Novel paired starter culture system for sauerkraut, consisting of a nisin-resistant Leuconostoc mesenteroides strain and a nisin-producing Lactococcus lactis strain.

Nisin-resistant Leuconostoc mesenteroides NCK293 and nisin-producing Lactococcus lactis subsp. lactis NCK401 were evaluated separately and in combination for growth and nisin production in a model sauerkraut fermentation. Strains were genetically marked and selectively enumerated by using antibiotic-containing media. The growth and survival of L. mesenteroides were similar in the presence and absence of Lactococcus lactis subsp. lactis. The growth of Lactococcus lactis subsp. lactis was not inhibited, although the maximum cell density was reduced and the population decline was more pronounced in the presence of L. mesenteroides. Nisin was detected within 24 h, and levels were relatively constant over the 12-day test period. The maximum cell populations and nisin level achieved could be altered by changing the initial cell ratios of L. mesenteroides and lactococcus lactis subsp. lactis. Isogenic nisin-producing and nisin-negative Lactococcus lactis subsp. lactis derivatives were used in combination with nisin-resistant L. mesenteroides to demonstrate that nisin levels produced in mixed culture were sufficient to retard the onset of the growth of nisin-sensitive, homofermentative Lactobacillus plantarum ATCC 14917.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).     

Locating nisin-producing Lactococcus lactis in a fermented meat system

S.C. STRINGER, C.E.R. DODD, M.R.A. MORGAN AND W.M. WAITES. 1995. Antibody-linked probes were used to locate nisin in a fermented meat system. Free nisin or nisin bound to susceptible cells or food components was not detected. Colonies of nisin-producing Lactococcus lactis were stained at all times during growth. The position of nisin-producing L. lactis colonies was noted and compared with the location of spoilage organisms or the distribution of areas with a fermented meat appearance. No relationship between the distribution of starter culture and the location of spoilage organisms or areas of fermentation was observed. In addition to the presence of L. lactis , a rapidly fermentable sugar was also required to obtain a fermented appearance and to reduce the levels of spoilage organisms.     

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).     

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation.

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.     

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.     

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.     

A nisin bioassay based on bioluminescence.

A Lactococcus lactis subsp. lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods.     

乳链菌肽细胞分子传感器的构建与应用

【背景】乳链菌肽主要是由乳酸乳球菌生产的一类多肽,对革兰氏阳性菌有抑菌作用,是目前联合国粮食及农业组织/世界卫生组织唯一批准使用的天然食品防腐剂。但是其产量低、缺乏简便高效的检测方法,限制了其研究和应用。【目的】构建一种可输出肉眼可见红色荧光的细胞分子传感器,以期能简单方便地检测样品中的乳链菌肽,同时应用该传感器筛选乳链菌肽生产菌株。【方法】用Golden-Gate克隆方法构建含乳链菌肽诱导启动子和下游红色荧光蛋白基因(两种)的载体,转入Lactococcus lactis中。用细胞传感器筛选可能的乳链菌肽生产菌株。【结果】构建的两种乳链菌肽细胞分子传感器都能对2?200 ng/mL乳链菌肽有灵敏的响应,可用于定量测定。两种传感器的最大荧光强度和表型也有所不同。利用细胞传感器确定了Lactococcus lactis ATCC 11454乳链菌肽的产生,同时排除了一个能产其他抗菌化合物的菌株。【结论】构建的细胞分子传感器能特异性地响应乳链菌肽,并能简单快速地筛选乳链菌肽菌株。     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.     

An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.     

Effects of Lactococcus lactis on composition of intestinal microbiota: role of nisin

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.     

Combined effect of bacteriocin-producing lactic acid bacteria and lactoperoxidase system activation on Listeria monocytogenes in refrigerated raw milk

The bactericidal activity of three bacteriocin-producing lactic acid bacteria alone and in combination with milk lactoperoxidase (LP) system activation against Listeria monocytogenes in refrigerated raw milk was studied. After 4 d at 4°C, the population of L. monocytogenes in milk inoculated with bacteriocin-producing Lactococcus lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Enterococcus faecalis INIA 4 was reduced by 0·21–0·24 log units. Activation of the LP system did not enhance inhibition at this temperature. After 4 d at 8°C, L. monocytogenes levels in the non-activated LP system milk inoculated with L. lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Ent. faecalis INIA 4 were reduced by 1·87, 1·54 and 1·11 log units compared to control milk, whereas in the activated LP system milk, this reduction was 1·99, 2·10 and 1·06, respectively. The higher nisin production by L. lactis subsp. lactis ESI 515 in milk with activated LP system than in non-activated LP system milk was responsible for the more pronounced decrease of L. monocytogenes counts in the former.     

乳酸链球菌肽发酵动力学的研究

提出了在恒定不同pH的发酵条件下,乳酸链球菌SM526的菌体生长、底物消耗、乳酸及Nisin产生的动力学模型。菌体生长、乳酸及Nisin产生用逻辑方程描述,而底物消耗是菌体生长和乳酸产生速率的函数。模型表明,乳酸链球菌SM526菌体生长和乳酸产生的最佳pH为7．0,而Nisin产生的最佳pH却为6．5。     

Behavior of psychrotrophic lactic acid bacteria isolated from spoiling cooked meat products

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10 degrees C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10 degrees C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4 degrees C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10(8) CFU/g at 10 degrees C after 7 to 12 days.