Biological Products ; Biotechnology ; history ; China ; Food Preservatives ; History, 20th Century ; History, 21st Century ; Nisin

The objective of this study is to propose a more accurate and faster MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay (MCA) for quantitative measurement of polypeptide bacteriocins in solutions with nisin as an example. After an initial incubation of nisin and indicator bacterium Micrococcus luteus NCIB 8166 in tubes, MTT was added for another incubation period. After that, nisin was quantified by estimating the number of viable bacteria based on measuring the amount of purple formazan produced by cleavage of yellow tetrazolium salt MTT. Then MCA was compared to a standard agar diffusion assay (ADA). The results suggested a high correlation coefficient (r(2)=0.975+/-0.004) between optical density (OD) and the inhibitory effect of nisin on a bacterial strain Micrococcus luteus NCIB 8166 at a range of 0.125-32 IU/ml. The MCA described in this study was very quick. Quantification of nisin took only 7-8 h and the detection limit was at the level of 0.125 IU/ml when compared to 12 IU/ml and 24-28 h for ADA. The MCA provides an accurate and rapid method for quantification of nisin in solutions and is expected to be used for quantification of other antimicrobial substances.
Bacteriocins ; analysis ; metabolism ; Colorimetry ; methods ; Immunodiffusion ; Micrococcus luteus ; metabolism ; Nisin ; Regression Analysis ; Tetrazolium Salts ; analysis ; Thiazoles ; analysis ; Time Factors

Polyhydroxyalkanoates (PHAs), as a novel class of biopolymer, are attracting more attention due to their diverse material properties and environment-independent biodegradability. Here we report the preparation of PHA exhibiting efficient antibacterial activity by embedding Nisin, a food additive generally recognized as safe, into poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a type of PHA with high biocompatibility. We first prepared Nisin-containing PHBHHx films using solvent casting method. Confocal laser scanning microscopy analysis showed that a well-mixed integrated structure of the films with an even distribution of the Nisin particles in the PHBHHx matrices. Then the antimicrobial activity of PHBHHx/Nisin films against Micrococcus luteus was quantified on agar plate by measuring the size of inhibition zone. Cultivation in liquid media further confirmed the releasing of Nisin from the films and the long-time antibacterial activity. Results showed that the threshold of Nisin concentration for long-time and effective inhibition against bacteria growth is 25 μg/g. These results altogether establish a technological foundation for the application of PHA in biomedicine and food industry.
3-Hydroxybutyric Acid ; chemistry ; Anti-Bacterial Agents ; chemistry ; Caproates ; chemistry ; Micrococcus luteus ; drug effects ; Nisin ; chemistry ; Polyhydroxyalkanoates ; chemistry

Nisin is an antimicrobial peptide widely used in food industry. In this study, Nisin A production in Lactococcus lactis ATCC 11454 was improved by overexpression of Nisin A structural gene nisA through introducing a shuttle expression vector pMG36ek-nisA and an integrated vector pDG780-nisA into the host strain. The differences of growth profiles and Nisin A production level between the two obtained genetic engineering strains FMM1/FMM2 and the parent strain were investigated. Our results show that while the growth profile (the growth rate, biomass and pH) of FMM1 was similar to the parent strain, its Nisin A production increased 31%. In contrast, the biomass of FMM2 was notably lower than the parent strain, while its yield of Nisin A enhanced slightly. The transcription level of genes involved in Nisin A biosynthesis in both engineering strains was further detected by RT-PCR. We found that all the 11 Nisin A biosynthetic genes in FMM1 and FMM2 had a higher transcription level than those in the parent strain, and these genes exhibited more significant increasing degree of transcription level in FMM1 which hosted the autonomous replicating nisA gene. These data suggest that expression of nisA may act as a rate-limit factor in Nisin A biosynthesis. In conclusion, this work provides a new method to improve Nisin A production by increasing the transcription level of nisA, paving the way to further large-scale industrial production of Nisin A.
Bacterial Proteins ; genetics ; Genes, Bacterial ; Genetic Engineering ; Genetic Vectors ; Lactococcus lactis ; genetics ; metabolism ; Nisin ; biosynthesis ; genetics ; Transcription, Genetic

The possibility of heterologous expression of human Stearoyl-CoA Desaturase (scd1) was investigated. The scd1 encoding sequence was inserted into the pNZ8149 to generate the pNZ8149-scd1 expression plasmids. Then we introduced the pNZ8149-scd1 construct into the Lactococcus lactis NZ3900 to investigate its enzyme activity. The results show that heterologous expressed SCD1 enzyme resulted in a 92%-169% increase in the C16:1n-7 and a 53-127% increase in the C18:1n-7 (P<0.05). The SCD1 enzyme was capable of producing n-7 fatty acids in Lactococcus lactis efficiently. It also suggests that the fatty acid desaturases can be heterologous expressed in Lactococcus lactis to produce the helpful fatty acids.
Electroporation ; Humans ; Lactococcus lactis ; genetics ; metabolism ; Mutagenesis, Insertional ; Nisin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Stearoyl-CoA Desaturase ; biosynthesis ; genetics

According to the knowledge gained from engineering of nisinZ, using plasmid pHJ201 DNA as template, NisinZ was mutated by site-directed mutagenesis, NisinZ mutant T8S contains Serine at position 8 instead of Threonine, NisinZ mutant N27K/H31K contains Lysine at position 27 and 31, respectively, instead of Asparagine and Histidine and NisinZ mutant T2S/ H31K contains dehydrobutyrine and Lysine at position 2 and 31 instead of dehydroalanine and Histidine. They are cloned into pMG36e and expressed in L. Lactis NZ9800, the expression products of these mutants purified by Sephadex CM-25 and Sephadex G-25 chromatography, some properties of NisinZ mutants (T8S, T2S/H31K and N27K/H31K) were studied. The results showed that the spectrum of antimicrobial activity and solubility of these mutants had not been changed, their antimicrobial activities were found to be slightly lower than that of the wild-type NisinZ. but mutants T8S and T2S/H31K showed higher stability, which were significantly more stable than wild-type NisinZ at 55 approximately 100 degrees C and pH7 approximately 9.
Anti-Bacterial Agents ; metabolism ; pharmacology ; Bacteria ; drug effects ; Electrophoresis, Polyacrylamide Gel ; Mutagenesis, Site-Directed ; methods ; Nisin ; analogs & derivatives ; genetics ; metabolism ; pharmacology ; Protein Stability ; Solubility ; Temperature

OBJECTIVETo isolate and identify nisin resistance determinant (NSR) gene from Lactococcus lactis.

METHODSThe Lactococcus lactis strains harboring NSR gene were isolated from different milk samples by selective culture supplemented with nisin and confirmed by PCR detection of 16S rRNA. Nisin resistance determinant gene was determined by PCR amplification, enzyme digestion and sequencing.

RESULTSThirty nisin-resistant Lactococcus lactis strains from fresh milk samples were obtained. Three of these strains contained NSR gene of about 1000 bp as determined by agarose gel electrophoresis and further confirmed by enzyme digestion and sequence analysis. The NSR gene was located on the plasmid of Lactococcus lactis.

CONCLUSIONComplete NSR gene, located on the bacterial plasmid, has been successfully isolated from nisin-resistant Lactococcus lactis strains from fresh milk.

Animals ; Anti-Bacterial Agents ; pharmacology ; Cattle ; Drug Resistance, Bacterial ; genetics ; Lactococcus lactis ; genetics ; isolation & purification ; Milk ; microbiology ; Nisin ; genetics ; pharmacology ; Plasmids ; genetics ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; genetics