was reached after 9 or 12 h of incubation at 37 and 30 degrees C, respectively, when culture entered the early stationary phase. Biochemical characterization showed that Bac217 retained antimicrobial activity within the range of pH 3 to 12 or after treatment at 100 degrees C for 15 min. Bac217 antimicrobial activity also remained unchanged after storage at 4 degrees C for 6 months or -20 degrees C for up to 12 months. However, Bac217 activity was completely lost after treatment with different proteolytic enzymes. BGBUK2-16 contains only one plasmid about 80 kb in size. Plasmid curing indicated that genes coding for Bac217 synthesis and immunity seem to be located on this plasmid. Bac217 exhibited antimicrobial activity against some pathogenic bacteria, such as Staphylococcus aureus and Bacillus cereus. Interestingly, Bac217 showed activity against Salmonella sp. and Pseudomonas aeruginosa ATCC27853. The inhibitory effect of BGBUK2-16 on the growth of S. aureus in mixed culture was observed. S. aureus treatment with Bac217 led to a considerable decrease (CFU/ml) within a short period of time. The mode of Bac217 action on S. aureus was identified as bactericidal. It should be noted that the strain BGBUK2-16 was shown to be resistant to bacteriocin nisin, which is otherwise widely used as a food additive for fermented dairy products.     

Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.     

Antilisterial and amylase-sensitive bacteriocin producing Enterococcus faecium SH01 from Mukeunji,a Korean over-ripened kimchi

A bacteriocinogenic strain identified as Enterococcus faecium SH01 was isolated from mukeunji, a Korean traditional over-ripened kimchi with antimicrobial activities against Listeria monocytogenes KCTC 3569 and Lactobacillus curvatus KFRI 166. The maximum bacteriocin titer (1,280 AU/mL) was detected at the early stationary phase and was maintained for 28 h with no activity loss. The bacteriocin activity, which disappeared after treatment with the proteolytic enzymes α-chymotrypsin, pronase E, proteinase K, and trypsin, was partially inactivated using α-amylase. The activity of bacteriocin SH01 remained after heat treatment (121°C, 15 min) and exposure to pH values from 2–12. The molecular weight of crude bacteriocin SH01 was 3 kDa. The bacteriocin production phenotype (Bac⁺) was linked to a 6 kb plasmid. Bacteriocin SH1 production was not induced by the co-presence of viable cells, heat treatment, or a cell-free indicator supernatant. The mode of action of bacteriocin SH1 was bactericidal.     

乳酸菌素生产菌的分离与鉴定

对乳酸菌素产生菌进行分离,得到4株理想菌株。利用高效液相色谱检测其发酵液,初步确定4菌株均为乳酸菌,其中Ⅱ32菌株产乳酸最多,对各指示菌的抑制作用最显著,对该菌株作进一步的研究。Ⅱ32菌株在MRS培养基上可以生长,最适生长温度为28℃,革兰氏染色呈阳性,无芽孢。通过API生化反应实验、16SrDNA测序可进一步确定该菌株是L·paraplantarum或L·paraplantarum。最终通过对recA-gen的PCR产物检测,鉴定菌株Ⅱ32为Lactobacillusparaplantarum(类植物乳杆菌)。牛津杯抑菌试验显示,该菌株的发酵液不仅对多数G+细菌的生长有较强的抑制作用,而且对大肠杆菌、沙门氏菌等Gˉ细菌的生长也具有明显的抑制作用,但对真菌没有活性。     

Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation

Autochthonous strains of lactic acid bacteria (LAB) have been isolated from traditionally homemade cheeses collected from specific ecological localities across Serbia and Montenegro. Genetic and biochemical analysis of this LAB revealed that they produce bacteriocins, proteinases and exopolysaccharides. LAB produces a variety of antimicrobial substances with potential importance for food fermentation and preservation. Apart from the metabolic end products, some strains also secrete antimicrobial substances known as bacteriocins. Among the natural isolates of LAB from homemade cheeses, bacteriocin producers were found in both lactococci and lactobacilli. Lactococcus lactis subsp. lactis BGMN1-5 was found to produce three narrow spectrum class II heat-stable bacteriocins. In addition to bacteriocin production, BGMN1-5 synthesized a cell envelope-associated proteinase (CEP) and shows an aggregation phenotype. Another isolate, L. lactis subsp. lactis BGSM1-19 produces low molecular mass (7 kDa) bacteriocin SM19 that showed antimicrobial activity against Staphylococcus aureus, Micrococcus flavus and partially against Salmonella paratyphi. Production of bacteriocin reaches a plateau after 8 h of BGSM1-19 growth. Bacteriocin SM19 retained activity within the wide pH range from 1 to 12 and after the treatment at 100 degrees C for 15 min. Among collection of lactobacilli, the isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8 produces heat-stable bacteriocin SJ (approx. 5 kDa) polypeptide. It retained activity after treatment for 1 h at 100 degrees C, and in the pH range from 2 to 11. In addition to isolates from cheeses, bacteriocin-producing human oral lactobacilli were detected. Most of them showed antimicrobial activity against streptococci, staphylococci and micrococci, but not against Candida. Isolate BGHO1 that showed the highest antimicrobial activity was determined as L. paracasei. Interestingly, Lactobacillus helveticus BGRA43, which was isolated from the human intestine showed strong activity against Clostridium sporogenes, but it was not possible to detect any bacteriocin production in this isolate by using standard procedures. Further analysis of antimicrobial activity revealed that BGRA43 has a relatively broad spectrum. Lactobacilli resistant to nisin were also detected among natural isolates. They produce bacteriocins, which have no activity against nisin producing lactococci.     

面包乳杆菌(Lactobacillus panis)C-M_2细菌素的分离纯化及特性分析

本实验对面包乳杆菌C-M2所产的新型细菌素进行分离纯化和特性研究。通过乙酸乙酯萃取、阳离子交换层析和半制备液相三步分离纯化该细菌素。最终细菌素的比活力达到5 044.96 AU/mg,纯化倍数为79.8倍,但回收率仅为0.35%。通过液相色谱-串联质谱法分析,该细菌素的分子质量为863.52 D,氨基酸序列为MVKKTSAV,它是一种新型的Ⅱ类细菌素。该细菌素具有广泛的抑菌谱,可以抑制革兰氏阳性和阴性的食品腐败菌。该细菌素对热和p H值稳定,即使在121℃灭菌15 min,仍保留82.1%的抑菌活性,在p H 6条件下保留85.6%的抑菌活性。它能被多种蛋白酶失活,不能被脂肪酶和淀粉酶失活,这些结果表明该细菌素具有作为食品生物防腐剂的潜在应用价值。     

Purification and partial characterization of antilisterial bacteriocin produced by Pediococcus pentosaceus KJBC11 from Idli batter fermented with Piper betle leaves

Bacteriocins are natural antimicrobial agents mainly act against closely related bacteria and at times unrelated organisms including various food spoilage and pathogenic organisms. Bacteriocins from Lactic Acid Bacteria (LAB) have been widely used for food preservation because of their safety nature. In this study, bacteriocin from Pediococcus pentosaceus KJBC11 was purified with the recovery of 15%, using cell adsorption‐desorption technique, gel permeation chromatography, hydrophobic interaction chromatography, and reversed phase HPLC successively. Molecular weight of bacteriocin KJBC11 was determined to be 4.5 kDa using Tricine SDS‐PAGE. The bacteriocin KJBC11 showed strong inhibitory activity against various Gram‐positive and Gram‐negative pathogens. Bacteriocin KJBC11 activity was not altered when treated with amylase, lipase, and trypsin but inactivated by protease and proteinase enzyme treatment. It was heat stable (100°C, 1 hr) and exhibited strong antimicrobial activity within the pH 3–7. Its mode of action was bactericidal in nature as revealed against Listeria monocytogenes showing leakage of internal contents out of the cell. Bacteriocin KJBC11 could belong to Class IIa‐pediocin like bacteriocins based on its characteristics as well as the presence of pediocin gene in the genome of the isolate KJBC11.

Practical applications

Studies on Lactic Acid Bacteria and bacteriocin have attracted increasing attention for a long time, due to their established role in food fermentation and preservation. The antimicrobial properties of bacteriocin became a trademark approach to achieve food safety and to counter the menace of antibiotic resistant pathogens. This study revealed a potent bacteriocin with wide range of antibacterial activity against various foodborne and clinically important pathogens to have potential application in food preservation and biomedicine.     

一种抗猪链球菌细菌素的生物学特性研究

为了开发新型天然抗菌物质,本研究以猪粪便中分离出的一株产抗猪链球菌细菌素产生菌屎肠球菌LPL420P06为试材,对其产生的细菌素进行了理化特性研究。结果表明,该细菌素具有良好的热稳定性,在90℃、30min热处理的条件下仍能保持90%以上的活性;易被胰蛋白酶、蛋白酶K、木瓜蛋白酶、中性蛋白酶失活,表明该细菌素是一类蛋白类物质;在pH5～9范围内37℃、4h处理,活性保持稳定;抑菌谱实验结果表明,该细菌素不仅对单增李斯特氏菌、金黄色葡萄球菌、猪链球菌等多种革兰氏阳性病原菌有明显的抑制作用,而且对沙门氏菌、大肠杆菌等部分革兰氏阴性菌有不同程度的抑制作用,显示出该细菌素具有很好的开发应用前景。